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www.europeanpharmaceuticalreview.com Issue 4 2013
ELN: a component of a largerinformatics puzzle
Michael H. Elliott, CEO, Atrium Research
Cell based label-freeassays in GPCR drug discoveryNiklas Larsson, Linda Sundström, Erik Ryberg and Lovisa Frostne, AstraZeneca
The rapidmicrobiological
methods revolutionEmanuele Selvaggio, QA Batch Disposition &
Investigation Supervisor, Pfizer
1.877.CRIVER.1www.criver.com/emd
As keepers of the industry’s largest microbial database, our scientists handpick
Learn more at www.criver.com/emd
Charles River supports the pharmaceutical,
medical device, nutraceutical, personal care and
cosmetics industries with its Accugenix® microbial
identification services. Having identified more than
a million samples of unknown microorganisms
over the past decade, our methods have been
shown to increase the efficacy of microbial
identification platforms for environmental
monitoring applications.
While a number of microbial identification
systems are available to the pharmaceutical
market, these platforms tend to underperform
because they do not support the broad range of
microorganisms encountered in environmental
monitoring programs. The associated reference
libraries are often biased toward clinical, rather
than environmental, organisms.
In 2010, we began offering AccuPRO-ID®
microbial identification services, a polyphasic
approach utilising MALDI-TOF and AccuGENX-ID®
16S rDNA sequencing, assuring the highest
accuracy and reportable rate. Since then, we have
processed over 80,000 samples using the Bruker
Daltonics MALDI Biotyper system and continue
to optimise the platform to better accommodate
environmental isolates.
A key activity for achieving better
performance is the continuous expansion of our
reference library. The number of microbial species
frequently encountered in the environmental
arena is tremendous and much higher than
realised by most. The inclusion of these species
in any microbial identification platform’s reference
library is critical to improving reporting and/or
accuracy for nonclinical applications. Therefore,
we developed a systematic approach for adding
relevant microorganisms to the MALDI-TOF
reference library.
The species that most frequently fail to
generate an identification using the MALDI
Biotyper system are frequently analysed by
16S sequencing, enabling the addition of these
fall-through species to the Accugenix® MALDI-TOF
reference library.
To date, these library entries generate
identifications more than twice as frequently as the
entries provided by the manufacturer, confirming
that the most frequent fall-throughs are added to
the Accugenix® reference library first.
The additional library entries generate nearly
37 per cent of all identifications, increase the
number of identifications by more than 10 per cent
and effectively reduce the nonreportable rate by
39.4 per cent.
With continued updates to the Accugenix®
MALDI-TOF reference library, the
percentage of samples generating
identifications will continue to rise.
While low cost and rapid turnaround time
remain strong advantages for MALDI-TOF
microbial identification platforms, our data
demonstrate that building a reference database
inclusive of environmental organisms is essential
to its suitability for nonclinical applications.
Methods
All data presented in this study were generated
from unknown samples received for microbial
identification. Samples submitted for AccuPRO-ID®
MALDI-TOF identification were processed using a
direct formic acid lysis method and/or the extract
method (per Charles River SOPs and the
manufacturer’s instructions) and analysed using
the Bruker Daltonics MALDI Biotyper software.
Samples with a ‘match factor’ greater than 1.75
and separated by > .1 were ascribed probable
species-level identifications. All samples that failed
to produce an identification using the MALDI
Biotyper system were submitted for AccuGENX-ID®
16S rDNA sequencing, where sequence data were
manually assembled and compared against the
validated and proprietary Accugenix® 16S reference
library. Qualified data analysts determined
identifications and confidence levels. 16S-based
sequence identification is used as the reference
method for this study.
The impact of a customised MALDI-TOF library forenvironmental microbial identifications: reducing the
nonreportable rate by 39.4 per cent
Figure 1: Diversity of species routinely identified
Figure 2: Systematic approach for the inclusion of newMALDI-TOF library entries
Figure 3: New entries target the most frequentlyoccurring species
Figure 4: Number of library entries and frequency of additions
Figure 5: Impact of a customised database onreportable identifications
Figure 6: Source of library entries leading to MALDI-TOF identification
For more information about
Accugenix® microbial identification services,
please visit www.criver.com/emd
or contact us at [email protected]
With recent revisions to USP General Chapters 41 and 1251, the pharmaceutical industry
needs to update their SOPs for testing of weighing equipment.
Good Weighing Practice™ (GWP®) from METTLER TOLEDO helps you get in the fast lane to
ensure compliance with USP while saving on testing costs.
Contact the weighing experts and learn more about how USP General Chapters 41 and 1251
affect your weighing processes.
Choose the Fast Lane to USP Compliance with METTLER TOLEDO
www.mt.com/lab-usp
email: [email protected]
When it comes to large, global, multi-site companies, rolling out a new
informatics system can take up to three years from start to finish to implement
across the entire business. While this may seem archaic in today’s age and with
the technology available, pharmaceutical companies have the added
challenge of dealing with highly sensitive data, usually in very large quantities.
Making the transition to a paperless laboratory is a long-term investment and
strategy for a business, usually involving a large investment cost that may not
see immediate gains for the company’s bottom line. With the mergers
and acquisitions that have become a staple of the pharmaceutical industry
over recent years, the added problem of integrating two, three or more different
systems used by different companies then complicates matters further. Is it time for
Pharma to fully embrace cloud computing, which would enable companies to
integrate data much faster? While it seems that the industry recognises the need
for cloud computing, in reality it could take years for cloud platforms to be embraced
by an industry that manages to be both forward thinking and yet reluctant to leave
tried-and-tested methods behind. The end result though will drive productivity and
decision-making as the data becomes easier to handle, analyse and ultimately produce
results for the company.
In this issue of European Pharmaceutical Review, we look at two areas of the
pharmaceutical industry using informatics – biobanking and biologics development
– in the informatics in-depth focus, which begins on page 23. Michael H. Elliott, CEO of
Atrium Research and Editorial Board Member of European Pharmaceutical Review,
looks at the components that make up the larger informatics puzzle and how ELN is
just one piece of the puzzle, while Eva Bürén, Head of IT at the Karolinska Institutet
Biobank looks at how LIMS supports researchers using biobank samples.
If you would like to contribute to European Pharmaceutical Review with the
latest news, editorial contribution or general interest, please send me an email at
[email protected] with your request.
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 3 Volume 18 | Issue 4 | 2013
Registered Office as above.
Russell Publishing Ltd, is registered
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Number 2709148
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EDITORIAL BOARDAnthony Davies
Director Irish National Centre for High Content Screeningand Analysis Department of Clinical Medicine,
Trinity College Dublin
Sheraz GulVice President and Head of Biology,
European ScreeningPort GmbH
Matthew MoranDirector, PharmaChemical Ireland
Don ClarkPfizer Global Research & Development
Michael J. MillerPresident, Microbiology Consultants
Michael H. ElliottCEO, Atrium Research & Consulting
RUSSELL PUBLISHING LTD Founder Ian Russell
Managing Director Vivien Cotterill-Lee
Editor Helen Bahia
Deputy Editor Annie McKenna
Senior Publications Assistant Karen Hutchinson
Group Sales Director Tim Dean
Sales Director Freddy White
Senior Sales Executive Andrew Johnson
Production Manager Brian Cloke
Front Cover Artwork Steve Crisp
European Pharmaceutical Review (ISSN No: 1360-8606, USPS No: 023-422) is published bi-monthly (6 times per annum) byRussell Publishing Ltd, and distributed in the USA by SP/Asendia,17B S Middlesex Ave, Monroe NJ 08831. Periodicals postage paidat New Brunswick, NJ. POSTMASTER: send address changes toEuropean Pharmaceutical Review, 17B S Middlesex Avenue,Monroe NJ 08831.
European Pharmaceutical Review is published bi-monthly (six times per annum) and is available by subscription at £90.00 for a year, which includes on-line membership access.Back issue copies can be requested at £15.00 per copy.
European Pharmaceutical Review: Published by Russell Publishing Ltd, Court Lodge, Hogtrough Hill, Brasted, Kent, TN16 1NU, UK Tel: +44 (0) 1959 563311 Fax: +44 (0) 1959 563123 Email: [email protected]
ISSN 1360 - 8606Copyright rests with the publishers.All rights reserved©2013 Russell Publishing Limited
Helen Bahia, Editor
Investing in informatics
Welcome...
European Pharmaceutical Review is proud to be affiliated
with JPAG (Joint Pharmaceutical Analysis Group), a not-for-profit
organisation for pharmaceutical analysts. Free subscriptions are
available for members of JPAG. Please contact Karen Hutchinson
at [email protected] for further details.
We Analyze Endotoxin Data Every Day
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Biotechnica
2013
8–10 October, Hall 9 Booth D34
03 INTRODUCTIONInvesting in informaticsHelen Bahia, Editor
07 FOREWORDICHQ2(R1) Validation of Analytical Procedures: Challenges and OpportunitiesDave Elder, Joint Pharmaceutical Analysis Group and GSK
08 NEWS
09 INDUSTRY EXPERT PANEL
10 EVENTS
13 GPCRSCell based label-free assays in GPCR drug discoveryNiklas Larsson, Linda Sundström, Erik Ryberg and Lovisa Frostne, AstraZeneca
18 ION CHANNELSChloride ion channels andtransporters: from curiosities of nature and source of humandisease to drug targetsJonathan D. Lippiat, School of Biomedical Sciences,University of Leeds
35 SHOW PREVIEWMIPTEC
36 RAMAN SPECTROSCOPYRaman spectroscopy: an enabling tool for accelerating pharmaceuticaldiscovery to developmentChanda R. Yonzon, Atul Karande, Sai P. Chamarthy and Brent A. Donovan, Merck & Co. Inc
54 SIX SIGMAImproving operations andperformance: how Rottapharm isusing Lean Six Sigma principlesRichard Hayes, Continuous Improvement Manager,Rottapharm
58 SHOW PREVIEWDRUG DISCOVERY 2013
59 PROTEASESHow naturally occurring inhibitorscan facilitate small molecule drugdiscovery for cysteine proteasesSheraz Gul, Vice President and Head of Biology, European ScreeningPort GmbH
63 SHOW PREVIEWBIOTECHNICA
64 PRODUCT HUBWith Robert Mount, Managing Director, BMG Labtech
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 5 Volume 18 | Issue 4 | 2013
41 IN-DEPTH FOCUS:RMMS &ENVIRONMENTALMONITORING
Featuring articles from EmanueleSelvaggio, Pfizer on the RMMrevolution and Chris Delaney, NoonanServices Group, discussing how tocontrol contamination in cleanrooms
Contents
23 IN-DEPTH FOCUS:INFORMATICS
Eva Bürén, Head of IT, KarolinskaInstitutet Biobank looks at using LIMS for biobanking and Michael H. Elliott, CEO, Atrium Research looks at whether ELNs are a good match forbiologics development
Research is still your favorite?MAKE GREAT THINGS HAPPENOpportunities for natural scientists: Do you want to explore, research, change things? Welcome to Merck. With leading research in pharma-ceuticals, through analysis of microorganisms, all the way to liquid crystals for LCDs, Merck continues developing innovative products. We offer excellent development perspectives in challeng ing research
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The guidance covers typical criteria for re-
validation, including changes in API synthesis,
changes in the composition of drug product
or changes in the analytical procedure. However,
the guidance is silent with respect to the
requirements for method validation during
clinical trial development, the expectations for
analytical method technology transfer (TT) and
with respect to validation of extremely sensitive
and sophisticated methods, e.g. trace analysis of
genotoxic impurities. Consequently, there is
often regulatory confusion with respect to
applicability of the ICHQ2(R1) guidelines in
these areas.
There are surprisingly few publications on
the attributable causes for non-robust analytical
methodologies and yet this is a commonly
reported deficiency by regulatory agencies
across the globe. The EMA1 recently indicated
that, ‘During inspections, GMP inspectors
have noted that the root causes for out-of-
specification (OOS) results are sometimes
attributed to a lack of test method validation in
the context of transfer of analytical methods.
Such situations also occur frequently at contract
Quality Control laboratories.’ Therefore, despite
these methods being fully validated according to
ICHQ2(R1), they are still found to be non-robust
during routine QC testing; i.e. not fit for purpose.
Molnár2 attributed these issues to that fact
that ICHQ2(R1) was a statistically based guideline
with limited focus on the resultant chromato -
graphy, particularly critical resolutions. He
indicated that most methods are critically
dependent on a number of key parameters, e.g.
pH, stationary phase (type and batch), temp -
erature, mobile phase, buffer concentration,
etc and in a global economy, it is often difficult to
try and address all of this potential variability
within the existing robustness assessment
contained within ICHQ2(R1). He articulated
that, ‘Therefore, today, we need HPLC methods,
which are adjustable from location to location
to perform the analytical goal of correct
quantitation of the product components.’ This is
obviously only possible if the methods are
developed allowing adjustments between
certain clearly defined limits (termed ‘maps of
critical resolution’). his is the forerunner to apply -
ing Quality by Design (QbD) in the development
of methods and as long as the parameters reside
within pre-defined ‘proven acceptable ranges
(PARs)’, these parameters can be modified to get
the optimal resolution of key impurities (which
again are pre-defined).
A recent example of this QbD approach
was reported by Schmidt and Molnár3 for
the development and validation of a UPLC
method for a second generation anti-histamine
(ebastine) in API and drug product. The authors
utilised chromatography modelling software
(DryLab 4) to develop a Design Space, as per ICH
Q8, for the method, which they described as
‘a region in which changes to method param -
eters will not significantly affect the results.’
They then verified that the Design Space was
accurate with a relative error of prediction (REP)
of only 0.06 per cent. The method was fully
validated as per ICHQ2(R1). Specificity, including
baseline resolution for all impurity peaks could
be achieved in four minutes. This represents
a significant enhancement in productivity
(40-fold), versus the established Ph.Eur. method,
allowing an increased sample throughput of
360 samples/day.
The robustness of the method was assessed
by varying six critical parameters (+1, 0, -1); temp -
erature, ternary composition of the mobile phase,
flow rate, gradient time and initial and final
concentration of the mobile phase composition.
The resultant 729 experiments were statistically
modelled from the established Design Space and
demonstrated that the critical resolution factor of
2.0 can be achieved in all cases.
Additionally, in order to address the
robustness issue, some practioners across
the industry have also advocated the use of
pre-defined rugged stationary phases (rather
than random choice), and by first intent only
developing methods using these columns.
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 7 Volume 18 | Issue 4 | 2013
Foreword
Dave ElderJoint Pharmaceutical Analysis Group and GSK
ICHQ2(R1) Validation of Analytical
Procedures: Challengesand Opportunities
The International Conference on Harmonisation (ICH) guideline for the Validation of Analytical Procedures (ICHQ2(R1)) currently
covers validation procedures for the four most common analytical tests: identification tests, quantitative tests for impurities, limit
tests for the control of impurities and quantitative tests for the active moiety(ies) in APIs (active pharmaceutical ingredients) or
drug products. The key underlying concepts and strategies are equally applicable to other analytical methodologies; e.g. particle
size analysis, dissolution, etc. Typical validation parameters covered in the guideline include accuracy, precision, specificity,
detection limits (DL / LOD) and quantitation limits (QL / LOQ), linearity, range and robustness.
1. Concept paper on the revision of chapter 6 of the
EU GMP Guide Quality Control, 12th October 2010,
EMA/INS/GMP/632654/2010 http://www.ema.
europa.eu/docs/en_GB/document_library/
Regulatory_and_procedural_guideline/2010/11/
WC500099260.pdf Assessed on 07th May 2013
2. I.Molnár. Searching for robust HPLC methods –
Csaba Horvarth and the Solvophobic theory,
Chromatographia, 62 (2005) S7-S17
3. A.H.Scmidt, I.Molnár. Using an innovative Quality-
by-Design approach for the development of a
stability indicating UPLC method for ebastine in the
API and pharmaceutical formulations, J. Pharm.
Biomed. Anal., 78-79 (2013) 65-74
Reference
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 8
Sanofi appoints Carsten Hellmann and David Meeker tothe Executive CommitteeSanofi has announced the appointment of two
new members to the Executive Committee,
effective 1 September 2013. Carsten Hellmann
joins Sanofi from Chr. Hansen Holding A/S and
will take the position of Executive Vice President
Merial, our Animal Health Division, following
the decision of Jose Barella to leave the company
to pursue other opportunities. He will be based in
Lyon and will sit on the Executive Committee.
David Meeker, MD, currently CEO Genzyme
and member of the Global Leadership Team, will
join the Executive Committee as Executive Vice
President, Genzyme.
In his previous role, Carsten was Executive
Vice President, Global Sales, Chr. Hansen
Holding A/S, a position he took upon joining
the company in 2006. Chr. Hansen Holding A/S
is a global biopharmaceutical company that
specialises in natural ingredient solutions for
the food, nutritional, pharmaceutical and agri -
cultural industries.
Merial, the Animal Health Division of
Sanofi, is the world’s third-largest animal
health company and the leader in companion
animal products, rabies vaccines and foot-and-
mouth vaccines with sales of more than
two billion Euros. A fully-fledged member of
the Sanofi family since 2011, there are a number
of significant opportunities for Merial that
remain untapped, particularly in production
animals and emerging markets. As Head of
Merial, Carsten will be tasked with bringing the
opportunities to fruition.
David Meeker was appointed CEO,
Genzyme, in November 2011 following
the acquisition of Genzyme by the Sanofi
Group in February 2011. Under his leader-
ship, a new organisation was put in place
maximising Genzyme’s unique expertise and
know-how, while leveraging Sanofi’s scale
and capabilities. The new organisation incorp-
orates the Rare Disease business and the
Multiple Sclerosis franchise as well as associated
R&D, manufacturing and support functions.
With two new multiple sclerosis medicines,
Aubagio® and LemtradaTM, recently receiving
positive opinions from the European Medicines
Agency (EMA), the business is set on a trajectory
of growth.
www.sanofi.com
AstraZeneca and FibroGen collaborate to develop andcommercialise FG-4592, a treatment for anaemia inchronic kidney disease and end-stage renal diseaseAstraZeneca and FibroGen have announced that they have entered into a
strategic collaboration to develop and commercialise FG-4592, a first-in-
class oral compound in late stage development for the treatment of
anaemia associated with chronic kidney disease (CKD) and end-stage
renal disease (ESRD).
This broad collaboration focuses on the US, China and all
major markets excluding Japan, Europe, the Commonwealth of
Independent States, the Middle East and South Africa, which are covered
by an existing agreement between FibroGen and Astellas Pharma Inc.
The AstraZeneca-FibroGen joint effort will be focused on the
development of FG-4592 to treat anaemia in CKD and ESRD, and may be
extended to other anaemia indications.
FG-4592 is a small molecule inhibitor of hypoxia-inducible factor
(HIF), a protein that responds to oxygen changes in the cellular
environment and meets the body’s demands for oxygen by inducing
erythropoiesis; the process by which red blood cells are produced.
FG-4592 has the potential to address the considerable unmet medical need
for an effective treatment for anaemia that offers the convenience of oral
administration and an improved safety profile as compared to current
standards of care. At present, treatment options involve a combination
of injectable erythropoiesis-stimulating agents (ESAs) and iron
supplements. FG-4592 works through the body’s natural oxygen-sensing
and response system to help produce red blood cells. This can be
compared to the body’s natural response to conditions at high altitude,
where oxygen levels are low, which is to produce more red blood cells.
In Phase II clinical studies, FG 4592 met its primary objective of
demonstrating anaemia correction in treatment-naïve CKD patients not on
dialysis as well as maintenance of haemoglobin levels and anaemia
correction in patients on dialysis. FG 4592 has demonstrated this efficacy
combined with an acceptable safety profile in clinical trials, and has
been shown to achieve anaemia correction in the absence of intravenous
iron supplementation.
The companies plan to undertake an extensive FG-4592 Phase III
development programme for the US, and to initiate Phase III trials in
China, with anticipated regulatory filings in China in 2015 and in the
US in 2017. www.astrazeneca.com
Albiglutide USPDUFA dateextended bythree monthsGlaxoSmithKline plc has announced
that the US Prescription Drug User Fee
Act (PDUFA) goal date for albiglutide,
an investigational once-weekly treat-
ment for adult patients with Type 2
diabetes, has been extended by three
months to 15 April 2014 to provide time for
a full review of information submitted by
GSK in response to the Food and Drug
Administration’s requests.
GSK announced the submission of a
Biologics Licence Application to the US
FDA for albiglutide on 14 January 2013
and this was followed by the submission of
a Marketing Authorisation Application to
the European Medicines Agency (EMA) on
7 March 2013. The EMA filing is pro -
gressing to schedule. Albiglutide is not
approved for use anywhere in the world.
www.gsk.com
“Physical and chemical properties of materials are generally determinedin laboratories. How can these measurements be best employed topredict material performance at manufacturing scale? What are the gapsbetween academic knowledge and manufacturing practice?”John Comer, Chief Scientific Officer, Sirius Analytical Instruments Ltd: Among the
steps involved in pharmaceutical manufacturing, the more important include
sourcing raw materials, synthesising APIs, preparing formulations, manufacturing
tablets and packaging. Measured physical and chemical properties of materials play
a part in all these steps. Properties of raw materials must be checked to confirm their
quality. Industrial-scale synthesis must be optimised by chemical engineering
calculations involving measured values for physical and chemical properties of the materials.
As for formulation, once the formulation approach has been selected, the process must be scaled
up and again, calculations involving measured properties are of great value. Tablet manufacture
and packaging are highly automated but property measurement is still important, not least for
quality control and investigating the stability of a drug under storage conditions. There are
many gaps between academic knowledge and manufacturing practice, so I’ll focus on just one area,
the behaviour of weak acids and bases in solvents. This is an issue during process development
if solvents are to be used. Many APIs and molecular building blocks are ionisable molecules, and
gain or lose protons in response to changing pH. The pKa value expresses the pH at which half
the molecules in solution are charged and half are neutral. Scientists measure the aqueous pKa
value of APIs because it influences ADMET and pharmacokinetics. However, values change
considerably when drugs are dissolved in solvents. For example, the pKa of Ketoprofen, an
anti-inflammatory drug is 4.12 in water, but rises to 11.45 in pure isopropyl alcohol. Such
huge changes mean that aqueous pKa values cannot be used to optimise synthesis procedures.
Academics have demonstrated ways to measure pKa in solvents but the characterisation of each new
solvent involves a lot of experimental work. Industry could profit from better-measured values, but
only after more research is done.
“How are rapid and alternative microbiological methods going toreshape the industry's strategies for microbiology testing?”David Jones, Technical Services Director, Rapid Micro Biosystems: Three trends
seem to be appearing in the QC microbiology area. Test frequency and time to result
is changing; testing is becoming decentralised from the QC laboratory; and the tests
are becoming less technical to perform. Risk analysis is focusing test frequency on
the critical areas of the process while lower risk areas are tested less frequently.
Traditional methods tended to be a single time point taken during the production
cycle. New technologies and modified methods change this. Active air samplers can now take small
samples spread over the full production day to improve the data from the traditional method;
however, the traditional incubation is still required. New technologies improve on that to allow
continuous sampling of air throughout the production day and give real time data. Similar technology
can be used for water testing to continuously monitor the bioburden. Technologies to identify
contaminants are also becoming much faster with real time identification on single cells in under
15 minutes being possible.
PAT initiatives have driven testing closer to the production line. An automated active air sampler
for viable organisms can be attached to the filling line or cleanroom structure to test and generate a
result at the point of use. Small portable endotoxin test machines are available that allow a test to be
performed at site and give a result in 15 minutes. Automated testing equipment can now be placed in
the manufacturing area, to take the samples and perform the analysis without the need to transfer
samples to the QC lab. With the move to the line and the introduction of more automation,
test methods are becoming simpler to perform; machines interpret and report the data. As a result,
the QC staff doesn’t need a microbiologist to perform daily tests; rather, microbiologists can
concentrate on the critical tasks of interpreting the data and problem solving.
This edition of European Pharmaceutical Review’s Industry Expert Panel delves into
the gap between academic research and manufacturing practice, and reshaping
microbial testing strategies.
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 9 Volume 18 | Issue 4 | 2013
Industry Expert Panel
Paul WituschekVice President of Sales, Developmentand Clinical ServicesCatalent
Katherine BakeevDirector of Applications Support
B&W Tek, Inc
Tim FreemanManaging Director
Freeman Technology
Fredrik SundbergGlobal Director
GE Healthcare
Ian LewisMarketing ManagerKaiser Optical Systems
Joe GecseyLife Science Application Manager,HACH Particle Counter UnitHACH LANG
Harald StahlSenior Pharmaceutical TechnologistGEA Pharma Systems
Laurent LeblancPharmaceutical and CosmeticsCulture Media R&DbioMérieux Industry
Allen L. BurgensonManager, Regulatory Affairs
Lonza Walkersville, Inc.
John DubczakGeneral Manager,
Endotoxin and Microbial Detection
Charles River
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 10
September 2013
ELRIG Drug DiscoveryDate: 3 – 4 September 2013Location: Manchester, UK
w: www.elrig.org
EPR Workshop: Label-free assays for screeningDate: 10 – 12 September 2013Location: Hamburg, Germany
w: www.europeanpharmaceutical
review.com/workshops-events/
label-free-assays-september-2013
HUPODate: 14 – 18 September 2013Location: Yokohama, Japan
w: http://hupo.org/2013
Advanced Analytics for Therapeutic Proteins:from Research toManufacturingDate: 18 – 20 September 2013Location: Kloster Irsee, Germany
w: http://events.dechema.de/
analytics2013
3rd Conference Innovation Drug DiscoveryDate: 22 – 25 September 2013Location: Pisa, Italy
w: www.apgi.org/Pise2013
2nd InternationalConference and Exhibition on Biowaivers & BiosimilarsDate: 23 – 25 September 2013Location: Carolina, USA
w: www.omicsgroup.com/
conferences/biowaivers-biosimilars-
2013/index.php
12th Annual BiologicalProduction Forum 2013Date: 23 – 25 September 2013Location: Dusseldorf, Germany
w: www.biological
production.com?mc=EL
Cell Culture Asia Congress 2013Date: 24 – 25 September 2013Location: Singapore
w: www.cellcultureasia-
congress1.com/download-
agenda-marketing
MiptecDate: 24 – 26 September 2013Location: Basel, Switzerland
w: www.miptec.ch
The Future of European PharmaDate: 30 September – 1 October 2013Location: Brussels, Belgium
w: www.ispe.org/2013-flexible-
facilities-eu-conference
October 2013
BiotechnicaDate: 8 – 10 October 2013Location: Hannover, Germany
w: www.biotechnica.de
PDA 8th Annual Global Conference on PharmaceuticalMicrobiologyDate: 21 – 23 October 2013Location: Maryland, USA
w: www.pda.org
CPhL WorldwideDate: 22 – 24 October 2013Location: Frankfurt, Germany
w: www.cphi.com
November 2013
PEGS EuropeDate: 4 – 8 November 2013Location: Lisbon, Portugal
w: www.pegsummiteurope.com
Next GenerationSequencing Congress Date: 18 – 19 November 2013Location: London, UK
w: www.nextgenerationsequencing-
congress.com
World DrugManufacturing SummitDate: 25 – 27 November 2013Location: Dusseldorf, Germany
w: www.wdmsummit.com
December 2013
FT Global Pharmaceuticals andBiotechnology ConferenceDate: 3 – 4 December 2013Location: London, UK
w: www.ft-live.com/pharmabio
January 2014
SLAS 2014Date: 18 – 22 January 2014Location: California, USA
w: www.slas2014.org
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Register now at www.europeanpharmaceuticalreview.com/handheld-raman
Discover the benefits and applications of handheld Raman and how it can facilitatecompliance with PIC/S regulations for raw material identification.
This webinar will discuss the benefits and applications of handheld Raman, including thereasons the industry is interested in the technology, data integrity, method development, howsamples are scanned and the challenges that have been conquered. We’ll also discuss PIC/Sand how it is unifying the regulatory perspective globally, currently including 43 membercountries with applications of Brazil, Japan, South Korea and the United Kingdom under review.We’ll discuss how these regulations are moving towards 100 per cent container testing globallyand how handheld Raman technology can help to achieve it, as well as the importance ofvalidation of instrumentation and methodologies.
Supported by
Organised by:
Advantages of handheld Raman in thepharmaceutical industry
A European Pharmaceutical Review Date:Tuesday, 24 September 2013
Time:15.00 BST
Length:1 Hour
Sulaf AssiAssociate Lecturer in ForensicSciences, Bournemouth University
Presentation: Handheld Ramanspectroscopy for identification ofonline drug products
Katherine BakeevDirector of Analytical Services and Support, B&W Tek
Presentation: Impact of handheld Raman for meeting PIC/S requirements
SPEAKERS:
G protein-coupled receptors (GPCRs) are among
the most important target classes within the
pharmaceutical industry1. Of the currently
marketed small-molecule medicines, approxi -
mately 30 per cent target GPCRs2,3. Historically,
ligand interactions with GPCRs have been
analysed using binding assays providing both
affinity and kinetic data. This technology did not
however take into account functional aspects of
the ligands (for example agonism, inverse
agonism, allosteric effects or signalling path -
way). Many downstream signalling assays like
detection of cAMP, IP3, Ca2+-flux, β-arrestin
recruitment or ERK1/2 phosphorylation have
therefore been applied extensively, expand-
ing the knowledge of functional aspects of
ligands4 (Figure 1, page 14). These assays have
also made it possible to search for new drugs on
orphan receptors. The expanding knowledge
around signalling through different G-proteins,
G-protein independent signalling and signal
bias, have pinpointed the limitations with assays
based on detecting specific intracellular mess -
engers along one single pathway5. However
more recently, label-free biosensor tech-
nologies such as surface plasmon resonance
(SPR), resonant waveguide grating (RWG) and
cell impedance spectroscopy (CIS) have been
developed and applied in drug discovery,
providing an opportunity for the revelation
of pharmacology of greater physiological
and disease relevance than before, including
simultaneous capturing of signalling via
multiple pathways6.
In this review, we focus on the application of
RWG for measurements of DMR in intact cells
upon GPCR activation. DMR is a consequence of
morphological changes and changes in distri -
bution of cellular components after receptor
stimulation. This type of readout is still
somewhat of a black box, but consists of
elements such as protein recruitment, receptor
internalisation, reorganisation of the cyto -
skeleton, as well as altered cell adhesion
(Figure 2, page 15). Activation of all four major
GPCR coupling classes (Gαi/Gαo, Gαs, Gαq/Gα11
and Gα12/Gα13) can be captured with this
technology7, something which is unachievable
with most other platforms. Initial studies
suggested that signalling via different
G-proteins result in class-specific DMR kinetic
profiles8. Subsequent studies have however
revealed that kinetic signatures are cell type
specific and the picture is therefore more
G protein-coupled receptors are one of the major classes of therapeutic targets for a broad range of diseases. The most commonly
used assays in GPCR drug discovery measure production of second messengers such as cAMP or IP3 that are the result of activation
of individual signalling pathways. Such specific assays are unable to provide a holistic view of the cell response after GPCR
activation. This is now changing as label-free technologies and assays on whole cells have been developed that are unbiased
towards the specific downstream pathways and capture the integrated cell response. In this mini-review, we focus on the
application of one of these technologies, namely resonant waveguide grating (RWG) for measurements of dynamic mass
redistribution (DMR) in intact cells upon GPCR activation. Since the technology is sensitive and non-invasive, it is applicable to
most cell types, including primary cells with native receptor expression levels. We discuss how DMR assays have become an
important component of GPCR drug discovery screening cascades and may have the potential to improve the ability to predict if
compounds will be efficacious in vivo.
Cell based label-free assays in GPCR drug discovery
Niklas Larsson, Linda Sundström, Erik Ryberg and Lovisa Frostne
AstraZeneca
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 13 Volume 18 | Issue 4 | 2013
GPCRs
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complex than initially suggested. Nonetheless,
DMR provides a G-protein unbiased and path -
way sensitive technology as a powerful
complement to single pathway technologies in
the GPCR assay toolbox.
Examples of applications in the
drug discovery process
We and others have applied cell based label-free
DMR GPCR assays for different purposes in
various phases of early drug discovery (Figure 3,
page 16).
Hit finding
Ligands can be biased with respect to signalling
via different pathways, thereby making the
pharmacology observed highly dependent on
the assay selected9. The potential benefit of
capturing signalling via multiple pathways and
thereby cover compounds with biased
signalling makes DMR an attractive choice for
identification of novel chemical starting points.
However, only a limited number of large DMR
screens have been published so far, likely due to
the relatively recent introduction of the
technology, high plate costs and throughput
challenges including detection of slow
responses using kinetic reading. One published
example is a 100k screen for muscarinic M3
antagonists10. In this screen, a low hit rate
consistent with many other HTS formats was
observed. The output did not suffer from a high
rate of false positives. Most importantly, unique
hits that were not picked up in a corresponding
Ca2+ FLIPR® screen were identified and con -
firmed in a binding assay. Thus, this example
illustrates the potential of DMR to successfully
deliver a relatively clean but unbiased HTS
output including chemistry which is not
captured by single pathway technologies.
Iterative screening
Our own most common application of DMR is as
an integral part of screening cascades within the
lead generation phase, either as the primary
structure-activity-relationship (SAR) driving
assay or as an orthogonal assay. We define an
orthogonal assay as an assay that measures
activity on the same target as the SAR driving
assay but with an independent technology,
which is applied to strengthen confidence in
potency / efficacy measures from the SAR assay.
In this context, DMR assays are of particular
value where other screening friendly assay
alternatives are limited, for example, for GPCRs
such as GPR55 which signals via the Gα12/13
pathway7, for receptors lacking appropriate
radioligands, or for antagonist screens on Gαi
coupled receptors. In addition, DMR assays are
often technically straightforward to establish.
Some concerns about the biological validity
of DMR assays to drive SAR have been raised due
to the undefined / black box nature of the
readout. However, this can be mitigated by
control experiments to confirm target depend -
ence such as demonstrating absence of
compound effects in comparable cell lines
lacking the target. It is also crucial to establish
early correlations between DMR and pathways
specific or binding assay platforms to interro -
gate the chemical series of interest with respect
to potential signal bias. Such a correlation, which
is a prerequisite for application in screening
cascades, has been established both for
overexpressed and endogenously expressed
receptors and is our own experience as
exemplified by the bradykinin B1 receptor
(Figure 3B, page 16).
Translation to biologically relevant
cell models
It is often a key challenge within early drug
discovery projects to establish a link between
assays using recombinant reagents and more
biologically or disease relevant cell models.
For example, overexpression of receptors can
shift both the potency and efficacy of com -
pounds (often overestimate) due to spare
receptors, thus not reflecting the pharmacology
in a native tissue11. Moreover, native tissues also
ensure the right G-protein composition and
expression relative to receptor levels, and this
may even be tissue-specific. Switches in
G-protein coupling have frequently been seen
for receptors expressed in different cell lines,
but are not limited to engineered cells12.
For example, endogenous dopamine D1/D2
receptor responses couples to different
G-proteins in different cell types12 highlighting
the import ance of selecting disease relevant
cells also for in vitro screening.
The high sensitivity of DMR assays enable
measurements of signalling after activation of
endogenously expressed GPCRs. Most import -
antly, since DMR assays do not require genetic
manipulation of cells, even primary cells
from patients can potentially be employed.
An example using non-engineered cells is a
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 14
GPCRs
Figure 1: GPCR signaling pathways
Simplified illustration of ligand-receptor binding and activation of signaling pathways by different G-proteins.Binding of ligands to the receptor, exchange of GTP to GDP and phosphorylation followed by β-arrestin recruitmentis fundamental and applies to all G protein families whereas other processes are G protein specific. Commonly usedread-outs for studying GPCR activation are indicated by blue, italics; GTPγS, cAMP, Ca2+, IP1, β-arrestin and binding,all of which are based on technologies using labels (e.g engineered cell lines or assay reagents) for detection. DMRis a label-free technology covering all activities that lead to a cell response and can be applied on intact native cells
‘‘The high sensitivity of DMR assaysenable measurements of signalling
after activation of endogenouslyexpressed GPCRs’’
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1280 compound screen using human epider -
moid carcinoma A431 cells endogenously
expressing histamine H1 and β2 adrenergic
receptors13. With respect to cells from native
tissue, it has also been shown that DMR has the
potential to detect prostaglandin E1 (PGE1)
activation of EP2/EP4 prostanoid receptors in
primary human keratinocytes7. In our own lab,
we have shown that activation of Bradykinin B1
receptors (B1R) can be monitored in primary
human preadipocytes and that the DMR
response is target mediated as it can be reduced
by knockdown of B1R using siRNA, demon -
strating specificity of the response (Figure 3C,
page 16). In summary, DMR assays commonly
have sufficient capacity and sensitivity to
provide a relatively high throughput link
between assays on recombinant reagents and
disease models to ensure relevant potency,
efficacy and coupling mechanisms of hit or lead
series early on.
Molecular mechanisms of action studies
Molecular mechanism of action (MMOA) is
defined here as the interaction between a
compound and its target, resulting in a specific
response3. These specific molecular interactions
link structure to function to provide a bio -
logically meaningful response. MMOA studies
are important for selection of functionally
relevant compound series and are generally
performed on small compound sets during late
lead generation and lead optimisation.
A key application in this context is
characterisation of signalling pathways in
physiologically relevant cells. The relative
contribution of different pathways can be
addressed by co-treatment with G-protein /
pathway specific blockers such as pertussis
toxin (Gαi), cholera toxin (Gαs) and YM254890
(Gαq)7 with subsequent measurement of the
remaining signal after agonist stimulation.
When inhibitors are not available, dominant
negative G-protein forms or siRNA can be used
as alternative tools7. Bock e t a l have taken
mechanistic studies one step further by taking
GPCRs
Figure 2: The principle of Dynamic Mass Redistribution (DMR)
Receptor activation results in numerous cellular processes ultimately leading to mass redistribution within the cell.The DMR causes a change in refractive index which can be measured as a change in wavelength (picometer range)using resonant waveguide grating. Available platforms for detection of DMR are the Epic®, Enspire® (with Epicmodule) and SRU Bind® readers. A) Baseline measurement prior to agonist stimulation. B) Measurement afteragonist stimulation, capturing the DMR
advantage of DMR and dualsteric ligands (i.e.
an orthosteric ligand physically linked to an
allosteric modulator) for elucidating the
dependence of promiscuous G-protein activa -
tion upon conformational rearrangements
within the extracellular domain14.
With respect to other MMOA applications,
it is technically possible to assess conforma-
tional mechanisms (e.g. surmountable /
insurmountable, competitive / non-competitive
compounds) but here DMR assays do not offer
any obvious benefits compared to classical
techniques. In contrast, for determination of
efficacy, DMR assays have the potential to
provide relevant measures over the range
from full agonists via partial agonists to
inverse agonists12. This is of particular value in
cases where it is possible to measure on
native(like) cells with endogenous receptor
expression levels.
Concluding remarks
Plate-based label-free technologies like RWG
that can be employed for cell based applications
such as DMR assays have been available for drug
discovery for approximately five years and the
number of applications is still growing. Since
the technology is sensitive, non-invasive and
able to detect signalling via multiple pathways,
it can be applied on disease relevant cell
systems. These properties offer the possibility to
use the same type of readout for hit finding,
iterative screening, (including species and target
selectivity), and disease relevant cell models and
may help to improve the ability to predict if
compounds will be efficacious in vivo.
In the future, we expect label-free cell based
assays with sufficient throughput for early drug
discovery to be commonly applied in com -
pound screening on disease relevant cells,
generating valuable chemical starting points to
be developed into tomorrow’s medicines.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 16
GPCRs
Dr. Niklas Larsson obtained his PhD 1999
in Cell and Molecular Biology at Umeå
University, Sweden. Since 2000, he has been
working with early drug discovery projects at
AstraZeneca (Mölndal, Sweden) in different
positions such as Project Leader, Team Leader
and currently as Associate Principle Scientist
within the Discovery Sciences function. His scientific focus is drug
discovery and molecular pharmacology of GPCRs.
Linda Sundström has over 10 years’
experience in GPCR targeted drug
discovery. She received an MSc in Chemical
Engineering from Lund Institute of
Technology, Sweden during which she
completed a thesis work at the Institute of
Biotechnology, University of Cambridge, UK.
She is currently an Associate Principal Scientist at AstraZeneca
Discovery Sciences, Sweden where her primary focus is on GPCR
pharmacology and applications of cellular assays on GPCR targets in
early drug discovery.
Dr. Erik Ryberg obtained his PhD in
Biomedical Science at the University of
Aberdeen, UK in 2009. He has been working
with GPCR related drug discovery at
AstraZeneca for over 10 years in different
positions. He is currently an Associate
Principal Scientist within the Cardiovascular
and Metabolic Diseases function with primary focus on molecular
pharmacology of GPCRs in different phases of drug discovery.
Lovisa Frostne joined AstraZeneca
(Mölndal, Sweden) in 2003 after obtaining
her MSc in Molecular Biology at the
University of Gothenburg, Sweden. She
completed her thesis work at the Institute for
Molecular Bioscience at the University of
Queensland, Australia. During her 10 years in
AstraZeneca, her work has been focused around GPCR pharmacology
in early drug discovery. She now holds a position as Team Leader in
the Bioscience department within the Respiratory, Inflammation and
Autoimmune iMed.
Biographies
1. Ma P, Zemmel R. Value of novelty? Nat Rev Drug
Discov. 2002 Aug;1(8):571-2.
2. Hopkins AL, Groom CR. The druggable genome. Nat
Rev Drug Discov. 2002 Sep;1(9):727-30
3. Swinney DC, Anthony J. How were new medicines
discovered? Nat Rev Drug Discov. 2011 Jun
24;10(7):507-19
4. Zhang R, Xie X. Tools for GPCR drug discovery. Acta
Pharmacol Sin. 2012 Mar;33(3):372-84
5. Whalen EJ, Rajagopal S, Lefkowitz RJ. Therapeutic
potential of beta-arrestin- and G protein-biased
agonists. Trends Mol Med. 2011 Mar;17(3):126-39
6. Cooper MA, Halai R. What is label-free screening and
why use it in drug discovery? European Pharma -
ceutical Review. 2012;17(6):51-3
7. Schroder R, Janssen N, Schmidt J, Kebig A, Merten N,
Hennen S, et al. Deconvolution of complex G protein-
coupled receptor signaling in live cells using dynamic
mass redistribution measurements. Nat Biotechnol.
2010 Sep;28(9):943-9
8. Fang Y, Li G, Ferrie AM. Non-invasive optical biosensor
for assaying endogenous G protein-coupled receptors
in adherent cells. J Pharmacol Toxicol Methods. 2007
May-Jun;55(3):314-22
9. Kenakin TP. Cellular assays as portals to seven-
transmembrane receptor-based drug discovery. Nat
Rev Drug Discov. 2009 Aug;8(8):617-26
10. Dodgson K, Gedge L, Murray DC, Coldwell M. A 100K
well screen for a muscarinic receptor using the epic
label-free system--a reflection on the benefits of the
label-free approach to screening seven-trans -
membrane receptors. J Recept Signal Transduct Res.
2009;29(3-4):163-72
11. Kenakin T, Christopoulos A. Analytical pharmacology:
The impact of numbers on pharmacology. Trends
Pharmacol Sci. 2011 Apr;32(4):189-96
12. Peters MF, Vaillancourt F, Heroux M, Valiquette M, Scott
CW. Comparing label-free biosensors for pharma -
cological screening with cell-based functional assays.
Assay Drug Dev Technol. 2010 Apr;8(2):219-27
13. Tran E, Ye F. Duplexed label-free G protein--coupled
receptor assays for high-throughput screening.
J Biomol Screen. 2008 Dec;13(10):975-85
14. Bock A, Merten N, Schrage R, Dallanoce C, Batz J,
Klockner J, et al. The allosteric vestibule of a seven
transmembrane helical receptor controls G-protein
coupling. Nat Commun. 2012;3:1044
References
Figure 3: Application of DMR GPCR assays in the drug discovery process
A) Phases in the drug discovery process where DMR assays can be applied to GPCR targets and typical number ofcompounds screened / characterised. B) pIC50 correlation between a DMR antagonist and a radioligand bindingassay using HEK293 cells transfected with the bradykinin B1 receptor. DMR was measured with an Epic® reader fromCorning. C) Activation of the bradykinin B1 receptor in primary human preadipocytes. Concentration responsesafter addition of the B1 agonist Des-Arg10Kallidin to preadipocytes. Cells were pretreated with control siRNA orsiRNA to knockdown the bradykinin B1 receptor
‘‘DMR assays have the potential toprovide relevant measures over therange from full agonists via partial
agonists to inverse agonists’’
The first practical steps are the selection
synthesis, management and subsequent
screening of molecular libraries, either small
molecule or biological in origin. The second
critical area is the selection, development
and prosecution of bio-assays for primary
hit identification, validation and profiling.
In the context of drug discovery projects,
hits are the further optimised using multiple
criteria including structure activity relation-
ships, selectivity, physicochemical properties
and liability. Automation is a key enabler to
increase productivity, particularly in structural
based approaches to hit identification and
validation, for example, x-ray crystallography
and NMR spectroscopy.
The Practical workshop: Chemical biology,
drug discovery and screening is designed for
scientists at all levels (undergraduates, post -
graduates and laboratory based scientists within
academic and industrial research organisations)
engaged in early stage drug discovery who have
an interest in the development, validation and
utilisation of cell based assays for screening
against small molecule libraries. The Practical
workshop: Chemical biology, drug discovery
and screening is equally well suited to
technically focused staff from core facilities or
contract research organisations who may wish
to extend their expertise. The evening dinner on
the first day will offer the opportunity for the
participants to network and establish relation -
ships that would be mutually beneficial.
All participants will take part in the practical
sessions and these will involve the development
of screening compatible assays, pharmacology of
standard compounds and proof-of-concept
screen. Participants in this workshop will discuss
and demonstrate practically: (1) the appropriate
steps in selecting suitable assays in light of the
fact that a multitude of assay technologies are
currently available for a given target; (2) how to
select an appropriate technology; which criteria
should be examined during the early stage
chemical biology and drug discovery processes;
(3) whether a generic, flexible set of assay
methodologies or customised solutions should
be applied to the targets being investigated.
The specific aspects of the lectures will
cover general concepts in chemical biology and
drug discovery, the role of biochemical
and cell based assays for drug discovery
purposes, their advantages and disadvant-
ages and how to incorporate them into a drug
discovery workflow.
Workshop topics
The Practical workshop: Chemical biology,
drug discovery and screening will include the
following laboratory sessions:
1. General concepts for biochemical assays
exemplified by kinase and protease targets
2. Pharmacology of standard compounds,
signal stability, choice of liquid handling,
Z′ calculation and proof-of-concept screen
3. Cell viability assays
4. Label-free cell-based assay for GPCR targets
5. Pseudo-label-free kinase assays
The emergence of chemical biology has coincided with increasing numbers of exploratory molecular targets and mechanisms,
both therapeutic and non-therapeutic in origin. Screening using miniaturised microtitre plate format based assays remains the
most widely utilised methodology for identifying novel chemical matter capable of modulating target function in a meaningful,
biologically relevant manner.
Practical workshop: Chemical biology, drugdiscovery and screening
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 17 Volume 18 | Issue 4 | 2013
WORKSHOPPREVIEW Date: 23 – 25 October 2013 · Location: European ScreeningPort GmbH, Germany
Organised by: Hosted by:
Sheraz Gul is Head of Biology at the
European ScreeningPort, Hamburg, Germany
where he manages the assay development
and screening of academic targets. Prior to
this, he worked for GlaxoSmithKline for seven
years where he developed biochemical and
cellular assays for high throughput screening
as well as hit characterisation. In addition he has worked in academia
for five years on proteases and kinases. He is the co-author of the
Enzyme Assays: Essential Data Handbook. He is also involved in many
European drug discovery Initiatives involving government, the
pharmaceutical industry and academia (e.g. EU Framework 7 and
IMI). His research interests are directed towards maximising the
impact of HTS for drug discovery.
About the coordinator
The Practical workshop: Chemical
biology, drug discovery and screening
is approved by the Society of Biology for
purposes of Continuing Professional
Development (CPD) and may be counted
as 72 CPD credits if registered on the
Society of Biology CPD Scheme.
SPONSORS
To register your interest in attending, please visit www.europeanpharmaceuticalreview.com/workshop
EARLY BIRD OFFER
www.europeanpharmaceuticalreview.com/workshop
Register by 13 September 2013
to receive 20% DISCOUNT
This satisfies virtually every case from the
classroom to the research lab until we consider
chloride channels. We know that they exist, are
important and might make good drug targets in
several disease areas, but until recently, they
have remained somewhat stigmatised and
unfashionable in the world of therapeutics.
The lack of selective ligands has not helped
at all. The only exception to this has been the
GABA-A receptor that is coupled to an
intrinsic chloride channel that opens upon
binding of the inhibitory neurotransmitter
GABA. Benzodiazepines have been potentiating
this receptor ever since Valium became available
in the 1960s, providing sedative and anti -
convulsant effects. This introduces an important
concept: there are several gene families and
different protein types that can be described as
being chloride channels. This is in contrast to
what we understand of cation-selective ion
channels where there is little flexibility in the
protein structure that can form a pore that is
selective for potassium, sodium or calcium; the
diversity of these ion channels is brought about
by the variations in the protein domains distinct
from the pore that influence the opening-
closing behaviour. To be fair, there do not seem
to be many physiological reasons why an anion
channel should exhibit chloride-selectivity as
there is little physiological role in the membrane
transport of other halides or small anions; any
anion channel will by default be a chloride
channel from a physiological viewpoint. On the
other hand, potassium, sodium and calcium
membrane currents each play different
fundamental roles and selective membrane
Early in their undergraduate education, the student is introduced to various types of integral membrane protein: receptors,
adhesion proteins, ion channels, ion pumps and ion transporters. As they progress through their studies, they find out that
discrete gene families and protein structures are responsible for these different protein classes and there is never any reason to
consider that there might be any ambiguity in assigning any particular protein to its appropriate protein class.
Chloride ion channels and transporters: fromcuriosities of nature and source of human disease to drug targets
Jonathan D. Lippiat
School of Biomedical Sciences, University of Leeds
ION CHANNELS
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 18
© n
obea
stso
fierc
e / S
hutt
erst
ock.
com
‘‘GABA-A receptors are members of the cys-loop pentameric ligand-gated
ion channel families’’
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Memmert Generation 2012 awarded in the category of medicine/health + care
permeability to these cations is crucial to cellular
function and indeed life itself.
GABA-A receptors are members of the cys-
loop pentameric ligand-gated ion channel
families, which include several types of
excitatory and inhibitory neurotransmitter-
gated channels. The cystic fibrosis trans -
membrane regulator (CFTR) protein is a
nucleotide-regulated chloride channel. It is
a member of the diverse ATP-binding cassette
(ABC) transporter family and is the only one that
does not seem to transport any substrate across
a membrane, but rather functions as a chloride
channel. The CLC family of voltage-gated
chloride channels arise from another distinct
gene family and have a general structure unique
among membrane proteins. This family of
proteins have brought about many surprises
over the last 30 years and members of this family
will be introduced here and also how they might
be considered drug targets.
Other exciting developments from the
chloride channel field have emerged following
the molecular identification of proteins
underlying calcium-activated chloride (ClCa)
channels. Functionally, we have known about
their physiological roles for some time, but
recent molecular identification of the channel
proteins has allowed development of molecular
and pharmacological tools to probe and alter
their activity. This review focuses on the
pharmacological potential of targeting ClCa
channels and CLC proteins; their function and
subcellular localisation are summarised in
Figure 1 (page 20).
An introduction to CLC proteins
The nine members of this protein family
are CLC-1 to 7, CLC-KA and KB. The founder
member, CLC-1, is the voltage-gated chloride
channel of skeletal muscle and will be discussed
below. Many functional studies have been
carried out on a homologue with the honorary
title of CLC-0, isolated from the E l ec t r o pl a x
electric organ, which enables the ray to stun its
aquatic victims. Reconstitution of CLC-0 into
lipid bilayers and electrophysiological recording
of currents flowing through single ion channels
revealed an intriguing property. Usually, when
recording current from a small patch of
membrane containing a single ion channel, one
observes fluctuations between two current
amplitudes: one representing the closed
channel and the other representing the current
flowing through the open channel. The
fluctuations reflect transitions between open
and closed kinetic states. With CLC-0, however, it
was evident that the single ion channel protein
consisted of two equivalent pores that could
open independently1. This channel was
therefore described as being d o u b l e -
bar r el l ed (Figure 2, page 21). It thus came of
no great surprise that the crystal structures of
homologues from enteric bacteria revealed that
the protein complex was a dimer, with each
subunit poss ess ing an ion-conducting pathway2
(Figure 2, page 21).
The second surprise arose when detailed
functional experiments were carried out on
CLC-ec1 from E . c o l i . Rather than functioning
as a chloride channel, like its long-lost relative in
vertebrate skeletal muscle, CLC-ec1 is a 2Cl-/H+
exchange transporter, or antiporter, with strict
2:1 exchange stoichiometry3. This protein is
important for the survival of enteric bacteria in
ION CHANNELS
‘‘The fluctuations reflect transitions between open and
closed kinetic states’’
low pH4 and is likely to exploit a chloride
gradient to keep cellular pH at a tolerable level.
Prokaryotic CLCs might therefore be targets for
inhibitors to treat pathogenic E. coli or
Salmonella infection4. The discovery of ion
exchange behaviour naturally stimulated
further exertions to determine whether any of
this chloride-for-proton exchange existed in
mammalian CLCs, on the assumption that Cl-/H+
exchange might be the true function of
archetypal CLCs and those identified as chloride
channels might just be exceptions to the rule.
This seems to be the case as there is strong
evidence that CLC-3 through to CLC-7 function
as 2Cl-/H+ exchange transporters, primarily
residing in intracellular organelles, whilst CLC-1,
CLC-2, KA and KB are true plasma membrane
chloride-conducting ion channels5-9. Because
the archetypal CLC property is exchange
transport, being found across all forms of cellular
life, the latter subclass of proteins, the bona fide
chloride channels, can therefore be considered
‘broken’ chloride transporters, which have lost
the coupling of chloride transport to the
movement of a second substrate ion.
CLC-1
This founder of the CLC family is the voltage-
gated chloride channel of skeletal muscle and
serves to regulate the membrane potential
and repolarise the membrane following action
potentials to relax the muscle. In most tissues,
we would expect potassium channels to play
this role, which they do in cardiac and smooth
muscle, as well as regulating membrane
excitability in other cell types. The transverse
tubule system is an extension to the plasma
membrane (sarcolemma) and penetrates into
the contractile tissue. It is critical for the rapid
spread of electrical activity throughout the
muscle and orchestrates rapid and controlled
contraction. Potassium efflux from the muscle
cells into the confined space of the t-tubule
would raise the extracellular potassium
concentration and collapse this ion gradient,
which would lead to prolonged membrane
depolarisation. With chloride channels playing
the predominant repolarising role, this is
prevented. Loss of function mutations in CLC-1
lead to myotonia in man, goats, and mice (for a
recent review see10) and is characterised by
impaired muscle relaxation, consistent with the
loss of a repolarising membrane current.
With expression confined to skeletal muscle,
CLC-1 might be an attractive target for drugs
that control muscle contraction by increasing or
decreasing CLC-1 function and thereby reduce
or increase muscle excitability, respectively.
Compounds that increase CLC-1 function might
be able to treat myotonia, particularly in cases
where they could compensate for partial loss of
chloride channel activity. Inhibition of CLC-1
could reduce the threshold for muscle con -
traction and may be useful in cases of muscle
weakness or degenerative diseases such as
muscular dystrophy.
CLC-2
The inwardly-rectifying chloride channel, CLC-2,
has somewhat widespread tissue distribution.
It can be found in central neurones where it
regulates neuronal activity11-13. In astrocytes, its
subcellular targeting to cell junctions is regu -
lated by an interaction with GlialCAM (MLC1),
mutations in which disrupt this targeting and
cause megalencephalic leukoencephalopathy14.
Recent therapeutic interests involve CLC-2
expression in gut mucosa and lung bronchioles
where it plays a role in intestinal and lung
secretions, respectively. Lubiprostone, a drug
used clinically to relieve constipation was
proposed to exert its effect by activating CLC-2,
but this effect is controversial. Whilst this
compound activates CLC-2 channels in some
studies, in others it regulates CLC-2 trafficking
and increases CFTR function via prostaglandin
receptor activation15,16. However, there remains
sufficient evidence that increasing airway CLC-2
function could provide an alternative chloride
pathway in cystic fibrosis17.
CLC-KA/KB
These chloride channels are notably expressed
in renal epithelia and contribute to the permea -
bility of cell membranes to passive chloride flux.
They have a key role in the ascending limb and
distal tubules of the nephron, providing a
basolateral route for chloride reabsorption,
following transport from the primary urine via
the apical membrane. The CLCKB gene, which
encodes CLC-KB, is one of five genes that
underlie Bartter’s syndrome. Loss-of-function
mutations in CLC-KB result in defective chloride
reabsorption and so leads to a salt-wasting
disorder, which is associated with polyuria. One
of the main features of individuals that are
affected by CLC-KB mutations (Bartter’s type III)
is low blood pressure. A more severe form of the
disease is caused by mutations in the BSND gene
(Bartter’s type IV), which encodes Barttin, an
accessory protein important for the trafficking of
both CLC-KA and KB to the plasma membrane18.
This form of the disorder also includes sensory
deafness, which is thought to be brought about
by the loss of trafficking of both CLC-KA and
KB to epithelial membranes in inner ear.
Presumably, loss of either CLC-KA or KB activity,
but not both, can be tolerated by the auditory
system. This suggests that inhibitors selective for
either CLC-KA or CLC-KB, or partial inhibition of
both, may act as a novel loop diuretic with the
potential to lower blood pressure and with few
side-effects. This has led to the study of their
pharmacology and the development of novel
inhibitor derivatives with low micromolar
affinity19, which had diuretic effects when
administered to rats20. On the other hand, drugs
that activate CLC-KB channels may enhance
residual activity of defective channels in Bartter’s
type III patients.
CLC-7
Of the 2Cl-/H+ exchange transporter subclass,
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 20
ION CHANNELS
Figure 1: Diversity and subcellular localisation of CLC proteins and selected calcium-activatedchloride channels
Of the CLC family CLC-1, 2, KA, and KB are plasmamembrane chloride channels, whilst CLC-3 to 7 residein organelle membranes and function as 2Cl-/H+
exchange transporters. Members of the Bestrophinand TMEM16 families are candidates for plasmamembrane calcium-activated chloride channels. In addition, Best1 also has a regulatory role in theendoplasmic reticulum. Chloride channels areindicated by ‘Cl-’ at either end of the double-headedarrow and 2Cl-/H+ exchange transport indicated bydouble-headed arrows with ‘2Cl-’ at one arrowhead and‘H+’ at the other
‘‘One of the main features of individualsthat are affected by CLC-KB mutations
(Bartter’s type III) is low blood pressure’’
CLC-7 is a promising target for drug action and
its inhibition may be beneficial in osteoporosis.
Once more, this indication takes its origin from
observations of human disease caused by
loss-of-function mutations in CLC-7, which
cause osteropetrosis21. In this disorder, bone
remodelling by osteoclasts is deficient, which is
thought to be caused by defective acid and
enzyme secretion. This leads to dense bone,
which if reproduced by a CLC-7 inhibitor could
reduce the dissolution of bone and therefore
strengthen the skeleton of osteoporosis
patients22,23. In proof-of-concept studies,
pharmacological inhibition of acidification24 or
disruption of CLC-7 function by antibodies25
reduced bone resorption. However, CLC-7
inhibition may not be without complica-
tions because human and mouse studies
suggest that lack of CLC-7 function may also
be associated with neuronal storage and
de generation disorders because of reduced
lysosomal function26.
Loss-of-function human mutations
– lessons from CFTR
It is worth a brief pause at this point to consider
that some of the therapeutic indications for
novel CLC-targeted drugs aim to treat disorders
distinct and at the opposite end of the spectrum
to those that are caused by defective CLC
function. In addition to those described above,
Dent’s I disease is an X-linked kidney disease that
is caused by loss of CLC-5 function (see27 for a
recent review). All are rare inherited disorders
and a major protein defect involves either
reduced protein activity or trafficking to the
target membrane. In fact, many individual
mutations cause ER-retention and a lack of
protein maturation. A parallel could be made
with cystic fibrosis, with most of the affected
individuals possessing the ER-retained ΔF508
mutation. Recent and stratified approaches to
treating cystic fibrosis have been the result of a
dual-pronged attack to correct mutant protein
folding (CF correctors) and/or increase the
activity of plasma membrane CFTR chloride
channels (CF potentiators). Success is emerging
with the potentiator class28, which are effective
in patients with mutations that reduce CFTR
activity without loss of protein biosynthesis or
trafficking (e.g. G551D), however the effective -
ness of folding correctors (which is required in
the majority of cases) remains to be estab -
lished29. Although the diseases associated with
loss of CLC function are all rare genetic disorders,
we may one day be able to treat individuals with
myotonia, Bartter’s syndrome, Dent’s disease
and osteopetrosis, as well as cystic fibrosis
with drugs that correct the actual cause of
their disorder.
Calcium-activated chloride channels
Finally, mention will be made of calcium-
activated chloride (ClCa) channels, which have
clearly-defined physiological roles in a number
of cell types, yet their molecular identification
suffered a few false and stuttered starts. The
story involves four types of protein: members of
CLCA, Bestrophin, Tweety and TMEM16 gene
families. In all cases, their recombinant over -
expression resulted in the generation of
membrane chloride currents that are stimulated
by raising the intracellular calcium concen -
tration and, to varying extents, membrane
depolarisation. The journey for CLCA came to an
end when it was found that this was a protein
that was secreted, but also likely upregulated
the membrane expression of ClCa channels
endogenous to the expression system30. Tweety
and Bestrophin proteins do not exhibit all of the
properties of ClCa channels studied in major
tissues, although Best1 appears responsible for a
component of ClCa in sensory neurons31 and
may also be an important regulator of calcium
release from the endoplasmic reticulum32,33.
Three independent studies proposed TMEM16A
(also called Ano1) as a candidate for a major
component, if not in its entirety, for a calcium-
activated chloride channel34-36. Many subsequent
studies, facilitated by the generation of
molecular tools, have supported this. TMEM16A
is important for regulating membrane
excitability in vascular smooth muscle, is
upregulated in an animal model of pulmonary
hypertension and tone can be reduced by
channel inhibition37,38. In sensory neurones,
TMEM16A couples the presence of inflamm -
atory mediators to membrane hyperexcitability
and TMEM16A inhibition has antinociceptive
effects39. In animal models of asthma, expression
of this particular chloride channel is increased,
and its inhibition may have beneficial effects40.
It is also found in interstitial cells of Cajal of
the intestine and channel function is required
for the rhythmical contraction of smooth muscle
in the intestinal wall41. Furthermore, TMEM16A
activation may provide an alternative pathway
for epithelial chloride secretion in cystic
fibrosis42. Whilst there is still some debate
surrounding the precise function of the rest
of the TMEM16 family, ClCa function has
been also been ascribed to TMEM16B
(Ano2), which is thought to underlie the ClCa
channel in olfactory hair cells43,44. There may
also be roles for this class of ion channel in
cancer cell biology and its inhibition may
prevent cell proliferation45-48.
Concluding remarks
This review has highlighted the diverse roles of
chloride conducting or transporting proteins
and how their dysfunction is coupled to human
disorder or disease-like symptoms in animal
models. There is a severe lack of pharmaco -
ION CHANNELS
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 21 Volume 18 | Issue 4 | 2013
Figure 2: Structural and functional properties of CLC proteins
A Representations of CLC structure generated from the CmCLC X-ray data51 using Pymol software; amembrane side-view (left) with intracellularcystathionine-β-synthase (CBS) domains lying belowthe transmembrane helices and an extracellular view(right) looking down onto the transmembranedomains. The individual monomers are coloured redand blue and the location of ions at external andinternal anion binding sites in each monomer areshown in green. B Representations of single CLC ionchannel currents usually recorded by electro -physiology. CLC channel openings are shown to twoequivalent levels, indicating one or two pores open,whilst the typical open-closed behaviour of othertypes of ion channel are shown below. Cartoonsillustrating channel opening and closing transitions areshown next to the simulated currents
‘‘CLC-7 inhibition may not be without complications because
human and mouse studies suggest thatlack of CLC-7 function may also be
associated with neuronal storage andde generation disorders because of
reduced lysosomal function’’
logical reagents that inhibit, activate or improve
membrane trafficking of chloride channels and
transporters. Progress is being made with drugs
that reverse the defective function of CFTR
in cystic fibrosis, which will hopefully result in
medications that are specific to the different
types of inherited mutation. Inhibitors and
activators of TMEM16A calcium-activated
chloride channels42,49 are proving useful
laboratory tools and new compounds might
make effective drugs, particularly if they can
deliver tissue-specific effects. Molecules specific
for particular CLCs are probably the most elusive.
Understanding the structural basis of voltage-
gated CLC activation50 might identify protein
domains that are targetable by rational
structure-based drug design. Such tools will
enable us to put some of the novel therapeutic
ideas that have been reviewed and presented
here to the test.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 22
ION CHANNELS
Dr Jon Lippiat completed his PhD on the
structure, function and pharmacology of
potassium channels at the University
of Leicester. He studied pancreatic beta-cell
function and diabetes at the University of
Oxford before his appointment as Lecturer in
Pharmacology at the University of Leeds. His
research involves elucidating the structural and physiological
properties of several different types of ion channel and transporter
and their potential targeting by novel pharmacological reagents.
Biography
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2. Dutzler, R., et al., X-ray structure of a ClC chloride
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3. Accardi, A. and C. Miller, Secondary active transport
mediated by a prokaryotic homologue of ClC Cl-
channels. Nature, 2004. 427(6977): p. 803-7
4. Iyer, R., et al., A biological role for prokaryotic ClC
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5. Scheel, O., et al., Voltage-dependent electrogenic
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6. Picollo, A. and M. Pusch, Chloride/proton antiporter
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7. Leisle, L., et al., ClC-7 is a slowly voltage-gated 2Cl(-
)/1H(+)-exchanger and requires Ostm1 for transport
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9. Matsuda, J.J., et al., Overexpression of CLC-3 in HEK293T
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10. Tang, C.Y. and T.Y. Chen, Physiology and
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11. Ratte, S. and S.A. Prescott, ClC-2 channels regulate
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13. Rinke, I., J. Artmann, and V. Stein, ClC-2 voltage-gated
channels constitute part of the background
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14. Jeworutzki, E., et al., GlialCAM, a protein defective in a
leukodystrophy, serves as a ClC-2 Cl(-) channel auxiliary
subunit. Neuron, 2012. 73(5): p. 951-61
15. Norimatsu, Y., A.R. Moran, and K.D. MacDonald,
Lubiprostone activates CFTR, but not ClC-2, via the
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Commun, 2012. 426(3): p. 374-9
16. Cuppoletti, J., et al., SPI-0211 activates T84 cell chloride
transport and recombinant human ClC-2 chloride
currents. Am J Physiol Cell Physiol, 2004. 287(5): p.
C1173-83
17. Cuppoletti, J., et al., ClC-2 Cl- channels in human lung
epithelia: activation by arachidonic acid, amidation,
and acid-activated omeprazole. Am J Physiol Cell
Physiol, 2001. 281(1): p. C46-54
18. Estevez, R., et al., Barttin is a Cl- channel beta-subunit
crucial for renal Cl- reabsorption and inner ear K+
secretion. Nature, 2001. 414(6863): p. 558-61
19. Liantonio, A., et al., Molecular switch for CLC-K Cl-
channel block/activation: optimal pharmacophoric
requirements towards high-affinity ligands. Proc Natl
Acad Sci U S A, 2008. 105(4): p. 1369-73
20. Liantonio, A., et al., In-vivo administration of CLC-K
kidney chloride channels inhibitors increases water
diuresis in rats: a new drug target for hypertension? J
Hypertens, 2012. 30(1): p. 153-67
21. Kornak, U., et al., Loss of the ClC-7 chloride channel
leads to osteopetrosis in mice and man. Cell, 2001.
104(2): p. 205-15
22. Schaller, S., et al., The role of chloride channels in
osteoclasts: ClC-7 as a target for osteoporosis
treatment. Drug News Perspect, 2005. 18(8): p. 489-95
23. Zhao, Q., et al., CLC-7: a potential therapeutic target for
the treatment of osteoporosis and neurodegeneration.
Biochem Biophys Res Commun, 2009. 384(3): p. 277-9
24. Karsdal, M.A., et al., Acidification of the osteoclastic
resorption compartment provides insight into the
coupling of bone formation to bone resorption. Am J
Pathol, 2005. 166(2): p. 467-76
25. Ohgi, K., et al., Antibodies against ClC7 inhibit
extracellular acidification-induced Cl(-) currents and
bone resorption activity in mouse osteoclasts. Naunyn
Schmiedebergs Arch Pharmacol, 2011. 383(1): p. 79-90
26. Kasper, D., et al., Loss of the chloride channel ClC-7
leads to lysosomal storage disease and
neurodegeneration. EMBO J, 2005. 24(5): p. 1079-91
27. Lippiat, J.D. and A.J. Smith, The CLC-5 2Cl(-)/H(+)
exchange transporter in endosomal function and
Dent's disease. Front Physiol, 2012. 3: p. 449
28. Ramsey, B.W., et al., A CFTR potentiator in patients with
cystic fibrosis and the G551D mutation. N Engl J Med,
2011. 365(18): p. 1663-72
29. Clancy, J.P., et al., Results of a phase IIa study of VX-809,
an investigational CFTR corrector compound, in
subjects with cystic fibrosis homozygous for the
F508del-CFTR mutation. Thorax, 2012. 67(1): p. 12-8
30. Gibson, A., et al., hCLCA1 and mCLCA3 are secreted
non-integral membrane proteins and therefore are not
ion channels. J Biol Chem, 2005. 280(29): p. 27205-12
31. Boudes, M., et al., Best1 is a gene regulated by nerve
injury and required for Ca2+-activated Cl- current
expression in axotomized sensory neurons. J Neurosci,
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32. Kunzelmann, K., et al., Role of the Ca2+ -activated Cl-
channels bestrophin and anoctamin in epithelial cells.
Biol Chem, 2011. 392(1-2): p. 125-34
33. Barro-Soria, R., et al., ER-localized bestrophin 1 activates
Ca2+-dependent ion channels TMEM16A and SK4
possibly by acting as a counterion channel. Pflugers
Arch, 2010. 459(3): p. 485-97
34. Schroeder, B.C., et al., Expression cloning of TMEM16A
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2008. 134(6): p. 1019-29
35. Caputo, A., et al., TMEM16A, a membrane protein
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42. Namkung, W., et al., Small-molecule activators of
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43. Stephan, A.B., et al., ANO2 is the cilial calcium-activated
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44. Hengl, T., et al., Molecular components of signal
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47. Mazzone, A., et al., Inhibition of cell proliferation by a
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References
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 23 Volume 18 | Issue 4 | 2013
25 Using LIMS forbiobanking andimplementing LIMS for researchEva Bürén, Head of IT, Karolinska Institutet Biobank
29 Biologicsdevelopment andELN: A good match?Michael H. Elliott, CEO, Atrium Research
33 InformaticsRoundtable
Moderated by John Trigg, Director, phaseFour Informatics
Informatics
SPONSORS
© F
abio
Ber
ti / S
hutt
erst
ock.
com
LIMS without BoundariesBrowser independence
Database independenceHardware independence
Location independence
The KI Biobank operations have expanded
greatly over the last few years with new
technology for automated high throughput
sample handling in order to increase through -
put and quality of samples.
The IT department
The IT department at KI Biobank has three main
purposes. The first is to develop, maintain and
support systems for the high throughput
processes at the Biobank facility, such as sorting,
aliquoting and storing samples in an automated
way. The second mission is implementing a LIMS
system, SCARAB-LIMS, which support research
groups that store biobank samples in freezers at
different laboratories. The third purpose is to
develop and maintain an infra structure for large
scale prospective studies. The infrastructure is a
highly integrated service consisting of a CRM
system, web survey system, a LIMS system and a
database platform for safe storage of data.
Implementing LIMS for
research: background
The traditional approaches in data management
are lab notebooks, multiple spreadsheet files in
computer folders and standalone databases.
As a result of the Swedish Biobank legislation
from 2003, which stated that all samples stored
for more than two months must be traceable,
managed with quality and to protect the
integrity of the donor, the Karolinska Institutet in
a joint effort with Stockholm County Council
decided to implement a new LIMS system in
2007 as support for clinical researchers in their
daily operations with using biobank samples
within the healthcare system.
LIMS
A laboratory information management system
(LIMS) database application enables a secure
system that fulfils the legal demands for
traceability of a sample. The system has an
audit trail that contains information about
everything being done in the system and
by which operator. The LIMS system also
manages information about the donor and it is
always at the donor’s discretion to withdraw
consent. One of the most important options
for a LIMS system is the consideration of the
value of the sample collections and associated
data. It enables utilisation of sample collections
and easy access of samples and sample informa -
tion using request and withdrawal functions
and it is possible to share data and stimulate
research collaboration.
SCARAB-LIMS
SCARAB, an acronym for Sample Collection
Administration Research and Biobanking – is a
LIMS from LabWare that has been configured
at KI. In the system, a module called the biobank
template is used. The template consists of
standardised functions for biobanking and
Karolinska Institutet Biobank is a core facility at KI established in 2004 to support medical research in short and long-term storage
of valuable human biology samples. Today, the KI Biobank holds four million samples in freezers from approximately 140 different
studies. The studies vary from disease-specific case control studies with the aim to identify biomarkers for complex diseases to large
prospective cohorts with the aim to investigate genetic and lifestyle factors and their interaction on health and disease.
The service provided at the Biobank is focused on study planning, pre-analytical processes, sample storage and retrieval of
specimens. The facility has 20 robots for sorting, aliquoting, DNA extractions and withdrawal of samples. On a daily basis,
approximately 500 blood samples from different research studies are registered and processed into multiple aliquots and stored in
2D barcoded micro tubes in 96 SBS rack format.
Using LIMS for biobankingand implementing LIMS for research
Eva Bürén
Head of IT, Karolinska Institutet Biobank
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 25 Volume 18 | Issue 4 | 2013
IN-DEPTH FOCUS: INFORMATICS
© V
LADG
RIN
/ Sh
utte
rsto
ck.c
om
sample collections. The main functionalities are
planning a study, logging of samples manually
or by file, searching for samples or patients by
different criteria, request of samples and storage
location functionality. Back-end functionality for
logging samples integrated to a robot is also
developed in SCARAB-LIMS.
Information security and maintenance
of the system
Due to the large amount of confidential data
that is being collected, the biobank has a great
responsibility in terms of information security
such as confidentiality, availability, integrity and
traceability regarding the collection, storage
and release.
Several laws and regulations, such as the
Data Protection Act (1998:204), Ethical Review
Act (2003:460), Public Access to Information and
Secrecy Act (2009:400), and the Biobank in
Medical Care Act (2002:297) regulate the
security of data management, sample trace -
ability and the option for sample donors to get
access to stored data. Four of the core principles
within information security are;
� Confidentiality – sensitive information
and programs should be protected from
being accessed or disclosed to un -
authorised persons
� Accessibility – access to information and
information systems should be kept at a
level that the business can run efficiently
and smoothly. The level should meet the
needs and goals of the services and system
owners made based on business
requirements. This information will be
restored within a reasonable period of any
loss or interruption
� Accuracy – information processed is
accurate, current, complete and under -
standable, and presented so that it
meets the intended purpose. Information
should not be changed accidentally or
delib erately distorted
� Traceability – in the handling of information,
it should be possible to track who did what
and when.
The system operates via a centralised data-
base. The database servers are maintained by a
central IT department and are set up within the
department firewall.
It is possible to connect to the system via a
web browser but the login must be performed
using a citrix client in order to increase the
security. Backup of the system occurs every
15 minutes. As the database is used by diff-
erent research groups, it is crucial to implement
restrictions that define different levels of
access to data. A useful functionality of the
system is the possibility to allow specific data
to be accessible for a specific group, user or
defined role.
LIMS and biobanking processes
In the initial study planning phase, the customer
relations group at KI Biobank help researchers in
the overall logistic planning and set up of
medical research studies i.e. ethics, logistics,
storage and informatics.
The process starts with the integration of a
study at the Biobank. Information about the
donor ID, study ID and sample types are
collected and imported into the LIMS system.
LIMS then produces sample numbers and
referrals are created and sent to the partici-
pants of the study. The participants use the
healthcare facilities for the withdrawal of
the blood samples. The samples and referrals
arrive at the Biobank, the referral is scanned
as a first step and the information is imported to
the LIMS system. The samples are then sorted
and scanned by a robot. Every sample has
a unique barcode and the barcode is vali-
dated by the LIMS. All samples are loaded
onto a sorting robot that starts by scanning
the unique barcode of the sample, volume
measurements of the sample and tube
type detection by a camera-based system.
The data is sent to LIMS to match the barcode
and tube type against the imported data.
LIMS generates a working list for the robot
based on the data and send it back to the
robot. The tube and sample type determines
further processing of the samples.
LIMS can be configured to communicate
with laboratory equipment, including analytical
instruments and liquid-handling robots.
This not only allows data to flow directly into the
LIMS as it is generated, but also enables
the system to direct the workflow with specific -
ally developed functions. Features like these can
improve efficiency by saving researchers the
task of manually recording and entering data,
and can reduce data transcription errors.
The LIMS enables a powerful functionality
for searching data and for generating reports.
Many LIMS are also equipped with data-mining
and trending tools that can provide unique
insights into the data.
Standards and pre-analytical codes
LIMS enables implementation of standards and
codes to achieve a higher and comparable
quality of collected samples than what is
available today and includes both samples and
information on how samples are collected
and processed. The SPREC code, developed
by ISBER, describes the pre-analytical con-
ditions of a sample. The codes consist of
seven elements describing for example sample
type, storage condition, pre-centrifugation and
container type.
To enable sharing of data and samples
between biobanks more efficiently, BBMRI.se
has developed a standard for biobank sharing:
Minimum Information about Biobank Sharing
(MIABIS). MIABIS includes data describing
biobanks, studies, contact information and
experiment types.
LIMS in large prospective cohorts
LifeGene and EpiHealth are prospective large
population cohorts in which data on health
status, physical measures, lifestyle, exposures
and biological samples are collected. The aim of
these cohorts is to study the influence of genes
and lifestyle factors on health and diseases
across the lifespan on several tens of thousands
of individuals.
Both EpiHealth and LifeGene have collected
data using the same IT infrastructure, which is
built in modules with a number of services /
applications to support research studies.
The services include study administration for
managing participant contact information and
test centres, websites for interaction with
participants and the public, collection of data
through web-based questionnaires, LIMS for
managing physical measurements and registra -
tions of samples at test centres, referrals to
laboratories for analysis and a biobank and
finally the central repository for all information.
At the test centre, a LIMS is used for
sample registration, instrument integration and
registration of physical measurements. The LIMS
system is integrated with the central database
via a citrix solution. The system allows for
different instrumentation dependent on the
study requirements. Implementation of LIMS
based data entry ensures quality of measure -
ment data. When data input is manual, e.g.
measuring waist circumference, reference
values and other logical checks assure
quality. The interface with the LIMS is peda -
gogically designed to ensure that all activities
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 26
IN-DEPTH FOCUS: INFORMATICS
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LABVANTAGE PHARMATAKE YOUR LABORATORTT Y TO TT THE TTNEXT LEVET L
Validate and Go Live in a Fraction of the TimeLABVANTAGE Pharma is the only pharmaceutical LIMS on the market that reduces traditional implementation cost by up to 85% and implementation time by up to 75% with:
LABVANTAGE 6 Preconfigured for Pharmaceutical-Specific Functionality
Pre-validated Package Executed Based on GAMP 5 Best-Practices Guide
LABVANTAGEPharma PackageImplementation
As Soon as3 Months
Minimum 12 Months
Months
A Typical LIMS Implementation
Approximately a 75% savings in timeApproximately an 85% savings in cost
in the test are performed in the same order regardless of personnel
performing the tests. The system validates if anything is forgotten or
the results are out of the given boundaries. The LIMS records all
activities in the test rooms. This further ensures that time
requirements for handling of blood samples follows study protocol.
LIMS also has functions to log sample transport to either the biobank
or different labs. This gives full control of the time from sampling to
safe storage.
Obstacles when using LIMS
Most LIMS systems offer a wide range of functionality and it is often
difficult for the researcher to define requirements that fulfil their
needs and to make a requirement specification of which functionality
is needed. The user friendliness of the system can also be an issue for
researchers with little or no IT experience.
What information should be stored in LIMS?
Many research groups don’t have any other systems for clinical data
and large analytical data. The LIMS is favourable for sample
management which is the main purpose of LIMS and the strength of
the system.
What is the best choice; to purchase a commercial
system or an in-house development?
There are pros and cons for both alternatives and there is no
simple answer. In-house development put strong demands on IT
experience in programming which might be a favourable solution
for limited workflows. A commercial system provided by different
vendors enables most functionality to be implemented within a
much shorter time frame compared to in-house development.
It’s quite expensive but has a higher security inbuilt in the system.
The conclusion, based on experience at KI Biobank, is that a key
factor for a successful implementation of a new system is to
carefully evaluate performance, integration to other systems or
instruments, user friendliness, functionality to make reports,
support and price offered by vendors and off course the strategies
and long term planning for the business are critical factors for a
successful implementation.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013
IN-DEPTH FOCUS: INFORMATICS
Eva Bürén started working at the KI Biobank in 2009 as a system developer.
She was involved in implementing a laboratory information management
system (LIMS) for research groups with sample collections at KI. In September
2010, she became manager of the LIMS group. In March 2011, as a result of a
merge of three IT-groups into one, Eva became Head of IT at KI Biobank. The IT
group at the Biobank develops, maintains and supports the IT systems within
the organisation. In addition to being head of the group, she is also account
manager of the IT-services in the organisation.
Eva has a background in molecular biology and has mostly been working in research and
development at Uppsala University and in Biotech companies. In Uppsala, she was involved in an EU
project developing methods for gene modification of strawberries. She also has experience from
developing a gene expression method at Global Genomics AB and working as a section manager of a
clinical laboratory. Eva holds an MSc in Medical Science from Uppsala University.
Biography
KI Biobank has for historical reasons two different LIMS systems, LabVantage LIMS
from Software Point and LabWare LIMS from Labware.
Footnote
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www.idbs.com/epr2013
While informatics within the small molecule
community is relatively mature with well-
established tools such as those for structure
activity relationship (SAR) analysis, reaction
planning, modelling and chemical registration,
the rapid increase in biologics investment has
outpaced the deployment of suitable IT systems.
For those systems commercially available,
adoption has been slow. Few on the biologics
side realise the importance of material reg -
istration and inventory management. The
common practices of ad hoc cell line and protein
batch naming, individual methods of data
management through Excel and reporting via
PowerPoint not only lowers efficiency, it results
in an inability to link data entities and easily track
project progression.
Both biologics discovery and development
phases are highly data intensive. Combined with
increasing numbers of projects, attention to
Quality by Design (QbD), and the ever-present
pressure to reduce cycle time to progress into
the clinic, development teams have particularly
acute stress. This combines with the adoption of
new technologies and new methodologies
generating exponential data volume growth.
For example, pre-formulation groups deploying
screening automation for the examination
of drug-like properties, cell culture teams
experimenting with microscale reactors, and
purification departments rolling out automation
for column condition optimisation. As small
molecule teams learned years ago after
investing in chemical library synthesis and high
throughput screening, data management
practices must adapt to avoid causing a
downstream data analysis bottleneck.
Informatics and biologics development
In the past three years, Atrium Research has
examined the workflows of several biologics
organisations, from large multinationals to small
biotech’s. Through a Voice-of-the-Customer
(VoC) analysis, we have found a consistent
pattern to the top three pain points with the
current state of data management amongst
scientists and their managers:
1. Difficulties finding / locating support-
ing data and information. Because data
manage ment is generally left to individuals,
finding where data lives requires tracking
down the person who created it. Therefore,
decisions are made without the necessary
data / information being available
2. Manual processes for data assembly,
storage and reporting. Results in decreased
resource efficiency, longer cycle times and
induction of data errors
3. Lack of standards in entity naming / numb -
ering, data organisation, calculations or
process. Negatively impacts data analysis
and scientific decisions – cannot merge data
or trace lineage of batches. Cannot deter -
mine if changes in upstream processing
impact downstream results.
Study participants were queried on the
capabilities needed to improve job performance
in a future state. The top five responses were:
1. A comprehensive collection of process
development data and information
2. Enable search across experiments and
studies to retrieve both structured data
(e.g., assay results) and documents (e.g.,
study reports)
Sales of monoclonal antibody (mAbs) and antibody-drug conjugates (ADCs) are projected to rise to USD 58 billion by 2016, an
increase of USD 25 billion from 20101
. This contrasts with other sectors of the pharmaceutical market that are declining. IMS
Health, in their latest survey, suggests that the overall US drug market shrunk in 2012 for the first time ever2
. Combined with
longer useful patent life and the complexity and cost of creating biosimilars, it is no wonder pharmaceutical companies are
diverting a larger portion of their R&D expenditures to biologics.
Biologics development and ELN: A good match?
Michael H. Elliott
CEO, Atrium Research
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 29 Volume 18 | Issue 4 | 2013
IN-DEPTH FOCUS: INFORMATICS
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3. Integrate data, regardless of origin, to
provide traceability and lineage from cell
line development through purification and
formulations. Be able to merge data across
organisational boundaries to increase
process understanding
4. Decrease variability and improve quality.
Enable consistent global data formats,
calculations, and platform components
5. Reduce non-value added activities through
elimination of manual data tasks.
What about ELN?
Many companies ask if Electronic Laboratory
Notebook (ELN) technology is ‘the solution’ to
mitigating the pain points and achieving the
desired capabilities of the future state.
Unfortunately, the answer is ‘no’ when exam -
ining functional requirements across all use
cases. There are too many capability require -
ments to be solved by any one technology
alone. ELN will not provide – without major
compromises – an effective solution to biologics
registration, inventory control, antibody design,
document management or analytical sample
and results tracking.
The correct question is: ‘Can ELN help?’ The
answer to that is ‘maybe.’ ELN is a component
of the larger informatics puzzle and the oper -
ational gains it offers depends on the decisions
made prior to implementation, such as the
project objectives. The system selected also has
an impact on benefits afforded. There are many
flavours of products on the market: systems
designed as generic tools for unstructured
intellectual property protection, products
layered on top of existing LIMS platforms and
systems with an integrated database for
structured data management. What you want to
do, what you select and what you do with it
determines success of failure.
For a generic implementation, one could
decide to take existing Microsoft Office files
and deposit them into the ELN for long-term
retention. The files are stored in a consistent
manner and tagging them with metadata
enables limited search. This does not address
data integration across experiments, let alone
the genealogy of a purified batch nor does
much to decrease inconsistency between
scientists. However, this is the easiest ELN
incarnation to implement and has the least
impact on existing workflows. If other systems
are developed for managing all the structured
data that results from experiments across all
functions, then this manifestation works well.
This approach can also ease the transition to a
comprehensive, workflow-altering deployment.
Start small, get scientists used to the technology
and slowly migrate them to a consistent method
of operation across the organisation is not a bad
way to go. This is a more of a cultural decision
rather than one of technology, assuming you
have selected a system that has the necessary
capabilities to be switched on at a later date.
ELN systems based on a LIMS are less
straightforward to deploy. Laboratory Informa -
tion Management Systems (LIMS) tend not to
adapt well outside of the analytical function.
Not only does the standard sample > test >
result hierarchy not match the workflows very
well, the ability of most LIMS products to easily
adapt to dynamic designs of experiments is not
present. In very late stage development
(i.e., clinical manufacturing), ELN variants that
are either based on a LIMS or have LIMS-
like design (known as ‘laboratory execution
systems’) can succeed as the workflows and
parameters are more fixed. But, this raises the
question of whether a company wants two
systems – one for the clinical manufacturing and
one for early and mid-stage development.
The systems that have had the greatest
success in the market (as measured by benefits)
are those ELN products with an integrated
structured data management component.
Though still not optimal due to their inherent
unstructured data design metaphor (i.e., paper
notebook replacement), these systems enable a
wholesale substitution of ad hoc spreadsheets
through the use of templates. Templates define
the structure for the data entry, calculations and
plots, supported by data dictionaries or
catalogues that establish a common metadata
vocabulary. Templates can be simple forms for
data entry and/or spreadsheets for data
aggregation and analysis. Manual data inte -
gration can be lessened: data can be integrated
directly from instruments such as fermenters
with near real-time analysis and visualisation.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 30
IN-DEPTH FOCUS: INFORMATICS
Figure 1: S88 – Recipe migration from platform models through clinical manufacturing
Not only do templates ease data entry, but
previous experiments can also be used as a
starting point for a new one where only a few
parameters need to be changed. A limited level
of cross-experiment data integration is possible,
though not nearly at the level of a data ware -
house designed for purpose.
With the level of change management
required to define consistency in terminology,
formats, workflows and calculations, these
types of ELN products are by far the most
difficult to manage and support. An advant-
age is that these systems are not as fixed as a
LIMS and therefore allow greater flexibility to
achieve the desired capabilities. But this is also
a curse, as this openness often means more
internal disagree ments over design con -
siderations. The effort involved to get a group
of scientists – who have their own Excel temp -
lates – to agree on a common format can
be considerable.
A mistake that template builders habitually
make is attempting to put an entire workflow
into a single template, feeling a large monolithic
version will be easier to support. An issue that
often arises is that any changes in the workflow
require a considerable re-architecture, making it
as complex as a LIMS to maintain. There are so
many triggers, extensions and integrations that
modifying one portion routinely will break
another. Our ELN user surveys highlight a trend
of an increasing frustration with vendors over
the complexities and maintenance of templates.
It is not unusual for the vendor support services
team to build the initial versions; they then have
to be called back at considerable expense to
update them later on. In many ways, several
ELN products are starting to look like the very
thing they replaced: big, unwieldy and complex
LIMS products.
The trend of biologics developers to use a
‘platform’ approach (i.e., a standard workflow),
may support a very fixed and limited set of large
templates. But as not all mAbs behave the same
way, changes in workflow and experiment
parameters are inevitable. Therefore, a break -
down of what a platform ‘is’ is necessary to mix
and match what is needed in the ELN workflow.
It is our opinion that the S88 standard can be
applied to ELN and platform biologics develop -
ment to create a modular approach that benefits
both the organisation and the technology.
S88 and ELN
ANSI/ISA-88 (S88) is the international standard
for defining production processes, emphasising
proper practices for the design and operation of
batch manufacturing and control3. It is a stand -
ard framework in use since the first version was
introduced in 1995. Multiple pharmaceutical
companies were involved in the development of
the standard and several are applying it to
biologics development to expedite technology
transfer and support QbD-like initiatives.
S88 provides a common vocabulary between
sites, scientists and engineers, but it is not a hard
and fast standard. It describes a flexible
framework to build models suited to the needs
of the operation.
The key concept throughout S88 is a ‘Recipe’
which is defined as: ‘the necessary set of
information that uniquely identifies the
production requirements for a specific product’.
It is a depiction of the process inclusive of
materials, steps and conditions. Recipes evolve
over time, starting with a set of process models
(i.e., platform components) that eventually turn
into equipment-specific Master Recipes for
clinical manufacturing (see Figure 1, page 30).
The first recipe, which may go through much
iteration during a development cycle, is a
General Recipe. The General Recipe takes the
process models and turns them into a recipe
that is specific to a particular product.
The beauty of S88 and the recipe concept is
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European Pharmaceutical Review
Volume 18 | Issue 4 | 2013
IN-DEPTH FOCUS: INFORMATICS
‘‘The systems that have had the greatest success in the market
(as measured by benefits) are those ELNproducts with an integrated structured
data management component’’
that everything is modular, enabling the
common process models to be reused and
mixed across many different recipes. This allows
for component ELN templates that support a
granular design approach. The procedure of
a recipe is built from stages, process operations
and process actions, which can be supported by
the ELN hierarchy.
In the example in Figure 2, a downstream
purification procedure has an ordered set of
stages: preparation, capture, intermediate
purification, polishing and packaging. The
capture stage has two anion exchange chrom -
ato graphy operations that are further broken
down into ‘Process Actions’ or specific pro -
cedural steps and their parameters. This
modularity enables mixing and matching of
process model templates at the action,
operation or stage level, depending on the
needs of the organisation.
An ELN template for the anion exchange
chromatography process action might have
sections for;
� Bill of Items with tables for the entry of:
� Materials (e.g., buffers, MSDS informa -
tion, lots numbers)
� Equipment (e.g., chromatography,
column, pH meters, spectrophotometer
for A280)
� Consumables
� Quality attributes (e.g., ranges for
analytical results)
� References
� Safety information
� An ordered set of steps for executing the
process action with fields for entry of data
such as loaded volume, buffer volume,
temperature, pH, etc. Calculated fields for
parameters such as elution concentration
� A sampling plan describing when samples
are taken and the assay required. There
would be an area for the entry of non-
structured results like a chromatogram.
Once the template is created, it can be
dragged to the ELN page from a library, allow-
ing the user to create experiments by
changing only a few parameters. Once the
parameters are optimised through a series of
experiments, the downstream recipe can
be merged with an upstream recipe to
create an overall process description. This
builds consistently documented institutional
knowledge documented, eases technology
transfer to clinical supplies and assists in the
creation of regulatory documentation.
Individual ad hoc approaches to data
management work for early stage biotech’s
but are not scalable. The increasing invest-
ment in biologics is forcing a change to a
controlled set of practices using common
process models across scientists and loca-
tions. The S88 standard can be applied in
biologics development to build modular
platform components; an ELN can be leveraged
to support the building of recipes though
modular template building. As a component of a
larger informatics puzzle, ELN can eliminate
many of the data management pain points,
helping to realise a future state of data
integration, consistency between scientists, and
improved efficiency.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 32
IN-DEPTH FOCUS: INFORMATICS
1. BCC Research; ‘Antibody Drugs: Technologies and
Global Markets’; Feb, 2012
2. IMS Institute for Healthcare Informatics; ‘Declining
Medicine Use and Costs: for Better or Worse?’;
May 9, 2013
3. http://www.isa.org
References
Michael H. Elliott is the founder and CEO of Atrium Research, a
scientific informatics market research and consulting firm. He can be
reached at [email protected]
About the author
Figure 2: Example of a Recipe for Downstream Purification
© a
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Externalisation has become a major
business strategy for Life Science
companies. How well aligned is
the laboratory informatics industry
to addressing the demands of
externalisation in terms of collaboration
support, access control and security?
Williams: As Pharma makes itself increasingly
‘virtualised’, there is a critical need for ‘inter-lab
informatics’ to support a network of partners
and IP producers. These are flexible data systems
which control and contextualise disparately
produced data so that it can be used effectively.
Modern, data centric electronic laboratory
notebooks (ELNs) are well aligned to support
this multidiscipline activity, managing data
security and access control. Legacy ‘digital
sticker-book’ ELNs with ‘single-login’ access are
being superseded with scalable systems with
strong ontology and workflow management,
enabling far flung scientists to move beyond
simple data capture and IP protection, towards
real-time collaboration.
Townsend: Over the last decade, the trend
towards centralised systems for laboratory
automation has led enterprise software vendors
to enable their products to support multiple
laboratories, across multiple sites which are
often spread around the world. LabWare
provides features in our LIMS and ELN products
to effectively manage data segregation, access
rights and allocation of functional privileges.
These tools can also be brought into play to
manage collaboration with external organisa -
tions. Furthermore, we also provide customers
with tools to export / import data in a variety of
popular data-interchange formats, which can be
used in cases where online access to systems is
not desirable or impractical.
Bailey: A sophisticated LIMS can work
outside of the organisation by interfacing with
collaborating systems so data flow seamlessly
between them, even within an extensive
workflow among multiple internal and external
users. Its state-of-the-art security will control
access to certain data so while one company can
track delivery of samples, another can access the
results of its tests. Since only certain businesses
implement a LIMS, the onus of this collaboration
is on the LIMS provider to facilitate.
Lab Informatics tools are increasingly
being influenced by consumer
technologies (e.g. mobile devices,
‘social’ tools). Do these technologies
have a place in the lab, and if so,
what role can they play?
Townsend: Mobile devices offer exciting oppor -
tunities for laboratories and adoption of mobile
technology is slowly but surely gathering pace.
For example, in LabWare we have seen how
mobile devices used for LIMS sampling opera -
tions can provide substantial benefits, including
for example use of GPS location tracking and
map technology. As for social tools, LabWare’s
own internet-based customer LIMS/ELN support
forums have delivered enormous value over
many years allowing customers from all corners
of the world to share experiences and knowl -
edge. In the same way, popular social tools are
able to deliver similar benefits as shown by the
growing number of lab informatics forums.
Bailey: Current consumer technology is
almost entirely web-based, which requires
the modern LIMS to run within a mobile
browser without losing the data sophistication
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 33 Volume 18 | Issue 4 | 2013
INFORMATICS ROUNDTABLE
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Moderator
John Trigg, Director, phaseFour Informatics
Glyn WilliamsVice President Product Delivery
IDBS
Peter BaileyCEO
LABVANTAGE
Nick TownsendDirector Life Sciences
LabWare
the user requires. Additionally, the smaller and
lighter these devices become, the more
convenient and desirable. For example, rather
than carrying notebooks, pens and traditional
stationery supplies, the user can simply carry a
tablet computer. In regards to social trends,
LABVANTAGE will soon unveil an exciting new
collaboration module that encourages users to
discuss tests and data while still providing
complete auditing and traceability.
Williams: The environment in and around
the laboratory is mobile, as are the people
who capture, review and share the data. But
standalone ‘m-informatics’ platforms do not
solve the problem. It is critical that mobile and
desktop applications access the same data.
Scientists love to think and talk about their
data. Social tools are vital if we are to capture the
full intellectual input of any scientist. Systems
enabling ‘comments’ and ‘tagging’ of data
encourage knowledge sharing and create
context and provenance for the data. This in turn
creates better information flow around the
organisation and increased scientific interaction.
Do you see ‘Cloud’ as a viable
implementation platform for
informatics tools? What do you see
as the benefits and drawbacks?
Bailey: LABVANTAGE understands the value of
the Cloud because we already use it as a daily
tool in our business. It requires no investment
into hardware and additional IT personnel and
there is no long term commitment. We host
distinct configurations for individual training
courses and for custom service projects. These
systems can quickly be created on the Cloud
instead of having to wait for requisitioned
hardware. Additionally, and almost identically
to using a third-party hosting solution, the
Cloud can provide secure, worry-free produc-
tion environ ments for organisations that
prefer to focus on the lab work rather than
the infrastructure.
Williams: ‘Cloud’ offers extensible comput -
ing power and centralised infrastructure for
collaborative working. But putting a desktop
application onto a Cloud does not make it a
Cloud application. The horsepower of the Cloud
can certainly benefit analytics systems and can
add value to truly scalable enterprise ELNs.
However, there remains corporate resistance to
the use of multi-tenanted applications for
storing high-value IP data. This may change with
improved stability of the Cloud and use of
sophisticated security. The best enterprise ELNs
are being developed with the Cloud in mind and
may revolutionise R&D informatics in the same
way as Cloud-CRM systems did for sales forces.
Townsend: The ‘Cloud’ is certainly a viable
platform for informatics but it has not yet been
widely adopted. We find that customers are still
unsure about how to ensure data security.
The benefits of using Cloud technology for IT
applications are well documented and the
opportunities for lab informatics applications
should be no different. Our technology is Cloud-
ready and we are open to work with customers
who wish to adopt the Cloud. However, this will
be driven by our customers as they gradually
take a view on data security and required
customer-vendor contractual arrangements
associated with moving and supporting
applications to the Cloud.
Laboratories often aspire to becoming
‘paperless’; how viable is that
aspiration, and what are the major
hurdles to be overcome?
Williams: The paperless laboratory is possible.
ELNS have already allowed scientists to replace
the paper notebooks in some companies.
Although there is more paper than just
notebooks to be considered, this shift is already
happening. Reducing transcription errors is a
major driver and integrating systems together
helps this process. There are cultural attach -
ments to paper but this is diminishing and must
be overcome. You only have to look at the move
in the newspaper industry toward digital
rather than print, to see where the science sector
is heading.
Bailey: LABVANTAGE has already taken the
paperless initiative. The fundamental of paper
documentation is record keeping, but to do so
‘paperlessly’ requires a method to input, file and
search. We input data using an electronic form
that mimics its paper counterpart, advanced
instrument integration that collects data directly
from instruments, barcodes, etc. All of this data is
stored permanently in the database with a
complete audit trail. Using both standard
reporting and the LABVANTAGE Ad Hoc Query
tool, users can easily retrieve the contents of
these documents. It’s not only paperless, but
also fast and accurate as well.
Townsend: It is feasible to achieve a
paperless state and it is realistic to achieve it with
LabWare LIMS with or without ELN, although
using LIMS with a LabWare ELN will typically
make going paperless easier. The effort to
achieve a truly paperless state should not be
underestimated. It requires carefully planning of
budget and resource to set-up the required
static data elements and implement data
capture templates that are convenient for the
end-user as they go about their daily tasks.
Laboratories usually interact with instruments
and other applications so ‘going paperless’ is also
dependent on the feasibility of providing
seamless and paper free integration.
What do you see as the biggest
challenges for the laboratory
informatics industry over the
next five years?
Bailey: The biggest challenge we see is keeping
pace with the ever-changing technology that
scientists use and the explosion of resulting data
being stored. In the past decade, both software
and hardware were dramatically improved,
which resulted in more data being stored. The
challenge for the LIMS is to discover more
effective ways to make sense of that data. In the
future, managing these global enterprise
databases and delivering business knowledge
will become the essential competence for the
LIMS vendor.
Townsend: The more mature lab informatics
products that are available on the market have a
much greater functional footprint compared to
that of a decade ago, and this footprint continues
to expand. Expectations of what lab informatics
solutions should provide are also growing.
Going forward, a key challenge is to ensure
expectations can be delivered, supp orted and
upgraded in a cost effective manner over the
long-term, especially when projects involve
integration of multiple products. Meeting
expectations will require customers and vendors
to work together and ensure projects are based
on sound product development strategies,
realistic goals and are executed well.
Williams: Informatics is the combination of
people with technology. Critical to success
therefore will be the ability of IT to help people
of many scientific disciplines cope with, and
share digital R&D. Systems must be available
on multiple platforms and bridge across
today’s legacy siloes. Key to this will be to make
enterprise data interoperable by smart data
management and ontology control. The impact
of consumer UI design also sets expectations for
how people interact with software: intuitively
and personally. The smartest companies have
already begun integrating many of these
capabilities. Those who have just started are
behind the curve.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 34
INFORMATICS ROUNDTABLE
This autumn, the scientific elite in pharma -
ceutical research will again come together at
MipTec, as they have done annually for over
11 years. The event has grown from its origins as
a specialist conference for laboratory auto -
mation into a leading global conference in the
life sciences sector. By offering a broad, diverse
and highly scientific conference programme,
MipTec plays a vital role in helping Pharma
and Biotech scientists successfully tackle the
significant challenges of today’s drug research
and development work. The exchange of the
latest scientific findings and technological inno -
vations, as well as the improving integration of
science and technology, will be at the centre
of this three day conference. For the organisers,
it is especially important to create an interactive
platform for researchers working in the life
sciences, who would otherwise rarely meet.
The conference
The 2013 conference theme is Personalised
Medicine, covered by internationally renowned
keynote speakers in the drug discovery and
life sciences field. The theme also interlaces in
the jam-packed three day dynamic programme.
BioValley’s Science Day, Strategy Day and
Connect Day explore facets where tech-
nology accelerates life sciences research, and
MipTec’s Nine Science Forums cover cutting-
edge developments that enhance drug
discovery: Medicinal Chemistry, Peptide
Thera peutics, HCS, NGS, Enzymology, Stem
Cells in Biomedicine, Natural Products and
Synthetic Biology as well as Customised
Therapies-Biomarkers and Biobanking and
Industry-Academic Collaboration Models.
The conference provides a platform
for networking and scientific exchange for
academic and industrial scientists. With more
than 100 exhibitors presenting state-of-the-art
equipment and services to advance biomedical
research, more than 100 scientific presentations
and 100 poster exhibitors, delegates will get
expert insights into the most recent develop -
ments and experiences in all dimensions of drug
discovery. Delegates will also have the oppor -
tunity to discuss strategic and business oriented
issues and learn more career options in many of
the exceptional satellite focused sessions.
Industrial Symposiums will
be organised by:
Bucher Biotec, Hamilton, Thermo Fisher,
Cellular Dynamics International, Toolpoint,
SiLA, PerkinElmer, Cellectis Bioresearch, BioTek,
Agilent Technologies and BMG Labtech.
Keynote Presentations
MipTec 2013 and the BioValley Life Sciences
week are proud to welcome the following three
keynote speakers at the conference:
Tuesday 24 September 2013Dr. Christoph Westphal, Founder of the
Longwood Fund (Boston, US). Lecture title:
Drug Innovation in Biotech: Alnylam, Momenta,
Sirtris, Verastem
Wednesday 25 September 2013Dr. Ralf Schumacher, Leader of the Large
Molecule Research Department (LMR) of Roche
Diagnostics (Penzberg, DE). Lecture title:
Strategies and Challenges for the Next
Generation of Therapeutic Proteins
Thursday 26 September 2013Dr. Kári Stefánsson, CEO deCODE Genetics
(Reykjavik, IS), Lecture title: Genetics of
Common Disease
Networking Events
Welcome ReceptionThis year’s conference will offer a Welcome
Reception on Tuesday 24 September 2013 for
exhibitors and delegates in Hall 4.1 in the
Congress Center Basel. Dr. Sylvain Cottens,
Global Head for the Center of Proteomic
(Novartis, NIBR) will welcome guests. Further -
more, the participants of MipTec and the
BioValley Life Sciences Week will have
the opportunity to network with colleagues
and opinion leaders in a relaxed atmosphere.
Poster Session & Apéro The MipTec Poster Session will be held on
Wednesday 25 September 2013. The authors of
the scientific posters will be available for
questions. Posters, recognised for their scientific
quality, will be nominated for poster prizes,
sponsored by Toolpoint and SLAS.
Industrial ExhibitionParallel to the scientific programme, the MipTec
exhibition will take place in Hall 4.1 in the
Congress Center Basel. Parallel to the MipTec
Drug Discovery Conference, over 900
square metres, exhibitors will showcase their
newest developments, products and services in
the field of laboratory and research reagents,
laboratory automation and instruments as
well as hard- and software for the computer-
supported analysis and medicinal chemistry of
industry leaders and decision makers in the
life sciences area.
The largest European conference on drug discovery, MipTec, continues to grow, taking place from 24 – 26 September 2013 and
offering a high calibre programme. Over 3,000 scientists from industry and academia are expected to attend the presentations
and scientific forums at MipTec which will be held jointly with the BioValley Life Sciences Week in the Congress Center Basel.
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 35 Volume 18 | Issue 4 | 2013
The detailed pre-programme is available on the
following websites: www.miptec.com;
www.lifesciencesweek.ch
ShowPREVIEW Date: 24-26 September 2013 · Location: Basel, Switzerland
Leading figures from drug discoveryand life sciences to gather in Basel
Within pharmaceutical development, Raman
spectroscopy has been widely used for non-
destructive quantification of actives and
excipients8. Moreover, Raman spectroscopy has
been proven to be a workhorse as a process
analytical technique for process monitoring9,10
(blending, granulation, compression, coating)
and understanding of process induced trans -
formation11. Raman spectroscopy has also been
used to address regulatory concerns over solid
state trans formations within the final dosage
form12. Due to its intrinsic properties such as
rapid analysis time, user-friendly interfaces, non-
destructive nature, Raman spectroscopy has
been used to tackle drug product counterfeit
and intellectual property rights issues13.
This review highlights a few representative
applications of Raman spectroscopy for in-
process characterisation of active pharma -
ceutical ingredients (APIs) and drug products.
Specifically, application of Raman spectro-
scopy is presented as a tool to monitor the
polymorphic state of an API in a slurry,
the content uniformity of a blend in a dry mixing
process, the end point of analyte dispersion in a
suspension and the measurement of a tablet
coating process. In each of these cases, Raman
spectroscopy has provided in-depth insights
into critical quality attributes of the pharma -
ceutical processes assessed, making the
pharmaceutical drug development workflow
monitorable in real-time.
API polymorph evaluation
Raman spectroscopy is a powerful technique for
characterisation of polymorphic forms of an API.
Implementation of in-line Raman probes allows
direct real-time monitoring of solid state
transformation of an API during the preformu -
Raman spectroscopy has emerged as the preeminent analytical tool for a number of applications within drug discovery and
development. Advances in the instrumentation, sensor fabrication and data analysis have enabled the wider acceptance of
Raman spectroscopy1,2
. In discovery, Raman spectroscopy is used to elucidate structural activity relationships3
and to optimise
reaction conditions and associated parameters (such as polymorph and formulation screening)4,5
that impact scale-up required
for the transfer of drug compounds from discovery to development6,7
.
Raman spectroscopy: an enabling tool foraccelerating pharmaceuticaldiscovery to development
Chanda R. Yonzon, Atul Karande, Sai P. Chamarthy and Brent A. Donovan
Merck & Co. Inc
RAMAN SPECTROSCOPY
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 36
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Table 1 %RSD comparison of Blend I and Blend II for the three components in a DPI
% RSD
Component I Component II Component III
Blend I 5.14 2.31 1.61
Blend II 3.35 2.20 1.02
lation phase of drug development. As shown in Figure 1, a slurry
experiment using 1:1 mixture of Form I and Form II in propanol:water
mixture at room temperature was performed to determine a stable
polymorph of the API. Real-time Raman spectra were collected
every two hours for three days (λex = 785 nm, Power = 400 mW, Tcollection
= 120 seconds). In order to normalise the spectra and monitor
changes over time, first derivative followed by standard normal
variate (SNV) was performed on the Raman spectra. Principal
component analysis (PCA) of the Raman spectra indicates that
Form I (1662 cm-1) converts entirely to Form II (1657 cm-1) within
around 20 hours (data not shown) at room temperature, there-
fore demonstrating that Form II is the more stable form of the API at
room temperature.
Quantitative determination of components in a
dry powder inhaler drug product
The traditional method for determining blend content uniformity of a
mixture is to sample the powder followed by off-line measurements.
A sample thief is used to sample small volumes of powder from the
blender and they are subsequently assayed using liquid chromato -
graphy to analyse the drug product14. This technique is not suitable
for rapidly testing hundreds of samples that are manufactured during
formulation screening and development due to time-consuming
sample preparation and lengthy analysis time. In addition, it is
technically challenging to sample only a few milligrams of powder
using a sample thief, a unit dosage for a dry powder inhaler (DPI),
without disturbing the blend.
In this study, a DPI blending process was used to demonstrate
the use of Raman spectroscopy as a robust and rapid screening
method to measure content uniformity of the powder blends8. The
DPI blend of interest contained three components that have distinct
Raman spectral fingerprints, making the system an excellent
candidate for quantitative analysis of all the three components.
Partial least squares (PLS) calibration models were constructed to
quantitate all three components. DPI blends from two intermediate
blending processes (blend I and II) were evaluated. Raman spectra
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013
RAMAN SPECTROSCOPY
Figure 1: Real-time transformation of an API from Form I to Form II at room temperature
‘‘The traditional method for determining blend contentuniformity of a mixture is to sample the powder followed
by off-line measurements’’
were collected from blend I (n = 35) and blend II
(n = 80). Figure 2 shows the content uniformity
of the batch normalised to 100 per cent for each
component. The relative standard deviation
(RSD) measured for all three components are
five per cent or less for both blend I and II
(Table 1, page 36). As the blending process
continues, the blending %RSD improves for
blend I versus blend II, therefore demon strat-
ing that Raman can be a useful technique
in determining an end point for blending. In
Figure 2 the dotted lines represent the dose
content uniformity (DCU) US specifica tion
(80-120 per cent)15 of a DPI. The data show
that the content uniformity of both blends is
within the suggested DCU specifica tion,
indicating a uniform batch. If a blend would fail
to meet the DCU specification, this method
allows one to verify expediently whether it is the
blending process that is at issue and correct it
prior to making the drug product.
Process monitoring of a nasal spray
drug product
Raman spectroscopy inherently has a minimal
interference due to water, making the method
an excellent candidate to monitor processes
in an aqueous formulation such as an aqueous
nasal spray. In this study, Raman spectroscopy
was implemented to determine the homo -
geneity of an analyte for a nasal spray product
during batch manufacturing16.
The analyte used in this experiment has a
distinct Raman spectral region of interest, which
does not interfere with other ingredients in the
formulation. The PhAT probe was used to collect
the Raman spectra from the top of the vessel so
that at t = 0 minutes, the Raman spectrum of
the drug substance in the mixture was the
most intense. Raman spectra were collected
every 15 seconds with five second accumulation
times. As the analyte is charged into the vessel,
the signal is the highest and as the analyte is
dispersed, the signal decreases to reach a steady
state, implying that the API in aqueous medium
is uniformly dispersed. Figure 3A is the 3D plot
of the real-time Raman spectra of the analyte
signal, which decreases over time. PCA was
performed on the Raman spectra after fitting
them to second order polynomial baseline
correction in order to remove the varying
background (Figure 3B).
To determine the optimal processing
time for analyte dispersion, a running RSD
was calculated on every two consecutive
principal component 1 (PC1) data points.
The steady state of the dispersion process
was considered to be achieved when the RSD
remained below three per cent for fixed number
of minutes.
Tablet coat thickness measurement
Coating processes are important unit operations
associated with the manufacture of many solid
dosage forms. It serves to impart numerous
advantages to a dosage form such as odour /
taste masking, physical / chemical stability and
modified / sustained release. The thickness and
uniformity of coatings provide information on
the quality of the coated tablets. Direct measure -
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 38
RAMAN SPECTROSCOPY
Figure 3: A) 3D plot of the Raman spectra B) PCA of the Raman spectra
Figure 2: Content uniformity of three components of two intermediate processes – Blend I and II. Unit dose level (~1 mg)
‘‘Raman spectroscopy inherently has a minimal interference due to water,
making the method an excellentcandidate to monitor processes in an
aqueous formulation such as an aqueous nasal spray’’
‘‘Coating processes are important unit operations associated
with the manufacture of many solid dosage forms’’
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Drug Products• Formulation Development• Raw Materials ID• Blending• Granulation• Drying• Tablets / Gelcaps• QA / QC
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ment of coat thickness is seldom carried out;
instead, indirect measurement, such as estima -
tion of coating thickness by weight change,
is practiced.
In this study, Raman spectroscopy was used
to quantify the tablet coating thickness as a
direct measure17. Placebo tablets (normal,
circular, biconvex, six millimetres in diameter)
made by direct compression were used for
coating. Hypromellose contained in the coating
formula has a prominent Raman peak at
approximately 1480 cm−1 where aliphatic ether
deformation of the polymer occurs18. Acquisition
of Raman spectra was carried out at an
excitation wavelength of 785 nanometres.
Each tablet was placed in a sample holder on a
mounting stage at the focal distance of the
probe and scanned on both surfaces using a
beam size of 300 μm. Acquired spectra were
subjected to spectral pre-processing such as
Savitzky–Golay smoothing and SNV algorithm
in order to remove sampling variations.
Raman spectra were correlated against changes
in coating thickness as coating progresses.
As the coating level increased, the intensity due
to the tablet core decreases and the intensity
due to the coating increases (Figure 4A).
Reference thickness measurements of these
tablets were obtained using optical microscopy.
Raman spectra were collected from 25 tablets to
generate PLS calibration model and inde -
pendent Raman spectra were used for
prediction (Figure 4B, page 40). The root-mean-
square error of calibration (RMSEC) and root-
mean-square error of prediction (RMSEP) are
2.16 and 2.73 μm respectively, indicating the
sensitivity and accuracy of Raman technique for
the coating thickness prediction. Measured
RAMAN SPECTROSCOPY
Figure 4A: Raw Raman spectra of blank tablet core and tablets coated to 0.4, 0.8, 1.2, 1.6, 2 and 3% (w/w) coatingweight gain (LP)
‘‘As the coating level increased, the intensity due to the tablet coredecreases and the intensity due to
the coating increases’’
(reference values using optical microscopy) and
Raman-predicted coating thickness values
indicate no statistical significant difference.
Overall, this work demonstrates that Raman
spectroscopy is a viable analytical technique to
measure coating thickness with similar accuracy
compared to that of optical microscopy.
Conclusion
Raman spectroscopy is a powerful technique
that is widely used within drug discovery and
pharmaceutical development. Raman spectro -
scopic methods in conjunction with the
application of in-line probes and/or usage
of chemometric methods are widely used in
a plethora of pharmaceutical applications.
In this review, a few examples of in-process
monitoring, which resulted in better under -
standing of the process and product critical
quality attributes, were discussed. These
examples further demonstrate that applications
of Raman spectroscopy in the pharmaceutical
industry can lead to significant optimisation of
workflows and understanding of in-process
manufacturing steps.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 40
RAMAN SPECTROSCOPY
Dr. Chanda Yonzon is an Associate Principal Scientist at Merck
Manufacturing Division. She received her PhD in Chemistry from
Northwestern University, Evanston, IL under the supervision of
Richard P. Van Duyne. She has extensive experience in vibrational
spectroscopic techniques and has applied these techniques to
understand several pharmaceutical drug product dosage forms and
their processes. She has supported numerous projects in preclinical to
commercial and supply stage. She has authored over 25 publications
and presented her research at several conferences.
Dr. Atul Karande is currently a Senior Specialist engineer at Merck’s
Respiratory product development group, where he is part of the drug
product development team. He received his PhD in Pharmaceutics
from National University of Singapore. He has thorough
understanding of quality by design concept in manufacturing of
different pharmaceutical dosage forms. He has supported inhalation
and intranasal projects in preclinical to commercial stage at Merck
Research Laboratory and Merck Manufacturing Division. Dr. Karande
has several original research articles and conference presentations
within pharmaceutical sciences. Before joining Merck, he worked at
Meggle GmbH as a Technical Application Manager focusing on
optimisation of lactose characteristics for improvement of dosage
form performance.
Dr. Sai Chamarthy received an MS degree in Pharmaceutics from
Duquesne University and a PhD in Industrial and Physical Pharmacy
from Purdue University. Dr. Chamarthy is currently an Associate
Director at Merck’s Respiratory product development group, where he
is part of the drug product development team. Dr. Chamarthy is the
current Chair for the Inhalation and Nasal Technology AAPS Focus
Group (INTFG) and an executive committee member for the Process
Modelling and Simulation Focus Group (PMSFG). Dr. Chamarthy has
published over 20 papers in peer-reviewed journals. His research
has also led to over 15 podium and 30 poster presentations at various
national and international conferences.
Dr. Brent Donovan is an Executive Director leading the respiratory
product development department within Merck Research
Laboratories in Summit, NJ. He leads a team of scientists that are
engaged in analytical and formulation development of dry
powder inhalers, metered dose inhalers and nasal spray products.
His group has been working on developing novel methodologies
for the characterisation of inhalation products as well as
supporting numerous projects in all phases of CMC development. He
received his PhD in Physical Chemistry from the University of
Michigan. Dr. Donovan has several published original research
articles within chemistry and pharmaceutical sciences. He previously
worked at Schering-Plough for nine years prior to the merger with
Merck in 2009.
Biographies
1. Corredor, C.C.; Jayawickrama, D.; McGeorge, G.; Both,
D. “Monitoring of blending uniformity: form
conversion and fluid bed drying by near infrared and
Raman spectroscopy” American Pharmaceutical
Review, 2010, 13(1), pp 66-72
2. Widjaja, E.; Kanaujia, P.; Lau, G.; Ng, W. K.; Garland, M.;
Saal, C.; Hanefeld, A.; Fischbach, M.; Maio, M.; Tan, R. B.
H. “Detection of trace crystallinity in an amorphous
system using Raman microscopy and chemometric
analysis” European Journal of Pharmaceutical
Sciences, 2011, 42(1-2), pp 45-54
3. Pivonka, D. “Vibrational analysis of structure activity
relationships (SAR) in molecular binding Applied
spectroscopy” Applied Spectroscopy, 2004, 58(3),
pp 323-331
4. Heinz, A., Strachan, C.J., Gordon, K.C., Rades, T.,
“Analysis of solid-state transformations of
pharmaceutical compounds using vibrational
spectroscopy,” J. Pharm. Pharmaco., 2009, 61(8),
pp 971-988
5. Xie, Y., Cao, W., Krishnan, S., Lin, H., Cauchon,
N.,“Characterization of mannitol polymorphic forms in
lyophilized protein formulations using a multivariate
curve resolution (MCR)-based Raman spectroscopic
method,” Pharm. Res., 2008, 25(10), pp 2292-2301
6. Zhou, G.; Guenard, R.; Ge, Z., “Infrared and Raman
spectroscopy for process development” From
Applications of Vibrational Spectroscopy in
Pharmaceutical Research and Development, 2007,
pp 185-211
7. Yong Z.; “Model for Raman spectroscopic monitoring
of process-induced pseudomorph interconversion”
American pharmaceutical review, 2009, 12(4),
pp 56- 62
8. Yonzon, C.R.; Donovan, B.A., “Raman spectroscopic
method for content uniformity of a dry powder
inhaler,” Amer. Pharm. Rev. 2008, 11(7), pp 70-76.
9. El Hagrasy, A.; Chang, S.; Desai, D.; Kiang, S.
“Application of raman spectroscopy for quantitative
in-line monitoring of tablet coating”, American
Pharmaceutical Review, 2006, 9(1), pp 40-45
10. Wikstroem, H.; Lewis, I.R.; Taylor, L.S. “Comparison of
sampling techniques for in-line monitoring using
Raman spectroscopy”, Applied Spectroscopy 2005,
59(7), pp 934-941
11. Xie, Y.; Cauchon, N. “Raman scattering as a probe for
properties of active pharmaceutical ingredients in
tablet formulations” American Pharmaceutical Review,
2012, 15(2), pp 26, 28-31
12. Gao, Q.; Lew, A.; Takahashi, L.H.; Cassella, J.V. “An
investigation into the morphology of loxapine in a
thermal aerosolization process from crystalline to
amorphous”Journal of Pharmaceutical Sciences, 2011,
100(4), pp 1407-1415
13. Kwok, K.; Taylor, L.S. “Raman spectroscopy for the
analysis of counterfeit tablets”, From Infrared and
Raman Spectroscopy in Forensic Science, 2012, pp
561-572
14. Muzzio, F.J.; Robinson, P.; Wightman, C.; Brone, D.
“Sampling practices in powder blending. International
Journal of Pharmaceutics”, 1997, 155, 153-178
15. U.S. Department of Health and Human Services, Food,
and Drug Administration, Center for Drug Evaluation,
Research, “Guidance for Industry: Metered Dose
Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug
Products” 1998
16. Pu, Y.; Medendorp, J. P.; Yonzon, C.R. “Real-time
monitoring of active ingredient dispersion in a
pharmaceutical aqueous suspension using Raman
spectroscopy” Journal of Raman Spectroscopy, 2011,
42(11), pp 1994-1999
17. Cahyadi, C.; Karande, A.D.; Chan, L.W.; Heng, P.W.S.
“Comparative study of non-destructive methods to
quantify thickness of tablet coatings” International
Journal of Pharmaceutics, 2010, 398(1-2), pp 39-49
18. Romero-Torres, S.; Perez-Ramos, J.D.; Morris, K.R.;
Grant, E.R. “Raman spectroscopic measurement of
tablet-to-tablet coating variability” Journal of
Pharmaceutical and Biomedical Analysis, 2005, 38(2),
pp 270-274
References
Figure 4B: PLS analysis of tablet coating thickness
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 41 Volume 18 | Issue 4 | 2013
43 The rapid microbiological methods revolutionEmanuele Selvaggio, QA Batch disposition & Investigation Supervisor, Pfizer
46 Controlling contamination in the pharmaceutical industryChris Delaney, Cleanroom/GMP Specialist, Noonan Services Group
52 RMMs RoundtableModerated by Jeffrey W. Weber, PAT Project Manager, Process Analytical Sciences Group & Chairman, Pfizer Rapid Microbiological Methods Steering Team, Pfizer
RMMs &Environmental
Monitoring
SPONSORS
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Shut
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For many years after that first experiment
until the second part of the 20th century, all
microbiological methods relied upon growth
methods. The discovery of several species
that were hard to cultivate (for which suitable
in vitro culture conditions have not yet been
defined) as well as the existence of injured and
viable but non-culturable stressed cells (which
cannot be recognised as ‘alive or dead’ by
the cultivable / non-cultivable dichotomy),
became stimulating factors for the research of
alternative techniques.
Although growth-based method provide
the laboratory with critical information about the
amount and the type of organisms, from an
industry perspective the time to results are
usually much longer than desired. In addition, in
the case of critical events such as massive
contaminations, environmental microbial
excursion and test failures, real time / rapid
technologies may have a key role to maintain the
expected quality level to patients and may help
from a quality risk management standpoint.
As per the Eur.Ph. 5.1.6 definition, alternative
methods for the control of microbiological
quality can be divided into three categories:
� Growth-based methods, in which a detect -
able signal is usually achieved through a
period of subculture
� Direct measurement, in which individual
cells are differentiated and visualised
� Cell component analysis, in which the expr -
ess ion of specific cell components offers an
indirect measure of microbial presence.
Usually, the main difficulty is finding the best
technology which fits both the specific
laboratory requirements and project constrains.
Indeed, nowadays there are several tech -
nologies which can be utilised to address any
type of request depending on the application
(sterility testing, bioburden, microbial identifi -
cation, air monitoring etc.). In order to find the
best solution, it must be pointed out that it is
mandatory to consider the particular factors
that make every single project unique.
Points to consider when starting with a
new project are:
� Purpose of the project (cost saving;
lead time reduction, process knowledge
improve ment, quality improvement, etc.)
� Drug product characteristics (Filtera-
bility, Antimicrobial effect, Colour, Matrix
influence, etc.)
� Budget constrains
� Personnel skills
� Laboratory space and ancillary equipment
� Vendor support and experience.
The technology of choice is as important as the
vendor of choice. In fact, over the last decade,
the overall number of vendors available has
been exponentially growing. As the majority of
rapid micro methods rely upon either universal
cell biochemical pathways or common macro -
molecular cell components, nowadays both
chemical instrument vendors and consolidated
micro laboratory vendors are competing for
the market.
Microbiology was officially born in 1676 when a Dutch tradesman and scientist from Delft, the Netherlands, observed bacteria
and other microorganisms for the first time using a single-lens microscope of his own design. Almost two centuries later,
a German biologist called Robert Koch founded modern bacteriology and microbiology. In the 1850s at the University of Breslau,
Ferdinand Cohn's main research tool was a large and expensive microscope that his father had bought for him. In the 1850s,
he studied the growth and division of plant cells and he proved that the use of liquid media was disadvantageous for isolating
pure culture. He was determined to find an alternative technique and introduced the gelatine liquid culture media to be poured
on sterilised glass plates to solidify for the first time.
The rapid microbiologicalmethods revolution
Emanuele Selvaggio
QA Batch disposition & Investigation Supervisor, Pfizer
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 43 Volume 18 | Issue 4 | 2013
IN-DEPTH FOCUS:RMM’S AND ENVIRONMENTAL MONITORING
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It cannot be denied that vendors play a key
role in the development of new method-
ologies across the site when deciding what
instrument to buy. Vendors are involved at
several stages of the project, e.g. feasibility
studies, design qualification, IQ/OQ, PQ and
sometimes even during filing (if any). There-
fore, lack of experience as well as reliability
issues would inevitably decrease initial expecta -
tions and chances to complete the project on
time and could even affect the possibility for
the project to be successful. Consequently, the
choice of technology is strictly connected to
the vendor of choice.
Whatever the technology is, there are some
common characteristics which should be
addressed by most rapid micro methods; they
are usually better detectors (more sensitive,
requiring fewer microbial cells) and allow the
laboratory to get faster and more accurate
results and to improve precision due to the
automation of most of the critical steps.
Depending on specific cases, RMMs are an
opportunity to reduce the cost/assay and to
improve the overall quality level.
An example of a cost saving project is
related to MALDI-TOF mass spectroscopy.
MALDI TOF technology is about to revolutionise
timing and costs currently related to microbial
identification in pharmaceutical microbiology
laboratories. This technique has been well-
known for years by chemists but only recently
has been successfully developed for microbial
identifications within GMP contexts.
In the pharmaceutical industry, the need for
timely and accurate microbial identifications is
critical for the characterisation of micro -
organisms isolated from the aseptic area as well
as from final product samples.
The ability to explain aberrant results and
provide information about potential sources of
microorganisms is important to maintain
control over the manufacturing process. A wide
range of microbial identification technologies
currently exists and varies in labour intensity,
cycle time and cost.
MALDI-TOF technology combines cost
savings goals with rapidity. In fact, for isolated
colonies (from agar plates), less than five
minutes is required to get a reliable (accurate
and reproducible) result, at the cost of about one
Euro each. This is an embarrassing fact con -
sidering that most common technologies need
hours if not days to get a result, and costs range
from 10 to 20 Euros per identification.
Whatever the process/assay is, the faster
results can be obtained, the more process
knowledge is increased. It must be noted that
compendia assays generate results which
are always delayed, hence most of the time
QA/QC cannot find the real root cause of an out
of limit specification.
Until a process is real-time monitored, it will
never be really under control so any potential
adverse event or unplanned deviation will be
managed too late to be properly addressed.
For these reasons, corrective and preventative
action plans are often ineffective. Further
analysis is usually required even weeks after
an event has occurred and the decision-
making processes are often too conservative
(batch disposition results in rejections due to a
lack of knowledge).
Real time technologies as well as rapid
technologies give those in charge of monitoring
the process quality an incomparable tool to
promptly react to process quality attribute
excursions, managing the present rather than
investigating the past.
Return of Investment calculations represent
a tough challenge for those scientists who
approach a rapid method for the first time.
Although a dedicated financial analysis is
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 44
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Mycoplasma DNA-Chip
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Germany (Main offi ce): Greiner Bio-One GmbH, [email protected] l Austria: Greiner Bio-One GmbH, offi [email protected]: Greiner Bio-One BVBA/SPRL, [email protected] l Brazil: Greiner Bio-One Brasil, offi [email protected] l China: Greiner Bio-One Suns Co. Ltd., offi [email protected] France: Greiner Bio-One SAS, [email protected] l Japan: Greiner Bio-One Co. Ltd., [email protected] l Netherlands: Greiner Bio-One B.V., [email protected]: Greiner Bio-One Ltd., [email protected] l USA: Greiner Bio-One North America Inc., [email protected]
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required case by case, a rough estimation of the
ROI can be made considering the following
aspects: manpower, disposables, reagents, costs
of goods and any related activity.
The points to consider are:
� Cost / assay
� Where it takes place
� Number of batches / session
� Disposables
� Reagents
� How many samples / assay
Manpower
� How long it lasts
� Number of single operations
� Time for single operations
� Skills required
Others
� Inventory impact (in term of ‘cash’
inventory costs)
� Warehouse (in case of renting)
Whatever the technology to develop is, an ROI
analysis always starts with a process mapping of
the current method to replace. Any single step
must be considered in terms of both manpower
and costs.
When designing the new process flow, ‘lean
laboratory’ principles as well as a DMAIC
approach are strongly recommended to get the
highest advantage from the new technology.
Indeed, rapid microbiology projects are often a
great chance to change the mind-set of
laboratory employees and to improve efficiency
and productivity.
In conclusion, rapid microbiological
technologies along with PAT are nowadays
the pillars upon which companies should
build the future structure required to
address latest BoH expectations (which are
progressively more demanding) and market
challenges which lead companies to an endless
budget dwindle.
IN-DEPTH FOCUS: RMM’S AND ENVIONMENTAL MONITORING
Emanuele Selvaggio received his degree in Organic Chemistry in
2003 from the University of Catania, Italy. During his career, he
worked in several departments such as Engineering Validation,
Quality Assurance and Technology. In 2008, he was certified as Green
Belt in accordance with the Lean Six Sigma Company Program and
has recently completed his Black Belt project. Since 2008, he has been
in charge of leading many OpEx projects related to several areas such
as Quality Assurance, Quality Control and Manufacturing/Pack
Processes. He was also recruited for several global projects such as
‘Validation Standardisation Project’ (Metrics team) and ‘Laboratory
Instruments Qualification Standard Protocol’.
Since 2009, he has been involved with PAT projects, particularly with
Rapid Microbiology projects (Bioburden, Identification and Cell count
based methods) and since 2011 he has been a permanent member
of the rapid micro methods steering team at Pfizer; he has conceived,
developed and successfully leaded a rapid micro project aimed at
reducing the sterility test cycle time from 14 days to less than one day.
He recently joined the Quality Assurance department as QA Batch
disp. & Investigation Spv.
About the author
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Most of the production in the pharmaceutical
industry is carried out in cleanrooms and
although the principle of cleanroom design
goes well back over 150 years in the hospital
setting, it is only since the 1950s that we have
really seen the development of the modern
cleanroom as we know it. This was driven firstly
by NASA’s space program and later by manu -
facturing needs for a clean environment to carry
out its processes. Cleanrooms are used in
practically all industries where particles can
adversely affect the manufacturing process.
These range from the semiconductor industry,
medical devices, pharmaceutical, biotech and
life sciences.
A cleanroom is a room in which the
concentration of airborne particles is controlled
and which is constructed in a manner to
minimise the introduction, generation and
retention of particles inside the room and in
which other relevant parameters such as temp -
erature, humidity and pressure are controlled as
necessary. The main function of a cleanroom is
to protect the manufactured product from
contamination and this is of utmost importance
to both the manufacture and the customer who
is in actual fact the patient. In the pharma -
ceutical industry, the manufacturer’s economic
survival depends on the safety and quality of the
product. The lives of patients and the reputation
of the manufacturer depend on the purity of the
product. Good Manufacturing Practice (GMP) is
widely accepted as the best way to conduct
business in the Pharma sector. GMP ensures
that products are consistently produced and
controlled to the quality standards appro-
priate to their intended use. GMP covers all
aspects of production from materials, premises,
equipment and the training and the personal
hygiene of staff.
Pharmaceutical cleanrooms are classified by
the cleanliness of the air. The method that is
most easily understood and universally applied
is the A to D version of the Federal Standard 209
in which the number of particles equal to and
greater than 0.5 millimetres is measured in one
cubic foot of air and this count is used to classify
the room. The most recent 209E version has
accepted a metric nomenclature. The newer
standard is TC-209 from the international
standards organisation. Both of these standards
classify a cleanroom by the number of particles
found in the cleanrooms air. The cleanroom
classification standards FS209E and ISO 14644-1
require specific particle count measurements
and calculations to classify the cleanliness level
of a cleanroom or clean area. In the UK, British
standard 5295 is used to classify cleanrooms.
Based upon both the US and EU GMP, con -
tinuous particle counting is a requirement for
sterile manufacturing. Other parameters and
controls in sterile manufacturing are temp -
erature, relative humidity, differential pressure
and air velocity and direction.
In the cleanroom, there are several sources
of contamination such as process equipment,
surfaces and of course personnel. Contamina -
tion can be surface or airborne contamination.
The three main types of contamination in a
cleanroom setting may be particulate, microbial
and rogue or cross contamination. Dust,
fibres and hairs are examples of particulate
con tamination, while microbial contamination
may arise in the form of mould, bacteria or yeast.
Rogue or cross contamination comes about if
materials or products are mixed. Operators are
typically the largest source of contamination in
cleanrooms and in order to counteract this
In Ireland and across Europe, the traditional manufacturing sectors have seen major declines in output and there has being a
fall in the numbers employed in this sector. However, one area that has seen growth is the pharmaceutical industry. In Ireland,
the economy is very dependent on the pharmaceutical industry and the industry currently accounts for over 50 per cent
of the country’s exports.
Controlling contamination inthe pharmaceutical industry
Chris Delaney
Cleanroom/GMP Specialist, Noonan Services Group
IN-DEPTH FOCUS:RMMS AND ENVIRONMENTAL MONITORING
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 46
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threat, it is important that the design is right and
also that proper controls are put into place.
These controls can cover areas such as gowning,
personnel flow and material flow. In the
remainder of this article, I am going to con -
centrate on the importance of having an
effective environmental monitoring programme
in place and how correct cleaning and sanitisa -
tion is essential in the cleanroom setting. From
my own personal point of view, after working in
some of the leading global pharmaceutical
companies, developing and improving con -
tamination control programmes these are areas
that I have a keen interest in.
Across the pharmaceutical, biotechnology
and medical devices industry, companies
have cleanroom operations of different sizes
and complexities. Manufacturers in these
industries have recognised that there is a
regulatory compliance to demonstrate clean -
room per formance and the control of product
bioburden by monitoring the environment in
their aseptic manufacturing areas. Environ -
mental monitor ing is a programme designed to
demonstrate the control of viable (living
microorganisms) and non-viable particles in
critical areas. Viable monitoring refers to testing
for the detection of bacteria, yeast and mould
and includes the monitoring of personnel,
surfaces and areas for microbial contamination.
A non-viable particle is a particle that does not
contain living micro organisms but acts as
transportation for viable particles. Non-viable
particles are monitored using particle counters
which do not distinguish between viable and
non-viable particles.
Companies must monitor their cleanrooms
to ensure that their desired quality standards
are met. Competition in the pharmaceutical
industry is intense and competitive and
manufacturers cannot afford to have even a
perception of any quality issue associated with
their products.
Results obtained from an EM programme
provide information on the performance of the
HVAC system, the effectiveness of cleaning and
IN-DEPTH FOCUS: RMMS AND ENVIONMENTAL MONITORING
Figure 1: Classification of cleanrooms: comparison of classification
sanitisation procedures and the effectiveness of
hygiene and gowning practices. Therefore, it is
critical that the programme is valid, justified and
compliant. In developing an adequate environ -
mental monitoring programme, there needs to
be a sufficient number of sampling locations
which are representative of the microbial
challenge to critical activities and are based on
the nature of the process being performed
and the impact of personnel and equipment on
microbial levels. The application of risk assess -
ment to environmental monitoring provides
information on the most appropriate locations
to monitor within the cleanroom with regard to
potential impact of activities on product
quality. The use of risk assessment approaches is
an important cGMP tool in microbiological
environmental monitoring. However, each suite
of cleanrooms or isolator will be subtly different.
Every aspect of the environment must be
considered and what level of monitoring best
suits the system decided, the techniques used
and the locations selected must be justified. Risk
assessments should be kept up-to-date and any
modifications to the area should be reflected.
The three areas that are usually monitored
in a cleanroom are personnel, air and surfaces.
Personnel are the biggest source of contamina -
tion in the cleanroom setting and account for
over 90 per cent of contamination in clean -
rooms. More and more companies are seeing
the benefit of providing a good training
awareness programme for their staff. The
environ mental programme must be designed to
highlight that the preventative measures such
as correct gowning, hand washing and personal
flow is being adhered. Personnel monitoring
employs contact plates to assess microbial
contamination of cleanroom personnel. The
contact plates monitor areas of the body that
may interact with the sterile field or exposure
areas. Personnel monitoring is a good indication
of how well personnel are gowning when they
enter the cleanroom. More and more companies
are using this technique, especially in their
aseptic process areas.
Some ways to measure air sampling in the
cleanrooms are air samplers (active air sampling)
and settle plates (passive air sampling). In air
samplers, predetermined volumes of air are
drawn in over a sterile media plate which is later
incubated to reveal the number of viable
organisms. With the settle plates, Petri dishes
containing sterile growth media are exposed
to the environment for a specific period of
time. Once the plates are incubated, viable
microorganisms that landed on the plates will
begin to grow.
The monitoring of surfaces in the clean -
room is another essential part of the EM
programme. This can be carried out by the use of
contact plates or swabs. The contact plates
which contain sterile growth medium are
pressed against the surface and then incu-
bated to see if there is microbial growth.
Likewise, the swabs which are sterile and stored
in a suitable sterile liquid are rubbed over the
test area that needs to be sampled. Swabs are
used for areas that are not flat or areas that
are hard to reach such as machinery. Environ -
mental organisms recovered from the
cleanroom areas should be identified to show
what organisms are present. Knowing this
will provide the organisation with important
information in monitoring and prevent
potential future contamination pitfalls. Alert
and action limits should be implemented
based on the company’s products, the intended
use of the cleanroom and the classification of
the cleanroom.
Prior to the notion of GMP regulations,
cleaning was never considered to be important.
It was the activity that was carried out last thing
in the day or by the newest employee. The
procedures were often brief and very often
limited to one sentence. But things have
changed dramatically over the years and now
regulators must position cleaning very highly on
their agenda when carrying out audits. Cleaning
is an essential element of contamination control
in the cleanroom. In my own opinion, I believe
that the cleaning process must be viewed as
important as the manufacturing process.
There are many decisions that need to be made
about the details of cleanroom maintenance
and cleaning. Applications and procedures
must be agreed upon by cleanroom managers,
quality depart ment and the cleaning team.
For an effective cleaning programme, there
must be a buy-in from all strands of the
organisa tion. More and more pharmaceutical
companies are out sourcing their cleaning and
maintenance programmes and some main -
tenance comp anies provide expertise advice
and support in drawing up such maintenance
and cleaning programs. In the pharmaceutical
industry, more and more emphasis is being
placed on an efficient and effective cleaning
programme. This is being driven by the need to
reduce the cost of maintenance and cleaning,
increasing prod uction time and also by the
importance being place on cleaning and
maintenance by the regulatory bodies. There
are many key elements of an effective cleaning
programme such as:
� What is regarded as clean
� How is clean managed
� What cleaning materials can be used
� When can the cleanroom be cleaned
� The frequency of cleaning that is required.
The disinfectants selected for use must be
appropriate so that the effectiveness of the
disinfectants is assessed and the scientific
rationale for their selection is documented
as required by USP <1072> ‘Selection of a
disinfectant for use in a Pharmaceutical
Manufacturing Environment’. For surface
challenge testing, the test organisms are
enumerated using swabs, surface rinse or
contact plate methods. Neutralisers that
inactivate the disinfectants should be included
in either the diluent or the microbiological
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 48
IN-DEPTH FOCUS: RMMS AND ENVIONMENTAL MONITORING
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media used for microbial enumeration or both. Like all cleanroom
developments, there have been great advances in the disinfectants
available and their effectiveness. A disinfectant must have a wide
spectrum of activity. It must have the ability to kill different types of
microorganisms. Currently, the majority of the pharmaceutical
industries use phenolics, quats or quat mixes for routine cleaning.
Therefore, rotation of disinfectant and a sporicidal is common in
the industry.
The USP indicates an acceptance criteria as follows, log reduction
(2 for bacterial spores and 3 for vegetative cells), pre-determined
contact time and recommended typical organisms to be selected.
EN 13697:2002 – Quantitative surface test of bactericidal activity –
describes the method to be used to determine the anti-microbial
activity of commercial formulation or active substances on bacteria in
the conditions in which they are used.
It is important that the right equipment and materials are
selected for cleanroom cleaning. Only equipment that has
proven cleanroom performance records should be selected for
use in cleanrooms. There are many products available now
for cleaning in all cleanroom classification. These include cleaning
and disinfecting solutions, cleanroom mops, cleanroom vacuums,
cleanroom wipes and cleanroom mop and bucket systems. From my
own experience, I have seen how an inadequate cleaning and
maintenance programme can lead to various levels of contamination
problems and potential loss in end user product quality. The saying
‘fail to plan and plan to fail’ is very appropriate when it comes to
setting up a cleaning programme in the pharma ceutical industry.
The effectiveness of the cleaning programme affects all sections of
the company and of course the end user, who is most important.
In conclusion, the main purpose of building a cleanroom suite is
to provide a vital element in the assurance quality of the product for
the end user which in the pharmaceutical industry is the patient. The
cleanroom and indeed the pharmaceutical industry has come a long
way over the brief 25 years that I have worked in it and the next
25 years promise to be more challenging and exciting. The develop -
ment of cleanroom technology is likely to continue in the coming
decades. The pharmaceutical industry is also expected to continue to
grow in the coming years and in recent years, cleanroom technology
has been applied to micro and nano systems processes and this also
looks like a high growth rate in the coming years. The development of
cleanroom technology will also be driven forward by the creation and
use of materials of the finest purity, and by the broad based use
of biotechnology. Given the scale of these challenges, cleanroom
technology looks set to remain indispensable to the pharmaceutical
industries in the coming years and it’s an exciting industry to be in.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013
IN-DEPTH FOCUS
Chris Delaney has spent over 25 years working in cleanrooms. He has
worked in the medical devices, semiconductor and pharmaceutical sectors.
After completing his degree in management, Chris began in the
manufacturing sector and worked in some leading companies in the medical
device and semiconductor industry. In 2004, Chris moved to the service
industry and joined Noonan service group where he now is GMP/Cleanroom
specialist in the Life Science section. Chris has worked in some of the leading
pharmaceutical companies and has developed many contamination control programs. Chris is also an
executive committee member of the Irish Cleanroom Society. He has also developed a number of
training courses on contamination and cleanroom cleaning.
Biography
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For more information about Rapid Micro
Biosystems and the Growth Direct™ System,
visit www.rapidmicrobio.com
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 51 Volume 18 | Issue 4 | 2013
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Product HUBJulie Sperry, Chief Commercial Officer atRapid Micro Biosystems discusses theirGrowth Direct™ System technology Rapid Micro Biosystems provides a range of products to aid faster detection of
microbial contamination in the manufacture of pharmaceutical, biotechnology and
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System, uses proprietary digital imaging technology that automatically enumerates
microcolonies days earlier than the traditional visual plate counting methods.
The system captures the native fluorescence (autofluorescence) that is emitted by all
living cells. By detecting microcolonies composed of a small number of cells, the
Growth Direct™ test can automatically report the number of microbes in a sample
days earlier than the current visual colony counting method. It is the first and only
automated system that addresses all key microbial QC applications and fits with the
current regulatory practices; a critical accelerator for adoption.
What are the perceived hurdles for
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Plummer: The greatest hurdle is the lack of well-
defined guidelines and regulatory expectations.
Existing documents aren’t in step with RMMs
evolution, either not considering RMMs or
not addressing distinctions between them.
Available guidance is open to interpretation and
requires adaptation to ensure test evaluations
and data analyses are applicable to the new
method. Pharma companies express reluctance
in making modifications without knowing if
their validation approach will find regulatory
acceptance. Exacerbating the uncertainty is the
ill-alignment of regulatory bodies in what is
required for validation. This makes imple -
mentation especially difficult for companies
who sell drug products across multiple
regulatory jurisdictions.
Herber: The biggest hurdle for potential
Rapid Micro Method users tends to be the
confusion regarding which new methods
adhere to existing compendia guidelines and
which methods do not. Our Charles River PTS™
cartridge technology for endotoxin testing
helps eliminate this confusion as it follows the
harmonised BET chapters, and thus is used
routinely for endotoxin determination in raw
materials, in-process samples and finished
product. Factor C and various in vitro assays do
not follow current regulatory guidelines and
would require additional validation as an
alternative method.
Stappert: Manufacturers of biologics are
quite reluctant in establishing RMMs due to the
lack of clear validation and/or acceptance
criteria for these methods.
Niccum: TSI’s BioTrak Real-Time Viable
Particle Detector utilises Laser Induced
Fluorescence (LIF) to determine if a particle is
viable in nature. This method does not rely on
culturability of microbial particles. LIF is more
sensitive and ‘sees’ more viable particles than
compendial methods. The highly sensitive, real-
time results offer significant advantages in the
market. To benefit from this technique, the user
community needs to be willing to trial these
innovative products by conducting evaluations
near existing compendial testing locations to
establish correlations. These can then be used as
support for identifying meaningful action and
alert limits.
What is the biggest challenge to
validating RMMs for manufacturing?
Stappert: Although RMMs may be validated by
the manufacturer of the different techniques,
an individual adaptation / testing must be
performed for every matrix / biologics pro-
duced / used by the manufacturer of biologics.
This task can only be achieved by a team play
between the RMM producer and the manu -
facturer of biologics.
Niccum: Existing guidance documentation
available in USP 1223, EP 5.1.6, and PDA TR33
focuses on laboratory based methods where
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 52
RMMS ROUNDTABLE
© Sergey Nivens / Shutterstock.com
Moderator
Jeffrey W. Weber, PAT Project Manager, Process Analytical Sciences Group & Chairman, Pfizer Rapid Microbiological Methods Steering Team, Pfizer
Carrene PlummerDirector, Quality and
Regulatory AffairsAzbil BioVigilant, Inc.
Ulrich HerberSenior European Product
Manager Endotoxin and MicrobialDetection Service
Charles River
Jörg StappertManager Biochip GroupGreiner Bio-One GmbH
Darrick NiccumSenior Global Product
Manager – BiotechnologyTSI
serial dilutions can be employed to generate
challenges comparing RMMs to traditional
culture plate count results. Aerosol challenges
are more difficult to create and control than
liquid samples. It is known that active samplers
have varying degrees of microbial capture
efficiency so the ‘truth’ data set has a high degree
of uncertainty. Thus, the numbers for the
‘standard’ method are highly variable and add as
much uncertainty as the aerosol instrument
under test.
Plummer: Many RMMs are vastly different in
functionality and performance from the
traditional methods they are intended to
replace. Data generated by these new methods
are oftentimes not directly comparable to data
obtained from traditional methods. Examples
include disparate units of measurement
(e.g., CFU vs. biologic count) and discrete vs.
continuous data streams. Determining what the
new data means and how to distil actionable
manufacturing decisions can seem over -
whelming. Azbil BioVigilant’s applications
engineers have assisted several customers early
in their implementation planning to address and
suggest practical approaches.
Herber: In addition to achieving rapid
turnaround time, one of the key challenges our
manufacturing customers face is maintaining
QA/QC oversight when testing is performed
outside of the central lab. Point-of-use testing
instruments on the production floor, like the
PTS™, must have built-in safeguards such as
password protection and the ability to be
externally controlled by the QA/QC group.
An invalid assay or an expired calibration
must result in the RMM instrument being taken
off-line until cleared for use by the quality
group. Prospective RMM users also should
ensure that their vendor provides support to
overcome these challenges.
What guidance is available for
statistical analysis of RMM compared
to compendial methods?
Niccum: We are eagerly awaiting the revision of
PDA’s TR33. Based on discussions with members
of the panel, there is significant emphasis on
providing appropriate statistical method
guidance for evaluating RMMs to compendial
method results. We anticipate additional
guidance when revisions to USP 1223 and EP
5.1.6 are released. Several courses sponsored by
ECA and PDA provide overviews of statistical
methods. It is imperative that users look beyond
the statistical values presented by instrument
vendors and understand the rational and
assumptions incorporated into the statistical
method utilised.
Stappert: General: ICH Q2B, USP1223, Ph.
Eur. 2.6.7/2.6.21; Specific: CLSI EP 17-A.
Plummer: Published monographs do exist
which can offer some initial, high-level guidance
on statistical approaches that may be suitable
for data analyses (e.g., EP 5.1.6 – Alternative
Methods for Control of Microbiological Quality
and its US counterpart, USP <1223> -Validation
of Alternative Microbiological Methods). These
monographs, however, do not define the
specific statistical methods that are best to apply
depending on the RMM and intended
application. It is highly recommended that RMM
evaluators seek advice from expert statisticians
to help define proper statistical analyses while
keeping the RMM, compendial method and the
Pharma customer’s validation goals in mind.
How can the Pharma industry
accelerate the adoption and
implementation of RMMs?
Plummer: Regulators have publicly expressed
their desire to see more widespread use of
RMMs and have stated their willingness to aid
end users in the implementation process.
The Pharma industry should be proactive and
take advantage of the open, informal dia-
logue that has been offered by the regulators.
Frequent communication with them, including
partici pation from both reviewers and inspec -
tors, is ideal. Working closely with regulators
fosters a smoother and faster transition to
RMM implementation.
Herber: The Pharma industry should
continue meeting with the regulatory bodies,
either during an industry conference or one-on-
one, to discuss RMMs and get agency buy-in
early on. The Pharma industry can speed up
RMM acceptance internally by better under -
standing the true cost of the conventional
microassay. Keep in mind, the conventional test
usually looks more attractive when just the
reagent costs are analysed, but it is only when
you look at hands-on technician time, data entry
and review, production efficiency, retest rate and
subsequent investigations that you can
establish a fair comparison between the
conventional and the rapid method costs.
Niccum: The Pharma industry includes
pharmaceutical manufacturers, instrument
vendors, regulators, and consultants. We must
develop open and honest dialogue where the
core RMM technologies are discussed and not
limit the dialogue to each instrument vendor’s
implementation of that technology. The
compendial methods have known capabilities
and deficiencies. The Holy Grail of a single
RMM suitable for all applications does not
exist. Each RMM has known capabilities and
potential deficiencies. Only through open
discussion and scientific analysis will appropri -
ate applications for each RMM be identified and
promulgated. This is especially true for real-
time methods where both the sampling and
counting functions are incorporated into a
single instrument.
Stappert: Firstly by getting clear guidelines
for validation of RMMs (as already established
since long time for the validation of IVD/clinical
products e.g., FDA_CDRH_HPV guidance) etc.,
followed by a close collaboration between the
RMM manufacturer and the RMM operator for
necessary matrix adaptations.
Are there practical RMMs being
developed to support environmental
and personnel monitoring?
Plummer: Most certainly. The Azbil BioVigilant
IMD-A system was developed with practicality
in mind. It requires no staining, no reagents,
and little operator intervention in order to
monitor the environment and warn users
when a microbial threat may be present.
The system also operates in real-time and
continuously, so immediate information is
available about the sampling environment.
These features allow actionable decisions and
remediation to be made as excursions occur
instead of retrospectively.
Niccum: Real-time viable particle detectors
are an example of a practical environmental
monitoring RMM. The technology is not at the
point where direct replacement of all com -
pendial active sampling and culture based
counting applications exist. This will occur
over time resulting from on-going use and
increased understanding of the technology.
With that said, there are many applications
where real-time viable detectors can be
employed offering significant financial and
quality benefits to pharmaceutical manu -
facturers. As one inspector mentioned, they are
currently another tool in the tool set which
should be used where applicable. TSI refers to
this as the innovation highway resulting in
greater acceptance by the user community.
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 53 Volume 18 | Issue 4 | 2013
RMMS ROUNDTABLE
Rottapharm can boast a rich portfolio of original
molecules and drugs, discovered and patented
by its own R&D department. From the original
glucosamine sulphate, an essential molecule
in the treatment of osteoarthritis, to pro-
glumetacina, a non-steroid anti-inflammatory
drug, up to the launching of the first transdermal
active matrix patch with estradiol an innovative
transdermal releasing system for hormone
replacement therapy in the menopause,
research has not stopped but in fact has
continued to grow and develop in a steady
manner. In the pharmaceutical sector,
Rottapharm|Madaus has over 13 projects in
its pipeline.
Rottapharm in Ireland
The Rottapharm manufacturing site in Dublin,
Ireland was officially opened in June 1999 and
commenced manufacturing operations later
that year. The Dublin site employs 150 people
manufacturing a range of capsules, sachets
and tablet formulations in addition to the API
Crystalline Glucosamine Sulphate. The comp -
any’s leading product DONA, the original
Glucosamine Sulphate, is manufactured in
sachet and capsule formats for markets
in Europe, Asia and South America. DONA is the
first drug of choice to be administered as early as
possible in the treatment of osteoarthritis.
Production output has grown steadily over
the years, with the Dublin site becoming one
of the most important manufacturing sites
within the Rottapharm|Madaus group of
companies. Figure 1 (page 55) shows the growth
in terms of percentage output from 2000 to
2012. The increase in growth from 2009 onwards
was as a result of a seven million Euro invest -
ment and facility expansion. The investment
involved the transfer of a number of new pro -
cesses from another facility within the group.
The project expansion resulted in an addition of
four packaging lines, two coating systems and
an encapsulation machine. Volumes of finished
product packaged increased significantly, as
a result and 35 new positions were created.
The project was completed in 2010.
Lean Six Sigma and the Operational
Excellence programme
In order to improve operations and per -
formance, the Senior Management team at
Rottapharm Dublin introduced a Lean Six Sigma
program in 2007. Six Sigma seeks to improve the
quality of process outputs by identifying and
removing the causes of defects and minimising
variability in manufacturing and business
From their headquarters based in Monza, Italy, Rottapharm’s long history of success began in 1961 with the creation of a small
laboratory for independent research. The company from its early beginning has continuously invested in research, innovation
and development of pharmaceutical products for distribution on a worldwide scale. After acquiring the German multinational
Madaus Pharma in 2007, the Group is currently one of the most significant and prominent pharmaceutical companies in Italy and
is present in over 85 countries worldwide. The field of research is above all the undisputed true mission of the company.
Improving operations and performance: howRottapharm is using Lean Six Sigma principles
Richard Hayes
Continuous Improvement Manager, Rottapharm
SIX SIGMA
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 54
processes. It uses a set of quality management
methods, including statistical methods, and
creates a special infrastructure of people within
an organisation (Black Belts, Green Belts, etc.)
who are experts in these methods. Each Six
Sigma project carried out within an organisation
follows a defined sequence of steps and has
quantified financial targets cost reduction
and/or profit increase. Rottapharm has run over
20 Six Sigma projects over the past five years.
Typically, these projects were completed on
difficult process problems encountered and by
the application of Six Sigma methodologies,
an effective means of problem-solving and
process improvement could be implemented.
These problems were tackled by teams using
the Six Sigma approach of Define the prob-
lem, Measure, Analyse, Improve and Control
(DMAIC problem solving process). The Six
Sigma DMAIC problem-solving approach con -
tinues to be used to date, supporting the overall
Operational Excellence programme within the
plant. The company has also introduced other
lean tools and concepts into the business in
order to remove waste and drive down costs
in the business. Some of these tools and con -
cepts are described below.
Total Productive Manufacturing (TPM)
The lean tool known as Total Production
Maintenance or Total Production Manufacturing
(TPM) is the main driver of the Operational
Excellence programme within the Rottapharm
Dublin facility.
TPM is a way of working in a cellular based
team structure with manufacturing teams,
made up of operators, technician and engineers,
supported by various Pillar Champions, usually
managers of a particular support function such
as materials, maintenance, safety, quality etc.
TPM focuses on equipment, the interaction of
people with that equipment and the waste
associated with the equipment and the various
interactions. TPM doesn’t just focus on equip -
ment alone; all other support departments are
integrated into the overall structure as their
contribution plays a key role in the success of
each project. All the waste associated with both
the actual manufacturing activity and the
support activities are targeted in order to keep
costs down and productivity up.
The TPM project at Rottapharm Dublin
began in May 2010 with 19 people trained on
the nine step TPM improvement plan. Two
separate shift teams focused on a manu -
facturing area while two other shift teams
looked at a sachet filling and packing line, each
team comprised six members. The teams took
20 weeks using one day a week every fortnight
to go through the nine step TPM programme for
their project. Each day that TPM was scheduled,
production activity was suspended and the
teams worked through a programme of activity
designed to improve the equipment uptime and
performance and remove waste associated with
that equipment and the associated interactions.
The success of the projects was immense;
particularly one associated with a packag-
ing line where a dramatic increase in OEE
was obtained rising from an average of
15 per cent to an average of 50 per cent.
Fourteen TPM projects have been rolled out to
date across the plant. The TPM programme is
broken into nine steps:
1. Collecting equipment history and perform -
ance information
2. Defining Overall Equipment Effectiveness
(OEE) measurement and potential and
reviewing progress
3. Assessing the six top losses and setting
improvement priorities
4. Carrying out a critical assessment
of the equipment
5. Carrying out a condition appraisal
of the equipment
6. Planning the refurbishment, spares and
manpower to carry out the work
7. Developing future asset care
8. Developing best practice
9. Problem solving and prevention.
At the end of the nine step TPM activity, the
teams present a close out to Senior Manage -
ment and then progress onto an ongoing
Continuous Improvement phase, the teams
meet every week for two hours reviewing
performance and performing various activities
focusing on improvement and reducing waste.
In order to ensure that progress is maintained
there are eight levels of assessment that each
team must achieve as the team progresses on
their journey. The teams start to become self
empowered and self managing of all activities
within their influence. The TPM tool is very much
a cultural change tool and the benefits of this
rather than as an equipment or maintenance
tool becomes evident after implementation and
using the tool over a period of time.
Total Productive Administration
In September 2012, based on the recognition
that the cellular based structure of TPM was
successful both in terms of achieving better
KPIs and developing a culture of Continuous
Improvement among cross functional depart -
ments, it was decided to introduce the concept
of Total Production Administration (TPA) into the
support functions at Rottapharm Dublin.
Four project areas were chosen, Quality
Assurance (QA), Quality Control (QC), Finance
and Materials. The teams selected were drawn
from members of those departments along with
members from other process and support
departments to provide an external insight to
the team and offer a new perception to the
project in question. The tool used by the teams
was process mapping whereby the teams used
the brown paper method to:
� Review existing process activities
� Remove non-value adding activities
� Develop a new improved process with the
wasteful activities removed.
In one example, the QC team reduced non-test
task activities from a total time of 39 hours to
SIX SIGMA
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 55 Volume 18 | Issue 4 | 2013
Figure 1: Production output expressed as percentage for year on year comparison
16.5 hours, a 58 per cent saving of time for a QC
analyst to perform more value adding tasks.
Paperless projects
The lean paperless projects, which began in
2009, have transformed the operations and
maintenance systems within the Dublin site.
Paperless maintenance involved the
installation of a Computerised Maintenance
Management System (CMMS) to support timely
and accurate information on maintenance
activities within the plant. The system was
installed and validated to CFR 21 standards
within six months. Data access and inputs are
performed by Rottapharm maintenance
personnel using military standard laptops.
The CMMS enables maintenance activities
to be logged as they occur. In conjunction with
an Overall Equipment Efficiency (OEE) system
integrated to the equipment, real-time
information on equipment performance can be
generated and a range of KPIs were developed
for performance review. The CMMS and OEE
systems are also linked which allows meter
based maintenance activities with actual
running times determining routine main -
tenance requirements.
The Manufacturing Execution System (MES)
is a software application which manages various
operations on the factory floor. The MES
implemented at Rottapharm includes numerous
interfaces with plant equipment. The project
started in the warehouse in 2009 and has now
been rolled out to over 95 per cent of the
manufacturing and all packaging activities. This
means that batch records are now electronic
rather than paper-based. Over 800,000 wet
signatures have been removed as a result of this
project. This ensures a reduction in processing
errors and allows a faster batch review time
through the use of ‘review by exception’ taking
place. Paper has all but disappeared within the
production and maintenance environment of
the company.
Energy reduction
Rottapharm has taken steps to improve the
energy efficiency of the business over the last
number of years. The primary reasons for this
have been to reduce the emissions of Green
House Gases, Carbon Dioxide (CO2) and also to
reduce the overhead costs of electricity and
natural gas. Green House Gas CO2 emissions
have dropped from a high of 4685.8 tCO2 in 2010
to 3207.3 tCO2 in 2012. Energy consumption
dropped by 44 per cent between 2010 and 2012
with relatively similar production output of
packs of 20.9 million in 2010 and 20.5 million
in 2012.
The energy awareness project team have
also introduced a number of energy reduction
projects over the last number of years, targeting
some of the significant energy users, which has
resulted in reduced consumption of electricity
and natural gas, these are listed as:
� The recirculation of air in the Heating Vent -
ila tion & Air Conditioning (HVAC) system
� The installation of occupancy and daylight
sensitive lighting controls in corridors
� The optimisation of the chilled water plant
by raising operating temperature and
improving control of the chilled water
plant units
� The optimisation of the Building Manage -
ment System (BMS) in order to control
heating requirements in times when areas
are unoccupied
� The phased installation of Light Emitting
Diode (LED) lighting in the facility.
The Green Shoots
Awards Competition
The Green Shoots Awards Competition came
about as a result of the need to promote energy
and waste awareness within the Rottapharm
plant. The competition was launched on World
Environment Day, 5 June 2012.
The competition was successful in engaging
employees in suggesting ideas for both energy
and waste reduction. Over 140 ideas were
submitted to the competition and through a
process of elimination this figure was short-
listed to 12 finalists. Each of the finalists was
assigned a sponsor to aid them in preparing
a more detailed explanation of their idea.
The 12 detailed entries were then judged by
Senior Management and members of the green
shoots awards committee. The overall winner of
the competition was selected along with the
category winners for the best energy and waste
reduction ideas. The winning energy reduction
entry was to examine what equipment etc.
could be switched off during off peak periods for
the whole plant. The waste reduction winning
idea was to install MES screens on the ware -
house turret trucks to speed up stock location.
The overall winning idea was establishing a CRS
standard Spectrum for all raw materials in the
QC laboratory.
The winners were announced on 19
December 2012 and all 12 finalists received a
prize. The ideas suggested, for energy and waste
reduction during the competition, have already
started to be implemented across the plant.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 56
SIX SIGMA
Rottapharm designed automation program for automatic enforcement of in process checks and the MESelectronic batch record screen
Figure 2: Rottapharm Dublin energy consumption in kilowatt hours (2009 to 2012)
Visit www.europeanpharmaceuticalreview.comto register now
Register now at www.europeanpharmaceuticalreview.com/nir-spectroscopy
The Process Analytical Technology (PAT) and Quality by Design (QbD) initiatives have been of interest forpharmaceutical manufacturing in the last few years. Implementation of PAT/QbD approaches involves monitoringand controlling critical process parameters that influence the critical quality attributes of the product. One of theprime processes in pharmaceutical solid dosage form is granulation and the drying process. With the PAT and QbDinitiatives, the FDA aims to increase the efficiency of the pharmaceutical production by real-time process analysisand control. Near Infrared spectroscopy (NIRS) is well accepted as a potential PAT analyser due to its rapid andnondestructive technique that additionally requires no sample preparation.
Metrohm is well-established in determining the moisture content by Karl Fischer titration in every conceivable typeof product. To provide advance solutions for pharmaceutical customers, Metrohm now is moving forward, bringingNIRS to help customers to optimise the use of raw materials and to consistently run production closer to targetspecifications with time- and cost-saving analytical techniques.
During this webinar, we will be showing the use of NIR in the process that allows for monitoring low levels ofresidual moisture and other process constituents to yield better process control and endpoint determination.Additionally, we will be focusing on:• Developing a robust and precise method• Minimising implementation efforts • Ensuring calibration model transferability between analysers.
Supported by
Organised by:
Real-time monitoring of themoisture content in differenttype of dryers by NIRspectroscopy as a PAT tool
A European Pharmaceutical Review Date:Tuesday, 8 October 2013
Time:15.00 BST
Length:1 Hour
Dr. Volker J. FrostHead of the CompetenceCenter Spectroscopy,Metrohm NIRSystems
SPEAKERS:
Wim GunsChief Sales Officer,MetrohmNIRSystems
Proposals for the annual ELRIG Drug Discovery
meeting were developed during 2006 and led to
the first meeting being held at the EMCC,
Nottingham. Over the last six years the meeting
has doubled in size, establishing itself as a
leading vendor exhibition in the UK. More
importantly, the Drug Discovery meeting has
become the premier show to attend within the
Life Sciences community, giving fantastic
networking opportunities to scientists from
Pharma, biotech and academia interested in the
challenges of pre-candidate drug discovery. This
year, ELRIG is once again taking the show back to
the Manchester Central Conference Centre
which is fast becoming the hub for the Annual
Drug Discovery event.
Over the two days a superb range of world-
class speakers will be presenting in eight
sessions covering a wide range of topical issues
for Drug Discovery including ‘Advanced Cell
Technologies’, Innovations in Assay Develop -
ment & Screening and also two sessions on
Macromolecules – as Therapeutics and as Tools
for Discovery & Target Validation.
Each meeting will feature an extensive array
of posters, poster taster talk, snapshot talks,
training, competitions and probably the best
exhibition for Drug Discovery products and
services in the UK, which incorporates a thriving
‘Innovation Zone’ for new companies promoting
exciting technologies to the market.
The conference programme will also
include what the organisers hope will be a
challenging and entertaining Dragon’s Den
event in which innovators will present new ideas
to a panel of industry leaders for discussion.
Finally there will be a range of exhibitor
organised meetings and training courses.
At the 2013 meeting, the keynote address
will be by Dr Ruth McKernan, CSO of Pfizer’s
Neusentis Unit, on ‘Practical applications of stem
cell-derived cells within drug discovery’.
In developing the programme for this
meeting, ELRIG has worked with the objective of
bringing together scientists from across the
field, whether they are based in academia,
the pharmaceutical or biotech industries, in
order to establish relationships to advance the
science of drug discovery in the UK and Europe.
As usual, attendance at the show is free –
thanks to the support of the meeting’s sponsors
and exhibitors. For more information, and to
register to attend, please visit www.elrig.org.
In 2013, ELRIG’s Drug Discovery meeting will be in its seventh year. Organised each year by ELRIG (European Laboratory Robotics
Interest Group) and supported by SLAS (Society for Laboratory Automation and Screening), this year’s event will take place from
3 – 4 September in Manchester, UK.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 58
ELRIG’s association with SLAS continues andprovides Students, graduates and post-docswith an opportunity to win the SLAS YoungScientist Award at the ELRIG Drug Discovery2013 Annual Conference and Exhibition.
The winner will be invited to present andparticipate in the Student Poster Competitionat the 3rd Annual SLAS Conference andExhibition, 18 – 22 January 2014 at the SanDiego Convention Center in San Diego,California, USA. SLAS Young Scientist Awardwinners receive a USD 500 cash prize,roundtrip coach airfare, shared hotelaccommodations and conference registrationfor SLAS2014.
ShowPREVIEW Date: 3-4 September 2013 · Location: Manchester, UK
© p
etar
g / S
hutt
erst
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com
Details of protease inhibitors in clinical use have
been reviewed and referenced by Abbenante &
Fairlie3 and up-to-date information relating to
clinical trials for a wide range of diseases,
including those that involve protease inhibitors,
can be identified using the National Institutes of
Health clinical trial database (ClinicalTrials.gov)
which currently contains more than 100,000
clinical trials from 180 countries and receives
over 50 million page views per month. Despite
the successes in discovering and developing
orally administered protease inhibitors,
significant challenges still remain with regards
to their safety profiles and demonstrable
efficacy in clinical trials. Nevertheless, the fact
that there are small molecule protease inhibitors
undergoing clinical trials confirms the view that
the protease target class are tractable for drug
discovery4. In this article, the roles of synthetic,
natural products and endogenous cystatin M/E
are discussed, in particular with respect to
facilitating cysteine protease small molecule
drug discovery.
Synthetic and natural product
low molecular mass cysteine
protease inhibitors
Most of the biochemical and structural studies
carried out on proteases have made use of the
model systems such as the serine protease
chymotrypsin and cysteine protease papain and
these have provided valuable insights into their
mechanism of action and specificity character -
istics5-8. The cysteine proteases contain an
essential highly reactive thiol group contributed
by a cysteine residue which is required for
catalytic activity. In addition, they also contain
an imidazole group contributed by a histidine
residue which is largely responsible for con -
ferring the abnormally low pKa of the cysteine
thiol group (3.4 in the case of papain) rather than
the usual pKa >9 associated with the dissociation
of low Mr thiol containing compounds such as
2-mercaptoethanol. The thiol groups in cysteine
proteases have an inherent propensity to react
with reagents such as iodoacetate9 and mercuri -
Cysteine proteases are expressed ubiquitously in the animal and plant kingdom and are thought to play key roles in maintaining
homeostasis. The aberrant function of cysteine proteases in humans are known to lead to a variety of epidermal disease states
such as inflammatory skin disease1
. In marked contrast, the serine proteases have been most widely implicated in disease
states including hypertension, periodontisis, AIDS, thrombosis, respiratory disease, pancreatitis and cancer2
, and a number of
their inhibitors have been approved for clinical use.
How naturally occurringinhibitors can facilitate smallmolecule drug discovery forcysteine proteases
Sheraz Gul
Vice President and Head of Biology, European ScreeningPort GmbH
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 59 Volume 18 | Issue 4 | 2013
PROTEASES
‘‘Most of the biochemical and structuralstudies carried out on proteases have
made use of the model systems such asthe serine protease chymotrypsin and
cysteine protease papain’’
benzoate10,11, however they lack specificity
features for cysteine proteases. Specificity for
cysteine protease inhibitors can be introduced
by the incorporation of features that are
complimentary with the binding sites of the
enzymes as exemplified by the irreversible
substrate derived inhibitors based upon
fluoromethyl ketones12, cyanogen bromide13-15
and 2,2'-dipyridyl disulphides16. Many cysteine
protease inhibitors also inhibit serine proteases
due to their similarities in the catalytic mech -
anism of action. However, as the catalytic site
thiol (of cysteine proteases) has a greater
nucleophilicity relative to the hydroxyl of the
catalytic serine (of serine proteases), this can
allow for selectivity towards cysteine proteases.
As protease enzymes have an inherent
propensity to degrade their respective sub -
strates, they are often synthesised in an inactive
form (pro-enzyme) in order to prevent aberrant
activity. This pro-enzyme subsequently under -
goes auto-catalytic processing to release
the pro-domain, which thereby results in the
generation of mature catalytically competent
protease17. The peptide sequences surrounding
the auto-catalytic cleavage sites of proteases are
often used to design protease substrates such
that they contain similar sequences18,19. However,
proteases usually undergo a conformational
change upon autocatalytic processing and the
specificity characteristics of the mature protease
may not directly resemble that of the pro-
enzyme, therefore it is not always the case that a
substrate designed on the basis of the auto-
catalytic cleavage site will be acted upon by the
mature protease.
The mechanisms by which low molecular
mass inhibitors act upon cysteine proteases
include (a) reaction with their catalytic site
thiol group resulting in the formation a
product which cannot undergo any further
reaction, (b) forming a reactive intermediate
that subse quently reacts with the enzyme
via a mechanism that is not part of its usual
catalytic act or (c) reacting with the enzyme
active centre via their usual mech anism and
under going further reaction at such a slow
rate that it is essentially considered to be an
irreversible reaction thereby rendering the
enzyme catalyt ically inactive. An ex -
tensively characterised low molecular
mass cysteine protease inhibitor is the
natural product alkylating agent
L-trans-Epoxy-succinyl-leucylamido
(4-guanidino)butane (E-64) that orig in -
ates from Aspergillus japonicas. This has
been shown to inhibit a variety of plant
cysteine proteases (including papain and
ficin), human cysteine proteases (cathepsin L20,
a protease from human breast-tumour tissue21,
and the calcium-dependent protease calpain
from chicken muscle22, but not to inhibit a
variety of serine proteases (trypsin, chymo -
trypsin, tissue kallikrein, plasmin and pancreatic
elastase) or aspartic proteases (pepsin and
Paecilomyces acid proteases). X-ray crystallo -
graphy studies of papain-E64 complex have
shown that the epoxide residue of E64 interacts
with the papain S1-subsite and the leucyl residue
is bound to the papain S2-subsite. A variety of
E64 derivatives have been synthesised and
tested in vitro against cysteine proteases,
of which CA-074 has been shown to inhibit
cathepsin B with an IC50 in the low nM range with
>1,000 fold selectivity against other related
cysteine proteases cathepsin L and cathepsin
H23,24. Collectively, these studies suggest that
E64 has been a valuable inhibitor for the study
of cysteine proteases.
The cystatins: endogenous
high molecular mass cysteine
protease inhibitors
As described above, proteases are often
expressed in vivo in an inactive form (pro-
enzyme). Upon cleavage of the pro-domain,
it usually dissociates from the mature protease
thereby rendering it catalytically functional.
The activities of mature cysteine protease
enzymes in vivo are regulated by a variety of
endogenous protease inhibitors such as
cystatins. Additional endogenous protease
inhibitors include the serine protease inhibitors
(serpins), a noteworthy example of which is the
myeloid and erythroid nuclear termination
(MENT), a stage-specific protein which has been
shown to inhibit the cysteine proteases
cathepsin L and cathepsin V25 and the tissue
inhibitor of metalloprotease26.
The cystatins are members of a superfamily
of evolutionarily-related proteins (each con -
taining more than 100 amino acid residues) that
can be divided into three major families, namely
Type-1 cystatins A and B (also known as stefins)
which are relatively simple in structure,
containing no disulfide bonds or carbohydrate
and are found intracellular as well as the
cytoplasm of cells as well as body fluids. Type-2
cystatins (C, D, F, G, M/E, S, SN, and SA)
containing two disulfide bonds and no
carbohydrate which are mainly extracellular
secreted polypeptides synthesised with a
significantly shorter (19 to 28) residue signal
peptide and are broadly distributed and found
in most body fluids, and Type-3, also known
as kininogens (L- and H-kininogens) which
are composed of many domains, disulfide
bonds and carbohydrate and these include
H-kininogen (high-molecular-mass, IPR002395)
and L-kininogen (low-molecular-mass) and are
found in a number of species27. The first human
cystatin was identified from the sera of auto -
immune disease patients and was shown to
inhibit the cysteine proteases papain, human
cathepsin H and cathespin B28.
In general, cystatins are competitive,
reversible, tight binding proteins that inhibit
cysteine proteases in a micromolar to pico-
molar range29. They are capable of rendering
their target proteases inactive via a stable
complex and preventing any additional
proteolysis30-33. These inhibitors act upon their
target proteases that have escaped or upon
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 60
PROTEASES
‘‘The activities of mature cysteineprotease enzymes in vivo are regulated
by a variety of endogenous proteaseinhibitors such as cystatins’’
© lc
ulig
/ Sh
utte
rsto
ck.c
om
PROTEASES
European Pharmaceutical Review
www.europeanpharmaceuticalreview.com 61 Volume 18 | Issue 4 | 2013
Entitled Practical Workshop: Cell based assays for screening, the third
workshop in the on-going series was a very successful event with a total
of 26 attendees across a wide range of the pharmaceutical industry, from
postdoctoral students to industry scientists.
Lectures included an introduction to drug discovery and the design
and development of biochemical and cell based assays for drug
discovery purposes, screening jargon and terms, a selection of assays to
ensure translation of hits between formats and data analysis and
reduction. During practical sessions, attendees were able to trial
equipment supplied by sponsor companies BMG LABTECH, Cellular
Dynamics, PerkinElmer and Promega to enhance their knowledge,
looking at general concepts for cell based assays, screening cell based
assays against a small molecule library and application of cell health,
cardiac hypertrophy and neurite outgrowth assays using human iPS cell-
derived cardiomyocytes and neurons.
As well as providing theoretical and practical knowledge of cell
based assays and screening, the workshop provided an opportunity for
professionals working in drug discovery to network, discuss their current
projects and keep up-to-date with the latest industry developments.
Participants were able to claim 72 Continuing Professional
Development credits if registered on the Society of Biology CPD Scheme.
Our next workshop, ‘Chemical Biology, Drug Discovery & Screening’,
takes place from 23 - 25 October 2013 at the European ScreeningPort
GmbH facility in Hamburg, Germany.
Please visit www.euopeanpharmaceuticalreview.com/workshop for
more information.
Sponsors
In conjunction with European Pharmaceutical Review, the European ScreeningPort GmbH hosted the third in a series of
workshops which examine, by way of practical sessions and lectures, the design and application of assays for screening
applications in drug discovery from 11 – 13 June 2013.
Practical Workshop: Cell based assays for screening
REVIEWHosted by: Organised by:
exogenous proteases of invading micro -
organisms. The absence of these endogenous
inhibitors has been implicated in disease states,
for example, cystatin C has been shown to
pro mote atherosclerosis in apolipoprotein
E deficient mice34. Each cystatin has a single
reactive site and binds to their target cysteine
protease in a non-covalent manner. Although
the cystatins have many common features, the
differences in their structures have a con -
siderable effect upon their abilities to inhibit their
target proteases. The chicken egg white cystatin
has been purified and extensively characterised
with regards to its bio-physical characterisation
and kinetics and mechanism of inhibition of a
variety of proteases35 and has been shown to
be composed of two major forms (Form A and
Form B, composed of 108 and 116 amino acid
residues respectively and containing two
disulfide bonds).
The role of endogenous cystatin M/E as
a cysteine protease inhibitor
There is evidence implicating the role of cysteine
proteases in the maintenance of epidermal
tissues36,37 as well as being down-regulated in
breast cancer38. The most notable example is the
characterisation of wild-type cystatin M/E and its
N64A and W135E variants against cysteine
proteases that led to the identification of key
residues of cystatin M/E that are responsible for
its inhibition profile. Although wild-type cystatin
M/E has been shown to inhibit legumain,
cathepsin V and cathepsin L with Ki with values
<2 nM, the N64A variant results in a significant
decrease in its potency towards legumain
(Ki >100 nM) whilst retaining similar activity
against cathepsin V and cathepsin L39. In the case
of the W135A cystatin M/E mutant, the potency
against legumain and cathepsin L is similar to
that of wild-type cystatin M/E, however, in the
case of cathepsin V, a significant decrease in
potency was observed (Ki >100 nM). The
homology model of cystatin M/E based upon
the crystal structure of cystatin D has led to the
identification of key regions within the protein
that can explain the inhibition profiles cystatin
M/E as well its variants39.
The studies of Grzonka et al40 involved the
characterisation of the potential of various
cystatins to inhibit papain and cathepsins B, H,
L and S and identified the key residues that are
responsible for the inhibition profiles against a
range of plant cysteine proteases (papain, ficin,
actinidin and cathepsin B).
Studies have shown that cystatin A, cystatin
B and cystatin C inhibit the cysteine proteases
cathepsin B, cathepsin H and cathepsin L with
Ki in the double digit nanomolar range.
Many of these enzymes have been implicated in
tissue degradation and excessive proteolytic
activity, leading to diseases such as arthritis,
stroke, Alzheimer’s and cataracts. The structural
basis for the inhibition of the cysteine protease
papain by chicken white cystatin has been
determined and shown to interact with the
S1-S3 subsite of papain and hairpin loops inter -
acting with the S1'-S2' subsite.
The use of cystatins to facilitate
small molecule drug discovery for
cysteine proteases
Considerable progress has been made in
relation to the understanding of the roles
cysteine proteases play in diseases. The
existence of a variety of endogenous protease
‘‘In general, cystatins are competitive,reversible, tight binding proteins that
inhibit cysteine proteases in amicromolar to picomolar range’’
inhibitors, notably the cystatins, have been
relatively under-exploited for the discovery of
inhibitors of proteases despite the extensive
kinetic characterisation of their mechanism of
inhibition of their respective protease target.
As a variety of natural cysteine protease
inhibitors have been identified with a range of
potencies, some which are relatively potent and
elucidation of their mechanisms of action,
identification of key binding interactions and
kinetics of inhibition can be used to facilitate
drug discovery. A comprehensive list of small
molecule cysteine protease inhibitors can be
found in the review of Otto and Schirmeiter41.
Recent examples of proteases against which
inhibitors have been developed, shown to be
efficacious in clinical trials and approved by
the Food and Drug Administration (FDA),
include Sitagliptin which inhibits the serine
protease Dipeptidyl Peptidase 442. Although
the results from these extensive studies can
be exploited in order to identify key inter-
actions for drug discovery purposes, it has
remained a considerable challenge to develop
suitable compounds with appropriate potency
and selectivity.
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 62
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Naval J (2001) Cladribine induces apoptosis in human
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Wilcox PE (1972) An x-ray crystallographic study of the
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15. Machleidt W, Thiele U, Laber B, Assfalg-Machleidt I,
Esterl A, Wiegand G. Kos J, Turk V, Bode W (1989)
Mechanism of inhibition of papain by chicken egg
white cystatin: Inhibition constants of N-terminally
truncated forms and cyanogens bromide fragments of
the inhibitor. FEBS Letters 243:234-238
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of low-molecular-weight thiols with some aromatic
disulphides. 2,2'-Dipyridyl disulphide as a convenient
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17. Hsu MF, Kuo CJ, Chang KT, Chang HC, Chou CC, Ko TP,
Shr HL, Chang GG, Wang AH, Liang PH (2005)
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19. Thomas DA, Francis P, Smith C, Ratcliffe S, Ede NJ, Kay C,
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CG, Tamai M, Hanada, K (1982) L-trans-epoxysuccinyl-
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References
Sheraz Gul is Head of Biology at European
ScreeningPort, Hamburg, Germany where he
manages the assay development and
screening of academic targets. Prior to this, he
worked for GlaxoSmithKline for seven years
where he developed biochemical and cellular
assays for High Throughput Screening as well
as hit characterisation. In addition, he has worked in academia for five
years on proteases and kinases. He is the co-authored of the Enzyme
Assays: Essential Data Handbook. He is involved in many European
Initiatives involving government, the pharmaceutical industry and
academia (e.g. EU Framework 7 and IMI). His research interests are
directed towards maximising the impact of HTS for drug discovery.
Biography
BIOTECHNICA visitors will be treated to a rich
array of displays revolving around the very latest
advances in technology and R&D, as well as the
latest biotech products and their applications in
the fields of medicine, food production, industry
and environmental protection. There will also be
a wide range of services for the biotech and
pharmaceutical sectors. BIOTECHNICA is con -
sidered to be one of the most important
platforms for bringing innovators together
with investors.
Four marketplaces dedicated
to sector trends
The 20th edition of BIOTECHNICA will boast a
new expo concept revolving around dedi-
cated marketplaces, each addressing a
specific theme or focus of interest in the biotech
sector. This new approach will make it easier
than ever for attendees to find what they
are looking for. Each segment will feature its
own forum with scientific talks and company
lectures, as well as networking services and
poster presentations.
The four marketplaces at BIOTECHNICA
are Personalised Medicine Technologies, Innova-
tion in Food, Industrial Biotechnology and
BioServices. The Personalised Medicine
Technologies Marketplace will focus on molec -
ular diagnostics and strategies for personalised
therapies. The forum discussions and talks
will explore next-generation sequencing,
cell-based assays, biomarkers, companion
diagnostics and molecular imaging. Keynote
speakers have been attracted from the top
German clusters in this field: Bio Deutschland,
the association of the German diagnostics
industry (VDGH), the association of pharma -
ceutical research comp anies (vfa) and the bio
section of the vfa.
The advances being made in bio-pharma -
ceuticals through the application of exciting
new technology will be the highlight at the
BioServices Marketplace, which aims to
establish valuable contacts between research
and development on the one hand, and
innovative biotech companies and the pharma -
ceutical industry on the other.
Featuring new topics and innovative marketplaces, BIOTECHNICA 2013 in Hannover will, from 8 – 10 October,
once again serve as the central hub for Europe’s entire biotech sector and its clientele. For the 20th time
BIOTECHNICA will bring exhibitors and trade visitors together to do business. As Europe’s leading trade fair for
biotechnology, the life sciences and laboratory equipment, BIOTECHNICA is the number one event in its field.
One of the keynotes of this year’s trade fair will be bio-economics. Switzerland – the first Partner Country to be
honoured at BIOTECHNICA – will also be a focus of attention.
For more information, please visit:
www.biotechnica.de
ShowPREVIEW Date: 8 – 10 October 2013 · Location: Hannover, Germany
BIOTECHNICA 2013 – Europe’s biotech hub
BMG LABTECH has a number of microplate
readers on the market, including the multi-
mode OMEGA range, which is popular in the life
sciences and the PHERAstar FS which has
become the gold-standard for the high
throughput screening market. “The CLARIOstar
is a mid-range reader that sits between the
OMEGA and PHERAstar FS,” says Mount, “and
because of its flexibility and performance, it can
fit into an individual research lab or a core
facility. It’s ideal for life science researchers, as
well as groups performing medium throughput
screening within pharmaceutical research. It’s an
exciting product with unique technology.”
So what makes the CLARIOstar so unique?
“It’s BMG LABTECH’s first microplate reader to
feature a monochromator and it is the very first
reader to use LVF technology,” says Mount.
“LVF technology has only just come to the
market generally and this is the first time it’s
been employed in a microplate reader.”
There are many benefits of the LVF
mono chromator over a traditional or grating-
based monochromator system and filters.
For example, the LVF monochromator not only
has selectable wavelengths from 320 to 850
nanometres, it also has continually variable
bandwidths selectable from 8 to 100 nano -
metres. This makes LVF monochromators
much more flexible than traditional mono -
chromators with limited or fixed bandwidths.
“This effectively allows the user to design
individual ‘filters’ for different assays, pro-
viding improved assay performance,” Mount
explains. “LVF mono chromators also avoid
stray light which again significantly improves
assay performance.” In addition, the LVF
mono chromator transmits more light than
a traditional monochromator, for various
tech nological reasons, which greatly im-
proves sensitivity.
“A key feature is flexibility; because the
wavelength and bandwidth can be selected in
LVF monochromators, fluorophores can be
scanned in both excitation and emission to
determine the optimal monochromator setting
or filter pairing to be used for the assay,”
Mount discloses.
Assay performance has always been central
to the design of microplate readers at BMG
LABTECH and filters traditionally provided the
best performance for most assays. “We didn’t
want to compromise the performance of our
microplate readers with the convenience of a
monochromator, but we now have the tech -
nology to produce a monochromator with
filter-like sensitivity,” says Mount. “The LVF
monochromator has opened up flexibility and
performance that hasn’t been seen before in a
microplate reader.”
Mount assures that this doesn’t spell the
end of filter-based microplate readers, though.
“The CLARIOstar has two additional detection
technologies, filters and a spectrometer.
Filters still have an important part to play in
micro plate reading; fluorescence polarisation or
time-resolved fluorescence assays, for example,
will always perform better using filters. And
absorbance assays benefit from the ultra-fast
UV-Vis spectrometer,” he says. “Other microplate
readers measure absorbance and fluorescence
assays using the same monochromator. Since
the CLARIOstar uses a dedicated spectrometer
for full spectrum absorbance measurements, it
does not compromise one mode for another by
using the same detection technology. Fast
absorbance spectrometers, which capture an
entire spectrum (220 to 1000 nanometres) in less
than one second per well, is a newer technology
that BMG LABTECH incorporated into their
micro plate readers over five years ago.”
The LVF monochromator is suited to various
applications; for example scanning new or
unusual fluorophores, where the excitation and
emission wavelengths are not known, and using
fluorophores in non-standard buffer conditions
where the pH may have an effect on the peak
maxima. “When multiplexing assays with
fluorophores or lumiphores of similar or
overlapping spectra, the ability to define
wavelengths and select any bandwidth from
8 to 100 nanometres allows the user to employ
‘filters’ that are away from peaks but are
sufficiently wide enough to allow enough
light through to make the assay workable,”
explains Mount. “Wavelength and bandwidth
can be the difference between an okay assay
and a fantastic assay.”
Launched in June 2013, the CLARIOstar is
already receiving positive feedback. “The
product’s tag line is ‘any wavelength, any
bandwidth, any assay’ and that’s literally it,”
Mount reveals. “This LVF monochromator,
together with the way we can use it with the
filters, is just completely novel.”
European Pharmaceutical Review
Volume 18 | Issue 4 | 2013 64
Tham
papo
n / S
hutte
rsto
ck
Established in 1990 in Offenburg, Germany, BMG LABTECH has spent the last 20 years producing and developing microplate
reading technology. The company has pioneered many new technologies in this field, including dual mode ABS/FI readers,
fluorescence polarisation, simultaneous dual emission detection, laser-based excitation, ultra-fast spectrometers and now linear
variable filter (LVF) monochromators. As Dr Robert Mount, Managing Director of BMG LABTECH Ltd., explains: “We could be
considered a niche company in that we only make microplate readers, however we choose to focus our efforts on serving the
market where we have the technology and expertise, and have no plans to dilute it by moving into other markets.”
Product HUBDr Robert Mount, Managing Director, BMG LABTECH discusses theirCLARIOstar microplate reader with LVF monochromator technology
Anything Is Possible.Any Wavelength. Any Bandwidth. Any Assay.
Alpha Technology is a registered trademark of PerkinElmer, Inc. HTRF is a registered trademark of Cisbio International.
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Advanced Detection for Fluorescence and Luminescence Assays: Continuously adjustable wavelengths (320 - 850 nm)
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Increased sensitivity over conventional monochromators
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Use monochromators, fi lters, or a combination of both
Fluorescence and Luminescence spectral scanning
CLARIOstar Additional Features: Full spectral absorbance with ultra-fast UV/Vis spectrometer
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Reagent injectors for kinetic or cell-based assays
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The CLARIOstar‘s monochromator has
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CLARIOstar® - High Performance Microplate Reader with
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