Melbourne July 2017/R. Strebel Real Time Microbial Detection · TOC, Conductivity, Microbial, Ozone, Sodium, Silica Process Analytics pH, Oxygen, CO 2, Turbidity Gas Phase Analytics

Real Time Microbial Detection

Melbourne

July 2017/R. Strebel

Global Pharmacopeia Acceptance of

Alternative Methods

Agenda

1 Introduction

2 Pharmaceutical Water Monitoring and Analysis

3 Why On-line Microbial Monitoring

4 Real-Time Detection Technology

5 Pharmacopeia acceptance of Alternative Methods

6 Validation Guidelines

2

Our Solutions across the Value Chain

We offer solutions along our customers' value chain to help them:

▪ Streamline processes

▪ Enhance productivity

▪ Reach compliance with regulatory requirements

▪ Optimize cost and waste

Confidential

Laboratory

Solutions

Process

Analytics

Industrial

Weighing

Logistics

Solutions

Product

Inspection

Retail

Weighing

Solutions

3

Process Analytics Solutions Overview 4

Confidential

Water Analytics

TOC, Conductivity, Microbial,

Ozone, Sodium, Silica

Process Analytics

pH, Oxygen, CO2,

Turbidity

Gas Phase Analytics

O2, CO, CO2, H2O, H2S, HCl

Reliable in-line and

real-time measurement

of key analytical

parameters to control

process parameters

and monitor product

quality.

Company Background

60+ years of Process Analytical Expertise

▪ Ingold founded in 1948, acquired by Mettler Toledo in 1986

▪ Thornton founded in 1964, acquired by Mettler Toledo in 2001

▪ Sensor/Analyzer manufacturer for pH/ORP,

Resistivity/Conductivity, TOC, DO and water quality

with focus on:

- Pharmaceutical and Biotech

- Chemical

- Microelectronics

- Power

▪ Extensive research

- Chemical and physical properties

- New technologies

- Product handling and asset management

▪ Active participation in ASTM, ISPE, PDA, Semi, EPRI, USP

5

Dr. Richard Thornton

Dr. Werner Ingold

Why is Water Important?

Water is the most widely used excipient in Pharmaceutical Manufacturing

and a significant part of all quality inspections▪ Water is multi-functional

- Raw material

- Solvent

- Ingredient

- Reagent

- Cleaning agent (hot water or steam)

- Sterile/Packaged waters

- Sterile Water for Irrigation

- Sterile Water for Inhalation

- Sterile Purified Water

- Bacteriostatic Water for Injection

- Water for Hemodialysis

“Availability of water will be a bigger challenge than climate

change” – Stanford University Business School

6

Agenda

1 Introduction






7

Pharmacopeia Bacteria Regulations (USP 40)

The regulated parameters can now be measured in real time !

Attribute USP 40* EP 8.2 JP 17 ChP5 IPWater For Injection

Total Aerobic (Microbial) (cfu/100 mL)2 USP

<1231>10 10 10 10 10

Conductivity (mS/cm at 25C) (USP <645>) 1.3 (3 stage)3 Same as USP 2.1 offline Same as USP Same as USP

TOC (mg/L) (USP <643>) 0.54 0.5 0.5 0.5 0.5

Bacterial Endotoxins (EU/mL) (USP 71) 0.25 0.25 0.25 0.25 0.25

Attribute USP 40* EP 8.2 JP 17 ChP IPPurified Water

Total Aerobic (microbial) (cfu/mL)2 -USP <1231> 100 100 100 100 100

Conductivity (mS/cm at 25C) - USP <645> 1.3 (3 stage)3 5.1 (1 stage) 2.1 offline6 Same as EP Same as USP

TOC (mg/L) - USP <643> 0.54 0.5 (optional) Same as USP Same as USP Same as USP

* USP40-NF35 is the current approved version under which you can find USP <1231> and <1223>

▪ Microbiological testing is considered to be harmonized, with the exception noted that the EP test is

written into the Production section, and the USP test is contained in a non-compendial general

information chapter

8

Bulk Water for Injection(s) - Today 9

Bulk Purified Water - Today 10

Pharmacopeia adoption of new technologies

▪ Chemistry/Organic tests

are:

- Qualitative

- Subject to bias

- off-line

- Carbon dioxide

- Calcium

- Ammonia

- Chloride

- Sulfate

- Oxidizable

substances

- Heavy metals

1840 - 1994 1994 - 1996 > 1996

▪ Testing to

compare on-line

instrumentation

versus wet

chemistry

▪ Chemistry tests

VS. conductivity

instrument

▪ Oxidizable

substances vs.

TOC instrument

▪ USP accepted

as compendial

tests

▪ Conductivity

instrument

▪ TOC instrument

2003 –

2006

▪ Adoption by

▪ EP

▪ JP

▪ ChP

▪ IP

11

Pharmacopeia adoption of new technologies

▪ Bacteria tests are;

- Plate Count

- Qualitative

- Subject to bias

- off-line

- Accepted to be correct

and accurate

▪ Traditional plate

counts vs. on-line

Real Time

Instruments

▪ USP <1223> now

encourages validation

of alternative

microbial methods

▪ Ph.EUR. 5.1.6.

“Alternative Methods

for control of

Microbiological

Quality”

▪ Accepted by

USP as a

compendial

Test?

▪ on-line, real-

time Instruments

▪ Accepted by the

other Global

Pharmacopeia’s

1890 - 2017 2017 to 20?? 20?? 20??

12

Relevant Sections of the USP

▪ Pharmaceutical Water Monographs

- Purified Water (bulk) Sterile Water for Inhalation

- Water for Injection (bulk) Sterile Water for Irrigation

- Sterile Purified Water (packaged) Bacteriostatic WFI

- Sterile WFI (packaged) Water for Hemodialysis

- Pure Steam

▪ Test Chapters

- 645 Water Conductivity 85 Endotoxins

- 643 Total Organic Carbon 71 Sterility

- 644 Conductivity – planned for the future 791 pH

▪ General Information

- 1230 Water for Health Applications - related to Water for Hemodialysis

- 1231 Water for Pharmaceutical Purposes (contains microbial limits)

- 1233 Instrumentation for Pharmaceutical Water – planned for future?

- 1644 Theory and Practice of Electrical Conductivity Measurements of Solutions

- 1223 Validation of Alternative Microbiological Methods – 2nd Supplement USP 38

13

Agenda

1 Introduction






14

Drivers for Alternative Methods

▪ Industry workgroup created to promote development and implementation of online

bioburden analyzers

▪ Members from Merck, Novartis, Amgen, Fresenius, Baxter, P&G, Roche, Sanofi, Lilly,

GSK and Pfizer

Focused on reducing risk & cost by:

▪ Reduced Labor: Less sampling & lab-based testing

▪ Better product quality/process understanding:

▪ Continuous monitoring-faster response to excursion

▪ Fewer investigations

▪ Real-time release

▪ Better product safety

▪ Energy Savings:

▪ Reduced sanitization frequency

OWBA - Online Water Bioburden Analysis

15

What is the Current Cost of Risk?

“Knowing the microbiological quality prior to use and reacting quickly to

out-of–specification trends reduces financial loss and regulatory risk.” OWBA

▪ False positives investigations

▪ Delayed product releases

▪ Release without completed microbial testing

leading to risks of product recall

▪ Excessive inventory waiting for test results

▪ Product contaminations due to undetected

real-positives

- USP <1223>:

"Studies on the recovery of

microorganisms from potable and

environmental waters have demonstrated

that traditional plate-count methods

0.1%–1% of the actual microbial cells

present in a sample"

16

Current method of microbial

detection:

▪ Costly

▪ Time consuming

▪ Unreliable results from

human errors

▪ Delay of product release

Costs of Microbial Sampling – from OWBA

Real-time microbial systems are faster and higher detection capability

▪ Microbial sampling and testing has been the

roadblock for pharmaceutical manufacturing

▪ The costs for sampling and performing standard

bacterial plate count tests is expensive and slow -

approx. >$100/sample

▪ Typically, sampling frequency is done daily to a

maximum of weekly at multiple locations through

out the water system (avg. minimum of 20 points)

▪ The Pharma industry estimates that over 80% of

positive results are false-positives caused by

human error

▪ Results are not obtained for 5 to 7 days and some

cultures take 21 days

▪ A Pharma facility has to decide the risk acceptance:

do they use the water or wait for the test results

17


FDA - Food and Drug Administration

Food and Drug Administration▪ FDA encourages use of new technologies

- Rapid Microbial Methods (RMM): help meet Quality by Design (QbD) principles.

- Regulatory mechanisms to implement RMMs are evolving.

▪ FDA Aseptic Processing Guidance (cGMPs)

▪ FDA Process Analytical Technologies-PAT

▪ FDA Strategic Plan for Regulatory Science

▪ FDA Senior Microbiologist Supports RMMs

▪ FDA works in partnership with USP

▪ FDA liaisons work with USP Committee

18

Traditional Microbial Detection

▪ A colony-forming unit (CFU) is a signal used to estimate the

number of viable bacteria or fungal cells.

▪ Counting with CFU’s requires culturing the microbes and

counts only viable cells.

▪ Duration and temperature of incubation are also critical

aspects of a microbiological test method.

▪ Classical methodologies using high-nutrient media are typically

incubated at 30°– 35° for 120 – 168 hours.

▪ Because of the flora in certain water systems, incubation at

lower temperatures (e.g., 20°–25°) for longer periods (e.g.,

7–14 days) can recover higher microbial counts when

compared to classical methods.

▪ Low-nutrient media are designed for these lower temperature

and longer incubation conditions (sometimes as long as 14 and

up to 21 days to maximize recovery of very slow-growing

oligotrophs or sanitant-injured microorganisms), but even high-

nutrient media can sometimes increase their recovery with

these longer and cooler incubation conditions.

19

https://en.wikipedia.org/wiki/Bacteria

https://en.wikipedia.org/wiki/Fungal

Microbial Detection Methods

▪ The visual appearance of a colony in a cell culture requires

significant growth. When counting colonies, it is uncertain if

the colony arose from one cell or a group of cells.

▪ Plating and culturing bacteria can be done by a

number of methods:

- The Pour Plate method wherein the sample is

suspended in a petri dish using molten agar

cooled to approximately 40-45 °C

- The Spread Plate method wherein the sample (in

a small volume) is spread across the surface of a

nutrient agar plate and allowed to dry before

incubation for counting.

- Membrane Filtration wherein the sample is

filtered through a membrane filter, then the filter

placed on the surface of a nutrient agar

All agar based methods request for an incubation

period of a minimum of 5 to 7 days.

Manual work and visual inspection are potential sources of error

20

Traditional vs. Real-Time Microbial Detection

▪ Current Plate Count Method can only culture viable cells in the growth phase

▪ Alternative Real-Time detection systems have the capability to detect all life phases

Plate Count

Real-Time Microbial Detection

Effective

Detection Range

21


EMA – European Medicine Agency

Food and Drug Administration▪ “Taking into account the speed at which organisms can proliferate, the use of rapid

microbiological test methods and systems should be employed in order to improve or

increase the probability of early detection and allow timely action to be taken.”

EMA/INS/GMP/489331/2016

22

Agenda

1 Introduction






23

Basic Principle of Operation

Combined Mie scattering and intrinsic fluorescence to detect bacteria

▪ Draw a sample through a flow cell into the interrogation zone

▪ UV laser light source is directed through the sample

▪ Scattered light is captured and collimated within a parabolic mirror

- the scattering of light determines the size of the particle

▪ The intrinsic fluorescence is also captured

- i.e. the amount of light emitted at a longer (than 405 nm) wavelength by a microorganism

▪ The two types of light (scattered/fluorescent) are separated and the data computed

▪ When a particle is detected at PD and PMT at the same time (within x μsec), then this is a

BIOCOUNT!

24

Metabolites in Bacteria that will Fluoresce

(Hill et al, Field Ana. Chem. & Tech, 3(4-5), 221,1999)

25

Particle and AFU detected at same time point 26

Agenda

1 Introduction






27

USP Recommendation for Microbial Monitoring

Real-Time Systems enable continuous measurement according to USP

recommendations

▪ USP < 1231 > Water for Pharmaceutical Purposes recommendation:

- Pharmaceutical water systems should be monitored at a frequency that ensures the

system is in control and continues to produce water of acceptable quality.

- The general information chapter endorses operating monitoring instruments continuously

in order that historical in-process data can be recorded for examination

- The new USP <1223> promotes and encourages the validation and development of

alternative microbial technologies

28

USP Guidelines for Microbial Monitoring

<1223> Validation of Alternative Methods:

▪ Acknowledges the limitations of the use of CFU as a standard signal for microbiological

methods. " the cfu signal then is prone to underestimate the number of cells in a sample”

▪ Provides guidance on selection, evaluation, and use of microbiological methods as

alternatives to referee methods.

▪ Encourages the validation and development of alternative technologies "provided that

proper technical and scientific attention is paid to the selection, qualification, and

implementation of the method.” USP <1223> states.

▪ Identify suitable alternative methodology

▪ Development of user specifications for the equipment selection

▪ Demonstration of the applicability of the method as a replacement for a standard referee

method

▪ Qualification of the method in the laboratory

▪ Provides Options for demonstrating equivalence

“Alternative methods and/or procedures may be used if they provide

advantages in terms of accuracy, sensitivity, precision, selectivity, or

adaptability to automation or computerized data reduction, or in other

special circumstances.” General Notices & Requirements

29

<1223> - Chapter for Alternative Microbial Testing

<1223> Validation of Alternative Microbiological Methods (2nd supplement

USP 38▪ "These chapter provides guidance on the selection, evaluation, and use of microbiological

methods as alternatives to compendial methods. To properly implement alternative

methods, one must consider a number of important issues before selecting the analytical

technology and qualifying that method with the actual product. These issues include, but

are not limited to, identification of suitable alternative methodology, development of user

specifications for equipment selection, demonstration of the applicability of the method as

a replacement for a standard compendial method, and qualification of the method in the

laboratory.

▪ This chapter outlines:

- User requirements

- Instrument qualification

- Validation of alternate technologies

- Method suitability

- The limitation of the use of CFU as a standard signal for microbiological methods

- Four novel options for demonstrating equivalence………"

30

European Pharmacopeia Alternative Methods

5.1.6 Alternative Methods for Control of Microbiological Quality

▪ The following chapter is published for information.

1. "General Introduction

- The objective of this chapter is to facilitate the implementation and use of alternative

microbiological methods where this can lead to cost-effective microbiological control and

improved assurance for the quality of pharmaceutical products. These alternative

methods may also find a place in environmental monitoring.

- The microbiological methods described in the European Pharmacopoeia have been used

for almost a century and these methods – for enumerating and identifying micro-

organisms – still serve microbiologists well. Over the years, these methods have been

invaluable to help control and secure the production of microbiologically-safe

pharmaceutical products. Nevertheless, conventional microbiological methods are slow

and results are not available before an incubation period of typically up to 14 days. Thus

the results from the conventional microbiological methods seldom enable proactive,

corrective action to be taken.

- Alternative methods for control of microbiological quality have been introduced in recent

years, and some of these methods have shown potential for real-time or near-real-time

results with the possibility of earlier corrective actions. These new methods can also

offer significant improvements in the quality of testing …."

31

The European Medicines Agency answers:

What testing should be employed during initial qualification and routine

operation sampling?

▪ Testing should be conducted in line with Ph.Eur. Monograph 169 ‘Water for Injections’

▪ Use of rapid microbiological methods should be employed as a prerequisite to the control

strategy to aid with rapid responses to deterioration of the system.

▪ Article 23 of Directive 2001/83/EC states: “...the authorization holder must, in respect of

the methods of manufacture and control...take account of scientific and technical

progress...”

▪ Quantitative microbiological test methods – in line with Ph.Eur. 5.1.6 monograph

‘Alternative Methods for control of Microbiological Quality’.

▪ Due consideration should be given to employing alternate methods for the rapid

quantitative determination of the contamination levels existing within the water system. The

validation of such system should be in line with the above referenced monograph.

▪ Use of alternative/ rapid microbiological test methods should be employed as part of the

overall control strategy for the system.

▪ Taking into account the speed at which organisms can proliferate, the use of rapid

microbiological test methods and systems should be employed in order to improve or

increase the probability of early detection and allow timely action to be taken.

32

Agenda

1 Introduction






33

Online vs Plate Counts – Why the Difference?

USP <1223> acknowledges and explains the difference between CFU and

AFU

▪ “It is important to understand that the cfu has always been an estimation of

microorganisms present, rather than an actual count.”

▪ "The appearance of a visible colony requires significant growth of the initial cells planted –

at the time of counting the colonies it is not possible to determine if the colony arose from

one cell or from 1'000 cells."

▪ “Studies on the recovery of microorganisms have demonstrated that traditional plate-count

methods reporting cell count estimates as colony-forming units (cfu) may recover 0.1%–

1% of the actual microbial cells present in a sample.”

▪ “Most of the rapid microbiological methods are, to some extent, direct cell count methods.

They, therefore, may provide a higher cell count estimate than the cfu method for a given

sample.”

▪ “Observations of cell counts that differ from cfu results are not a concern if the different

methods and their different signals of cell presence are equivalent to or are non-inferior to

referee methods in terms of assessing the microbiological safety of an article.”

▪ “Higher cell counts must not be considered as necessarily indicative of greater risk given

the inherent variability of standard growth methods.”

34

Correlation Sample <USP 1223>

▪ The CFU (Colony Formation Unit) has always been an estimation of microorganisms

present, rather than an actual count – colony may be from 1 cell or 1,000 cells (plate

count).

▪ Rapid microbiological methods are direct cell count methods, therefore a higher count is

expected.

▪ The difference between the CFU from plate count and AFU direct cell count is smaller

when the number of bacteria cells in the water sample is low. However, the difference is

proportionally much higher when the number of bacteria cells in the sample water is high.

35

0

20

40

60

80

100

1 2 3 4

Comparison test per USP <1223>

Bacteria

cells/sample

Plate count

(CFU)

Rapid Testing

Biocount

1 0 1

10 0 10

50 25 50

100 50 100

How to set new limits

Alert, Action and Out-of-Specification Limits based on Statistical Analysis

▪ Establish new Alert Level at 2 Std. Dev. of the average AFUs over test period

▪ New Action Level at 3 Std. Dev. of the average AFUs over test period

▪ Breach or Out-of-Specification Level at 5 Std. Dev. of average AFUs over test period

▪ These are only recommendations customer can decide on their own Levels

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126 131 136 141 146 151 156 161 166

AFU

Days

Reported AFU's

▪ Alert Level

▪ Action Level

▪ Out-of-Specifications Level

36

Conclusions

▪ "Current water sampling techniques are limited in their ability to identify changes in

ongoing water system performance restricting real trend analysis as grab samples only

provide a snap shot of the dynamic water system.” - OWBA

▪ “Any alternative microbial detection methods may be used provided it has been

demonstrated they are equivalent or superior to the Compendia Pharmacopeia method”.

- FDA Senior Microbiologist

▪ Traditional plate count method has been performed for 126 years since1890

- recognized as only an estimate of the actual microbial cells present

▪ The current microbial limits were established as a compromise

▪ Traditional plate count methods have a relative standard deviation of 35% or more

▪ Current plate count method has a level of detection of 0.1% to 1%

There is a better method – Real-Time Microbial Detection

Why On-line Microbial Monitoring? 37

Mettler-Toledo Thornton

Thank You!

Thank You!

Merci

Grazie

謝謝

ありがとう

Danke

고맙습니다

Gracias

Tak

Terima kasih

Dziękuję

Tack

Cảm ơn bạn

Cпасибо

Obrigado

Go raibh maith agat

σας ευχαριστώ

धन्यवाद

ודה

38

Documents

Melbourne July 2017/R. Strebel Real Time Microbial Detection · TOC, Conductivity, Microbial, Ozone, Sodium, Silica Process Analytics pH, Oxygen, CO 2, Turbidity Gas Phase Analytics