Singer and Sutton Best Micro Practices

7/31/2019 Singer and Sutton Best Micro Practices

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The feld o pharmaceutical microbiology is responsible or many key

objectives in ensuring patient saety and product quality. Quality

control, method development, process and product design, and product

stability are a ew o the objectives. The United States Pharmacopeia

(USP), other global pharmacopeias and some parallel industry specifc

compendia oer some standardized test methodologies and material

specifcations relating to microbiological quality and control. Howeverthese test methods assume signifcant operational knowledge on the

part o the laboratory practitioner and signifcant operational capabilities

o the laboratory itsel. It is imperative to have some basic knowledge,

experience and inrastructure that can support consistent use o these

methods. The USP inormational chapter Microbiology Best

Laboratory Practices was developed to serve a part o this purpose.

The proposed general inormation chapter about Microbiological Best

Lab Practices was frst published in 2003 (USP 2003) in the Pharmacopeial

Forum, ollowing the long standing USP Revision process o development

and writing standards by experts along with public comment. Ater

comments and urther revision o the drat chapter (USP 2004), it was

frst published as an ocial USP Inormational chapter in USP 29, 3 years

later (USP 2006).

The intent o the chapter was to address a perceived lack o clarity on the

parts o both industry and the regulators on the basic requirements o

inrastructure needed to support mandatory microbiological criteria and

tests in the USP. Chemistry had a lot o guidance and inormation, but

there was very little guidance or microbiological testing.

The question o laboratory variability was central to this concern.

Microbiologists work every day with variability in the detection, recovery

and growth o microbiological species. This variability can be thought o

in two categories, avoidable variability (variability due to poor practice)

and inherently unavoidable variability (variability due to limitations othe methods and the vagaries o dealing with biological samples - see

Jarvis, 1989). The goal o best practices would then, be to minimize

avoidable microbiological error.

Wha ae Be laba paccen Mcb?

Well, they are a way o developing control or, in analytical terms, having

a system suitability o the laboratory. It makes sense. Using and

benchmarking best laboratory practices plus good documentation

Microbiological Best

Laboratory Practices, USP

Value and RecentChanges to a Guidance

of Quality Laboratory

Practices

MiCroBiology

Author Name

Author Title

Author Company

Scott Sutton1, Ph.D. and Donald Singer2

1President, Microbiology Network2 Global Lead Manager, Microbiology R&D, GlaxoSmithKine

May/June 2011 | | 41


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practices ensures reliability o data. In our highly regulated industry,

assurance is paramount to control.

Note: we are not saying anything about Good Manuacturing Practices

(GMPs) although they certainly have a place in the discussion! We are

talking about laboratory quality and best practice that goes well beyond

21 CFR (Code o Federal Regulations) whatever (211, 612, 820, etc). The

goal o this is to minimize variability and erroneous results. A secondary

beneft is to provide a benchmark or the laboratory and or auditors o

laboratory unctions. Also, variability, inherent in microbiology, means

that microbiological data deviations will happen.

Wha n he ona Chae?

Lets take a look at the original USP chapter and its topics o discussion.

Figure 1 shows the integration o all the key topics:

Media preparation

Microbiological cultures

Lab equipment

Laboratory layout

Lab records

Interpretation o results

Training

Documentation

In the section about media preparation, the discussion included media

preparation, media storage, and quality control testing o media.

Te ona Chae

Media Preparation and Quality Control

The quality o work in a microbiological laboratory depends on the

quality o the culture media. It is essential to use the correct media or

the purpose at hand, although the correct media is not always obvious.

For example, water testing is commonly perormed with R2A agar, but

many acilities use TSA (Trypticase Soy Agar) or HPCA (Heterotrophic

Plate Count Agar) or this purpose. The recommendation is provided

that the choice o media should be consistent, appropriate and justifed.

An entire section is devoted to the question o media storage and the

eects this might have on the media quality. Excesses o heat and cold

are to be guarded against, as is the potential or dehydration o poured

plates. Some guidance is also provided in quality control or molten

media used in pour plates.

Maintenance of Microbial Cultures

Second only to media, saeguarding the stock cultures is the most

important component o a successul microbiology laboratory. These

must be handled careully at all times to avoid contamination.

The care o the cultures starts upon receipt. A careul stock culture

curator will confrm the identity o the received cultures, even i they

come rom as respected a source as a national culture collection.

Mistakes can happen. The use o an incorrect strain in a compendial test

could bring the results o weeks or months o work into question.

The chapter reinorces the compendial recommendation or the seed

lot technique in culture maintenance. Critical to this is the need to

go into your containers o stock culture only once, and in restricting

the number o passages. Now, it must be stated that there is nothing

magic about the number 5. This number o passages gained popularity

in the compendia through its use in the Sterility Test, and has been

maintained or consistency. The point to the practice is that a careul lab

will saeguard the purity and identity o their stock cultures by limiting

the potential or drit due to excessive transers.

Maintenance of Laboratory Equipment

This section was originally included more or the sake o completeness

than because o concerns peculiar to the microbiology laboratory.

Basic inormation on qualifcation requirements, documentation, etc.

was included.

Laboratory Layout and Operations

The need or this section stems rom the concern that too ew acilities

understand or plan or the separation o samples rom a microbiological

perspective. The success o a laboratory can be enhanced by the

thoughtul separation o samples likely to have contamination romthose that are expected to be sterile.

Training of Personnel

The chapter states plainly what should be common sense

in recommending that microbiologists and managers in the

pharmaceutical support lab should have academic training in

microbiology or allied health sciences. This recommendation is in line

with current best practice or biosaety as laid out in the 5th Edition o the

Center or Disease Controls (CDC) manual Biosaety in Microbiological

and Biomedical Laboratories (BMBL). (CDC, 2007)

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42 | | May/June 2011

Figure 1.


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May/June 2011 | | 43

Documentation and Maintenance ofLaboratory Records

These sections were included only or the sake o completeness,

although additional GMP Rules are added in this revision. It is nice to

see the rules written down somewhere.

One aspect o these sections should also be addressed, and that is the

expectations when dealing with contract laboratories. The list o required

bits o inormation or the lab write-up is designed to provide a minimal

amount o proactive documentation or GMP requirements. This is also

a reasonable expectation or GMP studies rom contract laboratories.

Many labs will accept little more than summary reports rom the contract

lab; it is the opinion o the authors that this is an ill-reasoned position

as it prevents adequate QAU (Quality Assurance Unit) review o the study

as required by 21 CFR210.3(b)12 & 15, 21 CFR211.84, 21 CFR211.87,

21 CFR 211.160 and 21 CFR211.165. While it might be argued that the

contract labs QAU ulflls this requirement, this assumes that the client has

complete and total confdence that the current Quality procedures and

policies o the contract lab meet or exceed their own.

Interpretation of Assay Results

This section was initially envisioned to provide inormation on laboratory

investigations. However, during the writing process it became clear

that the scope o this section was broader than merely investigations,

and so the current title was settled upon as the best choice.

A discussion o the inherent variability o microbiological data was

necessary in this chapter. One view o good laboratory practices could

be structured around determining practices that minimize variability

in the microbiology lab. However, because we are dealing with such

low numbers on plates (requently less than 20 CFU/plate) and the

real opportunities or human error in tests that may run over a month

to completion, the microbiologist must always be aware o the rolethat random chance has in the data and be on guard against over-

interpreting the results o a study.

ren n he Cuen ven

Chapter is a living inormational reerence, which means that

as the expert committee sees or hears o potential improvements, the

chapter can be updated. Since the ocial chapter was frst published,

and as part o a quality improvement plan or a USP chapter, both expert

committee and comments rom the public already have led to some

changes, in order to:

Keep the chapter updated

Improve clarity

Add more inormation that the public requested

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MiCroBiology

Now, lets take a look at the newly revised chapter, in Figure 2.

Some new relevant topics were added: Lab Resources, Sample Handling,

and Media Incubation Times.

In addition to the new topics, some modifcations were made to the

language used to improve clarity and accuracy within these topics.

Me Dea f he recen Chane

Introduction

Some clarifcations were added to the Introduction. The sentence about

key parameters relating to equipment was modifed, using the word

operation along with control to indicate the importance o equipment

perormance. When discussing data variability, as mentioned earlier, we

replaced the word known with inherent to enhance clarity about the

risk o variability in microbiology.

Media Preparation and Quality Control Testing

The revised chapter expands the discussion o media preparation as

well. The recommendations include accurate weighing o dehydrated

components, the use o high-quality (USP Purifed) water, as the

frst intent choice, completely dissolving the dehydrated media or

individual ingredients, and the need to control the heating o the

media to avoid damaging heat-labile components o the media. Some

recommendations on the labeling and packaging o media are also

provided. A general change in the chapter is apparent in this section,

with cross-reerences to various other relevant USP chapters added.

This cross-reerencing also raises expectations that the microbiologylab will be amiliar and compliant with these other chapters as well. For

instance, instead o explaining how to calibrate a balance, a reerence

was added or the USP General chapter about Weighing on an Analytical

Balance, USP .

The quality control o the media is a critical concern. Interestingly,

initially some o the most passionate commentary on the chapter dealt

with the excessive amount o space provided to media quality checks.

Since the initial release in 2003, however, the harmonized Sterility Tests

and the harmonized Microbial Limits Tests have both incorporated

stringent media quality checks. This section provides an opportunity

to provide additional general inormation on media growth promotion

testing which led to a signifcant expansion in revision.

The two statements about Sterility Assurance Level (SAL) related to media

sterilization were removed. The removal o SAL in both cases relates to a

belie that SAL was not developed or use with media sterilization.

Clariying the intent o the discussion about media sterilization, the

container size parameter was added as a key part describing what can

aect the rate o heating during a sterilization cycle. As a consequence

o sterilizing or heating conditions that can have multiple eects on

media, the discussion was enhanced by adding to the physical eects

parameters another issue, the potential o reduced growth promotion

or selective activity o a medium.

The chapter now clarifes what is meant by room temperature when

testing pH, by the addition o 200-250C. In addition, a reerence was added

or USP which discusses pH measurement and calibration. As we

know, there is an increased use o purchased media in our new world o

Lean Labs! A statement about purchased media was added relating to the

common storage at rerigerated temperatures beore pH testing.

The recommendation o Quality Control (QC) testing was updated

to include all prepared media. Because o the new harmonized

pharmacopeial chapters or Microbiological Enumeration and

Specifed Microorganisms , inormation was added to state the key

indicated parameters or QC testing o media:

pH

growth promotion

inhibition and indicative properties

This was meant to align better with these harmonized chapters.

The selection o challenge microorganisms or QC testing o media is

stated in the chapter. To clariy how selection is determined, a statement

was rewritten to indicate relevance, use and selection o the growth

promotion microorganisms, in particular environmental isolates.

Another sentence was added to the discussion to help clear up the

response relating to Growth Promotion ailures. Also added was a

statement that any ailed Growth Promotion tests would not negate any

positive recovery that occurs during testing.

Media used in aseptic or clean areas were recommended to be

100% pre-incubated and inspected frst, then ollowed by growth

promotion testing.

Microbiological CulturesSince the viability and identifcation o cultures used or controls and

QC testing are critical, this section intends to recommend parameters

to ensure integrity is the objective. We all oten use cultures that are

redistributed or reworked rom primary collections by secondary

suppliers. So, it is important to qualiy the secondary suppliers. This

statement was made twice in this section to stress its importance.

The discussion on microbial cultures was expanded in this revision to

make allowance or ready-to-use cultures and qualifed secondary

Figure 2.


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May/June 2011 | | 45

suppliers. A discussion o the reasons or minimizing the microbial

passages involved in culture maintenance was included in response to

some concerns rom the feld.

In this modern day o using polyphasic approaches to identifcation,

the original statement o accepting only genotyping was changed to

restate the main intent To determine purity and identity by your own

choice o approach.

A clarifcation was made to defne the type o change that can occur due

to increased transerring o cultures.

Lab Equipment

Originally, this section had a title o Maintenance

o Lab Equipment. The new section title,

Lab Equipment, allows or more general

recommendations instead o just maintenance

(such as cleaning, calibration, and use). Thus,

there is some new discussion about equipment

cleaning and sanitization and autoclave validation

related only to media.

Sanitization o equipment includes a statement

about regular cleaning o key routine equipment

where growth conditions can occur, and that

equipment which is di cult to sanitize should be

dedicated or specifc use situations.

Most lab equipment in the microbiology

laboratory is subject to the standard validation

practices o IQ, OQ, and PQ (Installation

Qualifcation, Operational Qualifcation, and

Process Qualifcation). As is common, periodiccalibration/maintenance may be required or the

particular equipment based on its nature, and

perormance verifcation checks should also be

perormed regularly. The requency will depend

on characteristics and use o the equipment

(urther inormation can be ound in USP

Analytical Instrument Qualifcation).

Laboratory Layout

Laboratory layout was an interesting part o

this chapter. It was originally written to talk

about separation o clean and dirty activities, in

microbiological terms, as well as containment

and su ciency o space or activities. From this

section, a new topic was extracted, that o Sample

Handling.

Sample Handling

This new section discusses sample sensitivity,

storage and transport. The importance o using

aseptic techniques or all sampling activities

is a necessary addition in this section. The

appropriate marking o samples to enhance tracking rom source to lab

is also included here.

This short section was added in response to questions about the

relevance o samples that are stored or extended periods beore testing,

or are transported to a distant acility or testing. As the test system is a

living system, it is expected to react to stimuli over time. The eects o

storage over time could have a signifcant eect on the test results.

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MiCroBiology

Mcbca Medaincuban tme

A new section was requested in the public comments to oer an

approach to answer the question, How do we determine end points o

incubation schemes?

Despite the sections small size, it has the potential or a large

impact on the processes in the lab as it provides recommendations

or determining how to interpret incubation times. These

recommendations would encourage documentation o time-in and

time-out proactive documentation o the incubator usage. This

short section is reproduced below:

Incubation times or microbiological tests o less than 3 days duration

should be expressed in hours: e.g., Incubate at 30o to 35o or 18 to 72

hours. Tests longer than 72 hours duration should be expressed in days:

e.g., Incubate at 30o to 35o or 3 to 5 days. For incubation times expressed

in hours, incubate or the minimum specifed time, and exercise goodmicrobiological judgment when exceeding the incubation time. For

incubation times expressed in days, incubations started in the morning

or aternoon should generally be concluded at that same time o day.

The purpose o these rules to consider is to simpliy and clariy a

response to this ongoing question.

Training of Personnel

Personnel training is a core parameter o our laboratories. Training is one

o the means o developing competency, and it may include achieving

certifcation by an accredited body. The importance o this parameter is

underscored by 21 CFR 211.25 Personnel Qualifcations.

Since competency is becoming a buzzword in our industry, and

microbiologists do have options to prove their competency, this

discussion is important to develop ways o meeting the regulatory

inspection requests as well as the quality improvement approaches

o the laboratories. One option or proo o competency is the NRCM

(National Registry o Certifed Microbiologists, American Society or

Microbiology) certifcation. The aspect o microbiology education and

training, theoretical and practical, being unique is strongly stated in

comparison to chemistry training.

This position is expanded with specifc instruction on the qualifcations

and competency o the laboratory management. As microbiology is

largely operator dependent, it alls on the management to train the

operators, review the data and establish procedures. This cannot be

done without adequate preparation. Discussing the lab management, a

new paragraph specifcally addresses this point:

Competency may be demonstrated by specifc course work, relevant

experience, and routinely engaging in relevant continuing education.

Achieving certifcation through an accredited body is also a desirable

credential. Further, it is expected that laboratory supervisors and

managers have a demonstrated level o competence in microbiology

at least as high as those they supervise. Expertise in microbiology

can be achieved by a variety o routes in addition to academic course

work and accreditation. Each company is expected to evaluate the

credentials o those responsible or designing, implementing, and

operating the microbiology program. Companies can thus ensure that

those responsible or the program understand the basic principles o

microbiology, can interpret guidelines and regulations based on good

science, and have access to individuals with theoretical and practical

knowledge in microbiology to provide assistance in areas in whichthe persons responsible or the program may not have adequate

knowledge and understanding. It should be noted that microbiology

is a scientifcally based discipline that deals with biological principles

substantially dierent rom those o analytical chemistry and

engineering disciplines. Many times it is dicult or individuals without

specifc microbiological training to make the transition.

In addition to the recommendation that the microbiology sta have

studied a relevant subject while in school, the proposed guidance

chapter points out a undamental link between training and the

units SOP system. It recommends that the SOP system should be

comprehensive and serve as basis o the training program. This proposal

also recommends that perormance assessments be done periodically

and should demonstrate competency in core activities o the lab.

Another area that impacts indirectly, but oten, on laboratory

competency and capability is resourcing. This new section was added

to mention the issues revolving around Resourcing.

Laboratory Resources

The importance o adequate resourcing cannot be overstated to

best lab practices. This is reinorced in 21 CFR 211.22(b), 211.25(c),

it was elt that including skills in budgeting and investigations were

important or Supervisors o Microbiology labs. Thus, in this new

section, the importance o budget management and investigational

skills is discussed.

Interpretation of Assay Results

The additions in this section are thoughts that are not ound in any o

the mandatory general Microbiology chapters. Expansions on lab

investigations are provided, as is a ormal recognition o the phrase

Microbiological Data Deviation (MDD).

This section o the proposed guidance document is intended to be both

a discussion o the limitations o compendial test methodologies and a

guide to developing methods o investigating test ailures. It discusses

the dierence between a test that has ailed, a test that should beinvalidated and a test that should be repeated or confrmation.

summa

All the changes mentioned above are benefcial to practice and

understanding microbiology in its role in quality assessment.

Restating the intent o the chapter, it is: To develop and ensure

microbiological laboratory eectiveness and data integrity.


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MiCroBiology

The original version published in 2006 was a good start on a di cult

subject, the revision published in 2010 expands on this work in several

critical areas. The laboratory should pay particular attention to the

inormation in this USP chapter not only or their internal policies, but

also as an aid to the audit and review o contract laboratories.

This general inormation chapter has been used successully to improve

the eectiveness, e ciency and inspection-readiness o a Microbiology

Laboratory. In these days o lean organizations, high perormance and

consistency o a laboratory can lead to sustainability. The chapter is

yours to gain the most beneft as a benchmark.

Acknwedemen

We would like to thank ellow members o and liaisons to the

USP Microbiology Expert Committees (2000-2015) who provided

ongoing insight and input in response to public comments as well

as assisting in developing the chapter. Special thanks go to Radha

Tirumalai, USP, or his guidance and support o the work by the

committees. A note o appreciation also goes to Axel Schroeder

(Concept Heidelberg) or his persistence and strong interest to share

USP topics in the European arena.

refeence1. CDC. 2007. Biosaety in Microbiological and Biomedical

Laboratories (BMBL) 5th Edition http://www.cdc.gov/od/

ohs/biosty/bmbl5/bmbl5toc.htm released to the web in

February, 2007.

2. Javis, B. 1989. Statistical aspects o the microbiologicalexamination o oods. IN Progress in Indust. Microbiol. 21.

Elsevier Scientifc Publishers B.V. Amsterdam.

3. USP. 2003. Good Microbiological Laboratory

Practices. Pharmacopeial Forum 29(3):842-850.

4. USP. 2004. Microbiological Best Laboratory



Practices. USP 29 Suppl 2 pp. 3804-3807.




Practices. USP vol 33 Reissue Suppl 2, pp. R-1100-R-1105.

Auh Bahe

Mr. Donald C. Singer is a Senior member o

ASQ and a ormer Chair o the Food, Drug and

Cosmetic Division. As a Certifed Specialist

Microbiologist (NRCM), Don is Global Lead

Manager, Microbiology R&D or GlaxoSmithKine.

He has been a Malcolm Baldrige Award Examiner

and a ormer National Director, Divisions, on

the ASQ Board. He has been a member o the

USP Microbiology and Sterility Assurance Committee o Experts since

2000. Don has convened and taught an ongoing series o workshops

or industry at the American Society or Microbiology General Meetings.

He has been a member o ASQ since 1982, is a Certifed Pharmaceutical

GMP Proessional and has a Six Sigma Green Belt. Don is an active

representative or ASQ in the USP Convention, with over 30 years o

research, quality control, quality assurance, and laboratory managementexperience in the pharmaceutical, cosmetic, ood, beverage, hospital

products and medical device industries. He is an author and editor o

books about the laboratory, including two Quality Press books, and he

has been an adjunct proessor o Biopharmaceutical QC Microbiology at

the University o Maryland Baltimore Campus.

Scott Sutton has over 25 years o experience in the the pharmaceutical,

medical device, cosmetics and personal products industries with

extensive publications and presentations. Consulting and training in

GMP, contamination control, investigations o MDD (OOS), laboratory

management and microbiology-related project management are

areas o special interest. His clients have included startups, generics,

established Fortune 500 companies, law frms and investment broker

houses. Scott has owned and operated The Microbiology Network

(http://www.microbiol.org) since 1996. This company provides

consulting and training services to industry.

Documents

Singer and Sutton Best Micro Practices