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Microbial Testing, Analysis and Monitoring:

Strategies and Technologies for World-Class
Pharmaceutical Production
Contents
4 Data Integrity Issues in Microbial Testing
by Cheryl Platco and Tony Cundell, Ph.D., Microbiology Consultants, LLC

12 Limitations of Microbial Environmental Monitoring Methods

in Cleanrooms
by Angel L Salaman-Byron, Janssen Research & Development

20 The Hottest Topics in Microbiology

by Karen Ginsbury, PCI Pharmaceutical Consulting Israel Ltd.

24 Biologics Production: Impact of Bioburden Contaminations

on Non-Sterile Process Intermediates on Patient Safety and
Product Quality
by Friedrich von Wintzingerode, Roche-Genentech

2
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Data Integrity Issues in
Microbial Testing
by Cheryl Platco and Tony Cundell, Ph.D.
Microbiology Consultants, LLC

• Testing and only reporting passing data.

Introduction • Fabricating data
A lack of data integrity often is “just fraud,” says Howard Sklamberg, FDA • Using previously generated data
deputy commissioner for global regulatory operations and policy. FDA
• Not following test procedures and sampling plans
relies on company information documenting adherence to cGMPs, he
explained at a July 2014 conference sponsored by the Food and Drug • Missing, altered or raw data not captured on test report or batch
Law Institute. Yet almost all recent warning letters cite evidence of altered records
and falsified records. If data are “knowingly incorrect, we take that very
seriously,” Sklamberg stated, expressing dismay that some manufacturers • Memorized or recorded data on loose pieces of paper
still fail to remedy record-keeping problems despite repeated warnings • Electronic records changed without an audit trial
from the agency. For Indian pharmaceutical companies supplying generic
drugs to the U. S. market this has meant import alerts excluding their drug
products from the U.S. (Wechsler, 2014).
Why Has Data Integrity Become a
Hot Topic?
What is a Lack of Data Integrity
Beginning in 2014 there was a marked increase in warning letters to Indian
Many people in the pharmaceutical industry are confused by the concept drug manufacturers addressing data integrity as the FDA uncovered
of data integrity. Data integrity is comprised of these following broad problems during regulatory inspections (Unger, 2017). This increase is
actions to hide test failures and/or manufacturing deviations:
illustrated in Table 1.
• Omission of data
Table 1. The Number of FDA Warning Letters Issued Addressing Data
• Errors in data recording
Integrity
• Changing data Calendar Year Number of Warning Letters Most Frequently
addressing Data Integrity Cited Country
• Deleting data
2008 3 None
• Destroying data 2009 5 None

These actions may be both unintentional and intentional, representing 2010 5 China
GMP violations that can have civil and criminal consequences to the 2011 4 None
company and seriously damage the company’s business. 2012 6 None

Examples of a lack of data integrity in chemical testing include: 2013 6 India

2014 10 India
• Not documenting activities or failing to document activities at
the time performed (pre- or post-dating) 2015 15 India

2016 39 China
• Testing and discarding failing data

4
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Is the Issue Limited to Indian and
Chinese Pharmaceutical Companies?
In 2016, the FDA issued 39 warning letters citing a lack of data integrity
(Unger, 2017). The national distribution of warning letters was China 13
(33%), India 9 (23%), U.S.A. 6 (5%), E.U. 6 (15%), Brazil 3(8%) and Japan
2 (5%).
The FDA completed a total of 5,615 GMP inspections of registered drug
and device establishments in FY 2015. Of these 4,055 (72%) were domestic
inspections and 1560 (28%) were foreign inspections. Based on the recent
U.S. GAO-17-143 Report on the FDA’s Foreign Offices and Drug Inspections
in FY 2015 there were 842 foreign drug establishment inspections of
which 527 (63%) were for GMP surveillance, 250 (30%) for pre-approval
and surveillance, 23 (3%) for preapproval only and 42 (5%) for cause. The
report highlighted the high frequency that an establishment may have
never been inspected. For example, in the FY 2017 catalog were 572 Indian
establishments with 33% never inspected and 535 Chinese establishments
with 45% never inspected. This suggests that Indian and Chinese drug
manufacturing facilities will continue to be cited for data integrity issues
into the foreseeable future unless they aggressively address data integrity
issues within their companies.
Data Integrity Directed Towards
Microbiological Testing
Many of the cited data integrity issues are not associated with
microbiology but with chemical analyses, especially High-Performance
Liquid Chromatography (HPLC). The norm for microbiological testing is
the visual inspection of media for the detection of microbial growth or
the enumeration of microbial counts. Typically the results are recorded
on paper worksheets or more recently into electronic notebooks. The
data, but not always the original microbial cultures, are checked by a
second person for completeness and the absence of recording and
arithmetical errors. The data are then entered into Laboratory Information
Management Systems (LIMS) and approved by a laboratory supervisor.
These laboratory procedures place a high reliance on the integrity,
experience, and training of the individual analyst, the ability of the peer
reviewer or supervisory staff to detect poor data integrity, and the culture
of the company to set the highest standard. This type of data could be
susceptible to falsification. Microbial methods rarely take advantage of
modern analytical technologies.
Contributing factors to reduced data integrity in the microbiology
laboratory are the subjectivity of scoring microbial growth in broth,
analyst-to-analyst variability in counting colonies on plates, and the
inability to perform aseptic manipulations with the contempora-
neous recording of data. Often LIMS data fields for many microbial
6
measurements such as <10 CFU/g, Log 10 counts, and absence of spec-
ified microorganisms test results are lacking or inconsistent. For most
microbiological tests, the absence of computer data collection, au-
tomatic calculations and generation of a finished report lack detail
and flexibility.
A broader discussion of the issue of data integrity in the area of microbiology
could be useful in terms of setting industry and regulatory expectations.
Microbiological data has historically been evaluated and recorded
manually by appropriately educated and experienced microbiologists
trained in the art of contamination detection and colony counting.
Microbiologists must use experience, expertise and judgment for test
interpretation, which may lead to subjective and variable interpretation
and documentation of test results. While the use of rapid technology,
digital image capture and automated plate readers can remove most of
the subjective data interpretations and solve data integrity issues, the
algorithms used to count colonies or detect contamination are subject
to validation limitations. Microorganisms grow at different rates, different
con-ditions, evolve to different colony shapes, sizes, and colors that must
be accommodated. The addition of a high quality digital image of a plate is
helpful in terms of data integrity (verification of test results, data archival,
data retrieval) and verification of the accuracy of results for the manual
recording of colonies. Microbiologists may still have to override electronic
results if spreading colonies, colonies embedded within other colonies
in plate counts, or product precipitation or flocculation in sterility tests
makes interpretations by instrumentation inaccurate or more challenging.
Risk Analysis
Many pharmaceutical companies are now evaluating data integrity using
standard risk assessment tools such as FMEA (Failure Mode and Effects
Analysis). In the opinion of the authors the tests which appear to be the
most critical, subjective, and prone to data integrity issues are the sterility
test, the gel clot Limulus Ambeocyte Lysate (LAL) bacterial endotoxin assay
and any microbial enumeration test which involves counting microbial
colonies which covers most of the other compendial tests. The sterility and
LAL gel clot assays are subjective in their interpretations. The enumeration
test accuracy depends on the interpretation of discreet colony counts by
the microbiologist reading the plates.
Using risk assessment tools, the authors believe that data integrity issues
can be classified as high, medium and low risk processes.
High Risk
• Analyst records incorrect data
• No second opinion for subjective interpretation of test results
• Analyst performs wrong method or wrong sample preparation
• Missed critical data or incorrect information
• Falsification or lack of authentic data
Medium Risk
• Real time data entry difficulties
• Incomplete data entries
• Chain of custody for samples issues
• Delay in testing, times not documented
Low Risk
• Improper recording of non-critical data
• Lack of real time entry of non-critical data (lot numbers, expiry
dates, and recalibration dates.)
GMP Requirements with Respect to
Data Integrity
Data integrity is addressed in 21 CFR 211.194 Laboratory Records where
the GMP requirements are defined. Recently four guidance documents
addressing data integrity have been written by the U.K. Medicines
& Healthcare Products Regulatory Agency (MHRA), Parenteral Drug
Association (PDA), U.S. Federal Food and Drug Administration (FDA),
and Pharmaceutical Inspection Co-operation Scheme (PIC/S). The
documents are:
• 2015 MHRA GMP Data Integrity Definitions and Guidance
for Industry
• 2016 PDA Elements of a Code of Conduct for Data Integrity in
the Pharmaceutical Industry
• 2016 Draft Guidance for Industry – Data Integrity and
Compliance with CGMP
• 2016 PIC/S Guidance Good Practices for Data Management and
Integrity in Regulated GMP/GDP Environments
Why These Documents
Inadequately Deal with Data
Integrity Issues in Microbiology
On April 14, 2016 the FDA published a Draft Guidance for Industry on
data integrity that emphasized computer data management systems
but not specifically microbiological testing. The document defined data
integrity as to the completeness, consistency and accuracy of data. They
used the acronym ALCOA which stands for Attributable (A), Legible
(L), Contemporaneously recorded (C), Original or a true copy (O) and
Accurate (A).
On August 10, 2016, a draft PIC/S guidance document was published
to provide industry with a “consolidated, illustrative guidance on risk-
7
based control strategies”. The new guidance from PIC/S was applied on
a 6-month trial basis by participating regulatory authorities. The authors
agree with the practical approach taken by PIC/S.
The PIC/S guidance defines and deals with:
• Data governance systems
• Organizational influences on successful data integrity
management
• Specific data integrity principles and enablers
• Data integrity considerations for paper-based and computerized
systems
• Integrity considerations for outsourced activities
• Regulatory actions in response to data integrity findings
The guidance wisely states, “Management should aim to create a work
environment (i.e., quality culture) that is transparent and open, one in
which personnel are encouraged to freely communicate failures and
mistakes, including potential data reliability issues, so that corrective and
preventative actions can be taken.”
The authors agree that the PIC/S draft guidance, unlike the other
regulatory documents, details the various deficiencies linked to data
integrity failures that may have varying impacts on product quality (i.e., a
risk-based approach to a loss of data integrity):
Critical deficiency (Impact to product with risk to patient health):
• Product failing to meet specification at release or within shelf
life. Reporting of a “desired” result rather than an actual out of
specification result when reporting of quality control (QC) tests,
critical product or process parameters.
Major deficiency (Impact to product with no risk to patient health):
• Data being misreported (e.g. original results “in specification,”
but altered to give a more favorable trend). Reporting of a
“desired” result rather than an actual out of specification result
when reporting of data, which does not relate to QC tests,
critical product or process parameters. Failures arising from
poorly designed data capture systems (e.g. using Post-its® to
record information for later transcription) No impact to product;
evidence of widespread failure.
• Bad practices and poorly designed systems which may result
in opportunities for data integrity issues or loss of traceability
across a number of functional areas production, QA, QC, etc.,
though individually, each violation “has no direct impact to
product quality”
Other (minor) deficiency (No impact to product with limited evidence
of failure):
• “Bad practice or poorly designed system, which results in
opportunities for data integrity issues or loss of traceability in a
discrete area. Limited failure in an otherwise acceptable system”

Application of ALCOA
Principles to Microbiological
Data Integrity Issues
Applying the ALCOA principles to microbiology testing may on the
surface seem to be a common-sense approach to cGMP compliance.
However, after applying risk-based principles to the discreet tasks relat-
ed to microbiological testing, documentation can be difficult and cum-
bersome. Approaches to applying the ALCOA principles can be minimal,
moderate or extreme. Extreme approaches may counteract good docu-
mentation practices. A risk-based approach to the criticality and func-
tionality of the task and its documentation should be used. Companies
also need to decide if “data” includes ancillary items such as media, re-
agent lot numbers, and expiration dates, instrument numbers and their
recalibration dates, and laminar flow hood numbers. Applying extreme
approaches to ALCOA principles for data integrity can result in a second
person having to shadow the microbiologist and witness every docu-
mented step. Microbial tests also involve the use of aseptic techniques
where real-time recording of the tasks cannot be performed without
jeopardizing the quality of the results. Manual documentation of hun-
dreds of environmental monitoring, water, or enumerations plates are
often batch read with “no growth detected” plates being separated from
“growth detected” plates. All documentation is batch recorded at the
same time. Do the ALCOA principles require each plate read, documen-
tation, and recording of a second person confirmation all have to occur
at the same time? Do the principles require that a second person have to
re-enumerate quantitative plates to confirm the accuracy of the colony
forming units? The authors believe not. For microbiology testing, many
tests are performed over days, even weeks. How do we document team-
work? Frequently multiple individuals work on the same test, perform-
ing dilutions, culturing organisms, counting plate colonies, and reading
or interpreting results at different times over a number of days? How are
ALCOA principles applied? These FDA 483 observations give an insight
into the regulatory thinking about data integrity as applied to microbi-
ology. Microbiologists can also evaluate how rapid technologies may be
helpful in remediating the problems.
Attributable
The first ALCOA principle is ATTRIBABLE. The data is linked to the
person performing the action. Only that person may sign for the work
completed. An FDA 483 observation was given to a company which
read: “While multiple employees participate in a process step that spans
more than one shift, only the last person involved is required to sign
the batch record for completion of the activity.” Another observation
read: “The inspection documented that all of your QC laboratory
computerized instruments (redacted) were found to be stand alone, and
8
laboratory personnel demonstrated that they can delete electronic raw
data files from the local hard drive. Your firm deleted multiple data files
acquired in 2013 allegedly to clear up hard drive space without creating
backups. Your QC management confirmed that there is no audit trail
or other traceability in the operating system to document the deletion
activity. Furthermore, your analysts do not have unique user names and
passwords for the computer and laboratory information systems; your
QC analysts use a single shared user identifier and password to access
and manipulate multiple stand alone systems.”
While these observations may not apply directly to microbiology
laboratories, there is a concern around the inability to attribute a task or
data set to a person or group of people. Often hundreds, even thousands
of microbiology plates are read. There may be multiple individuals all
working together to complete the task spanning more than one shift, or
even more than one day. How granular and frequent must the tasks be
documented?
The requirements for data integrity were not intended to limit laboratory
flexibility or agility. Problems can arise when personnel must repeatedly
log in and out of computers for each task completed to document
individual contributions to the team. Shared electronic notebooks can
help, but care must be taken to disallow and/or track overwrites and
accidental omissions. In some ways, paper documentation is easier
than electronic documentation. However, electronic documents can
be templated, time stamped, and changes tracked, which is a huge
advantage. The advantage of rapid technology automatic data capture
readers is that they can address many of the data integrity concerns. The
use of automated plate readers and rapid technology instruments may
come with some logistical issues since one must assure that functions
such as audit trails are turned on, and permissions for tasks appropriately
assigned in a manner that there is no conflict of interest. The ability
to reproduce/retain the raw data in a readable form for archival and
retrieval must also be achieved.
Legible
The second ALCOA principle is LEGIBLE. Companies have received
observations for obvious use of correction fluids, overwrites/scribbles on
handwritten data sheets, and not having appropriate change controlling
procedures in place. This includes access to and use of signature stamps
for electronic systems in lieu of handwritten signatures. General data
integrity principles have always mandated that data must be readable
throughout the data retention period and lifecycle, with permanent
and identifiable changes using permanent ink, neatly, and following
established date and time formats. Electronic data has additional
challenges. The rules apply to all raw data, meta data, and finished data
reports which are stored and backed up for archival and retrieval. Many
computerized systems store the raw data and use internal software
to access, process, and report the information. Computer systems
and programs are constantly being updated. Therefore, the retired
instrument programs may also need to be archived in a manner that raw
data can be retrieved and reprocessed. Computer operating systems
may not be able to run the specialized software after many years. This
conundrum has yet to be resolved.
Contemporaneous
The third ALCOA principle is CONTEMPORANEOUS. For microbiologists, this
may pose the most challenging principle. These challenges include:
• Signing/dating records at the time of activity completion
• Back-dating or pre-dating data sheets
• Limiting access to change date/time of electronic data
• Aseptic manipulations under laminar flow hoods or in isolators
including the opportunity to document a microbial test
A recent 483 observation was …. “Specifically, an operator performed
the in-process tablet (redacted) testing for the (redacted) mg tablet
batch #(redacted) without the batch record or a manufacturing form to
document the results contemporaneously. …your operator stated that
he records the two weights with (redacted) significant figures into the
batch record (located in another room) from memory”. Do tasks or data
have to be recorded during microbial testing when activity occurs and
how do we maintain aseptic technique while still complying with the
requirements for data integrity?
Original
The fourth ALCOA principle is ORIGINAL. This can be difficult for
microbiologists. Many microbiologists write the plate counts on the plate.
Many isolate zero count plates into one location to document all negative
or 0 CFUs later. The authors believe these are common practices, which
allow the laboratory operations to remain productive and flexible.
Original data is data as the file or format in which is was generated,
preserving the integrity (accuracy, completeness, content and meaning)
of the record
• Retain all raw data and printouts
• Original data must be recorded directly into GMP records
• Original data must be reviewed
• Electronic raw data files must be retained
A recent 483 observation stated: “Your firm repeatedly delayed, denied,
limited an inspection or refused to permit the FDA inspection. An FDA
investigator identified the presence of torn raw data records in the waste
9
area and asked one of your firm’s QA Officers to remove these torn raw
data records for the investigator’s review. This QA Officer presented the
FDA investigator with approximately 20 paper records, none of which
included raw data entries identified in the waste area earlier during the
inspection. The FDA investigator then revisited the waste area and found
that the raw data records had been removed and placed in a different
holding bag.” While this observation is an example of a more egregious
breach of data integrity, microbiologists struggle with exactly what is
original data. If plate counts are written on the plate and not recorded
directly onto a report, are the plates raw data?
Accuracy
The fifth and last ALOCA principle is ACCURACY. This article addresses the
Who, What, Where, When, Why and How data represents the complete set
of factual data and meta data.
• Data must reflect the action/observation accurately
• All data must be controlled and retained for the lifecycle
• Modifications to the data must be explained if not obvious
• Meta data includes units of measure, method details, audit trail,
date/time modifications
Recent FDA 483 observations stated: “Processing each result file
individually using different processing parameters generates the data for
assay. The second person reviewer is not required to review each of the
processing parameters used to generate results.” Another observation is
“Only one operator was assigned to evaluate sterility test on the last day
of the incubation period. To ensure reliability of the tests, your procedure
needs to specify steps that enable objective evaluation, such as assigning
two operators for evaluation on the last day.” This implies that the FDA
expects a second microbiologist check all sterility test media at the end
of the test time.
A third recent FDA 483 observation stated: “Not all laboratory data were
recorded accordingly. On 5/25/2017 an FDA microbiologist observed the
following: a. “The firm’s microbiologist read the settling plate at location
Vial Sealing Area Passive on 5/24/2017 and reported “0” CFU. A FDA
microbiologist observed 1 CFU for the same plate.”
The observation that the company reported no CFU and the FDA
investigator was finding 1 CFU is disconcerting and may be a serious
data integrity issue. The delay in examining the plates may have been a
factor - the plates were read 5/24/2017 and the inspection conducted
between 5/25 and 6/1/2017. Should one automatically assume that there
was falsification of the environmental monitoring results? The authors
believe that only the CFUs reported within the recommended incubation
time are valid. Since specifications are for a defined incubation period,
viewing colonies after the end of the incubation may not be valid. It is
possible that a slower growing colony was not visible at termination of
the test. It is possible that one opens the plates for a better view to read
the plates. One should not automatically infer error or fraud for data which
is acquired during a set period of time and the analytes are known to be
dynamic at the end of a tests’ duration.
Ultimately the maintenance of data integrity is the joint responsibility
of upper management, managerial and supervisory staff, and bench-
level microbiologists. The authors do not advocate employing a second
microbiologist to review all microbial test results, but potential failures
of critical tests should be reviewed contemporaneously, if possible, by a
second person. Test result reports should be reviewed by a superior for
accuracy and completeness. Out-of-specification results should be fully
investigated. Unlike the opinion of some FDA investigators, the authors
believe that negative sterility tests do not require a second person review.

Risk Mitigation
How can the risk be mitigated with microbial test methods? What should Alternative Microbial Test Methods
and should not industry do in response to data integrity issues? The
Are the risks different with alternative microbial test methods? Will this
first step is to acknowledge the limitation of the classical microbial test
difference drive the implementation of these newer methods? Alternative
methods, (accuracy and precision), their dependency on the technical
microbiological test methods usually depend on analytical signals that
capabilities, honesty of the microbiologists conducting the tests, and
are quantitative, more sensitive, and usually more reliable than classical
supervisors reviewing and approving the tests for data integrity (Table 2).
growth-based methods (Table 3). Many alternative methods generate
Table 2. Data Integrity Level of Classic Microbial Test Methods results in real time so can be described as rapid microbial methods (RMM).
The signals generating results may not be fully equivalent to colony-
Microbial Test Data Data Integrity Risk Mitigation
Generation Level forming units counted in a traditional plate count. Analytical methods
Microbial Colonies counted Moderate: Count Second person review; use are typically objective, do not rely the subjective scoring of colonies
Enumeration on a plate or subject to analyst of automated plate readers
membrane; variability; with electronic storage of
growing on a plate or turbidity of a broth, and can be captured electronic
turbidity in a Most susceptible to the image and archived which means they have inherent data integrity. These
Probable Number falsification;
(MPN) broth tube. impermanent record characteristics of alternative microbial test methods should help promote
Test for a Specified Characteristic Low to moderate: Second person review;
Microorganism growth on Atypical growth electronic storage of the
selective media pattern; subjective image
Table 3. Data Integrity Level of Alternative Microbial Test Methods
after enrichment interpretation;
culture. presumptive Microbial Test Technology Data Data Integrity Level
result that must
Generation
be confirmed
by microbial Microbial Automated Plate Colony Counting High
identification. Enumeration Counter

Sterility Tests Presence of Low to moderate: Second person review; end Test for a Specified RT-PCR DNA Extraction, PCR Moderate to High
microbial growth Growth detected point determinations, e.g., Microorganism Amplification and
in the liquid media as turbidity, ATP determination, gaseous Detection
surface pellicles, exchange, etc.
Sterility Tests ATP ATP level Moderate to High
floccular growth or
Bioluminescence CO2 production and
precipitation
Respiration O2 Consumption
Microbial Probability of Moderate: Utilization Second person review Detection of
Identification identification tests based in pH Flow Cytometry fluorescent-labeled
(Phenotypic) based on the and redox changes Solid Phase cells
pattern of chemical Cytometry
reaction
Microbial MALDI-TOF Mass Riboprotein charge- High
Antimicrobial Log reduction Moderate: Serial Second person review Identification Spectrometry mass fingerprints
Preservative of challenge dilution and plate (Phenotypic)
Effectiveness Test microorganism counts
Microbial Vitamin Automated Zone Zones of inhibition High
at specified time
and Antibiotic Readers on plates
intervals
Assays
Bacterial Gel Clot Visual Examination Moderate to Second person verification of Automated Turbidity in broth
Endotoxin Assay of gel formation high: subjective results by direct observation Turbidometers culture
interpretation of gel
Bacterial Endotoxin Kinetic Color change or High
formation
Assay Turbidimetric or turbidity due to gel
Note: Second person review of test vessels (plates, canisters, tubes) should be limited to alert and Chromogenic clotting
action level results, out-of specification results or ambiguous results only. Methods

10
their implementation in the pharmaceutical industry. However, many of
the data integrity issues associated with chemical assays would likely be
applicable to these newer microbial methods.
Concluding Statements on the Issue of
Data Integrity
The amount of recent regulatory activity in the area of data integrity and
the failure of four recently published guidance documents to address
microbiological issues is a concern to pharmaceutical microbiologists. The
challenge to ensure data integrity in the microbiology laboratory is unique.
Computerized record keeping is not as advanced as it is in chemistry
laboratories. In most cases contemporaneous data collection is not
possible due to the necessity of using aseptic techniques or contamination
control. Furthermore, the volume of plates to be read in support of
environmental and water monitoring makes second person review of
all plates impractical. The authors believe that the right response to this
challenge is that second person review be limited to alert and action level
results, out-of-specification results or ambiguous results only. Completed
work sheets should be diligently reviewed by laboratory supervisors and
laboratory test failures aggressively investigated. Ultimately, data integrity
can only be ensured by the maintenance of high ethical and professional
standards throughout a pharmaceutical company.
11
References
1. FDA Draft Guidance for Industry – Data Integrity and
Compliance with CGMP www.fda.gov/downloads/Drugs/
GuidanceComplianceRegulatoryInformation/Guidances/
UCM495891.pdf
2. Wechsler, J. Data integrity key to GMP compliance. BioPharm
International (September 2014) www.biopharminternational.
com/data-integrity-key-gmp-compliance
3. Unger, B. 2017 An analysis of FDA warning letters on data
governance and data integrity Pharmaceutical Online, Guest
Column July 14, 2017 www.pharmaceuticalonline.com/doc/
an-analysis-of-fda-warning-letters-on-data-governance-data-
integrity
4. U.S. Government Accountability Office GAO-17-143 Report on
the FDA’s Foreign Offices and Drug Inspections www.gao.gov/
products/GAO-17-143
5. PIC/S Guidance Good Practices for Data Management and
Integrity in Regulated GMP/GDP Environments 2016 www.
picscheme.org/en/publications
Limitations of Microbial
Environmental Monitoring
Methods in Cleanrooms
Angel L. Salaman-Byron
Janssen Research & Development
Introduction
Environmental Monitoring (EM) program requirements are currently
described in the 21 Code of Federal Regulation (CFR) 211.42, 21 CFR 211.46,
21 CFR 211.22,1 USP <1116> Microbiological Evaluation of Clean Rooms
and Other Controlled Environments,2 European Medicine Agency (EMEA)
Annex I3, International Standard Organization (ISO) 14644-1,4 ISO 13408,5
ISO14698-16 and Parenteral Drug Association (PDA) Technical Report #13,
Fundamental of an Environmental Monitoring Program (Revised, 2014).7
Regulatory agencies like the Food and Drug Administration (FDA) and
European Medicines Agency (EMA) requires pharmaceutical manu-
facturing companies to have an EM program and standard operating
procedures (SOPs) in place as an important part of the drug manufac-
turing control process to ensure product safety attributes. Regulatory
bodies have established the requirement for trending and identifying
contamination sources. Major observations have specifically been is-
sued to companies that lack adequate systems for monitoring envi-
ronmental conditions in aseptic processing areas and for not having
written procedures for EM including sampling frequency, sampling
locations and procedures for alert and action levels.
The ISO 14644-1 regulation specifies the total particulate counts allowed
for a clean environment to meet the defined air quality classifications.
Unlike microbial contamination where humans are the main significant
source, nonviable particulates can arise both from the environment,
humans and from processing equipment. Non-viable (total) air particulate
(i.e. total particles count) at rest and during normal operations is
conducted to confirm that the environmental quality in ISO-classified
areas is maintained. However, EM of total particulate does not correlate
directly on the bioburden of the environment.
12
ISO 14644-1 and ISO 14698-1 do not set limits or assign classification values
for viable airborne particles. The sampling of airborne viable particles is
dealt with in ISO 14698, however action levels are to be determined by the
user. Per ISO 14698-1, section 5.2 “Microbiological Alert, action and target
levels are set by the user as appropriate to the field of the application”.
These levels should be based upon target levels related to what product
is manufactured as well as the product requirements. These levels may be
adjusted based upon data collected during initial start-up and at intervals
established by the contamination control policy or monitoring plan.
Various regulatory bodies have guidance related to the manufacturing of
sterile medicinal products. These guidances, link cleanroom class based
upon non-viable particle levels to viable particle levels. These levels are
provided for active air sampling, settle plates, surfaces and on operators.
Every microbial EM program should have a combination of the following
methods: settle plates, contact plates and/or surface swabs, active air
samples, and rinse samples (i.e. equipment). Air sampling is meant to
assess the engineering/design controls performance as intended to
clear aerial contamination and meeting classification requirements for
total particulate per volume of air and personnel aseptic techniques
practices and hygiene. Surface sampling are mainly intended to assess
surface cleaning and sanitation effectiveness and personnel aseptic
techniques practices and hygiene. All these methods are based on the
ability of captured organisms to be visibly counted due to replication
in nutrient media under aerobic conditions and mesophilic incubation
temperatures. At present, nearly all these methods rely on the growth
and recovery of microorganisms, many of which are in environmental
stress and therefore may be difficult to recover. The lack of accuracy and
precision of the traditional enumeration methods and the restricted
sample volumes that can be effectively analyzed suggest that microbial
EM is incapable of providing direct quantitative information about
sterility assurance. All known methods only sample at a single point in
time, therefore would require repeat sampling to assess range of counts.
On the other hand, microbial EM recover efficiency is undoubtedly
influenced by many factors. Only viable microorganisms would be
detected by EM methods. The type of surface, presence of product
or chemical residues, organisms stress status, type of microorganism,
technique bias by technician during sampling are among the facts that
influence the recovery efficiency. The absence of recovery may give a
false impression that the air or the surface sampled was “clean” if most
of the microorganisms are non-cultivable or non-viable (incapable to
reproduce), have special nutritional requirements, or are slow grower
among other facts.2,7 As a fact, the absence of growth does not mean
the sample location is free of microorganisms and likewise a single
sample point excursion does not indicate that the area is not in a state
of microbial control. There is not a microbiological sampling plan that
can 100% prove the absence of microbial contamination, even when no
viable contamination is recovered.8
Microorganisms Recovery from Air
The distribution of microorganisms under the conditions of an air medium
is strongly tied in with the colloidal properties of microorganisms. The
13
length of occurrence of microorganisms in the air, diffusion, electrical
charge, and their mixing by air flow are based completely on the laws
of colloidal chemistry. A colloid, is a mixture in which one substance of
microscopically dispersed insoluble particles is suspended throughout
another substance. Unlike a solution, whose solute and solvent constitute
only one phase, a colloid has a dispersed phase (i.e. the suspended
particles) and a continuous phase (i.e. the medium of suspension).9 It is
assumed that bacteria in the dust phase are not bound to dust particles,
as occur with water, but freely suspended in air. Vlodavets (1964) reported
that bacterial dust particles settled down even faster than bacterial drops.
Initially a high concentration of microorganisms is created after the creation
of the bacterial drop aerosol (i.e. bacteria with a water-based solution).
Then, the concentration of bacterial in air gradually decrease. Decrease in
concentration is dependent mainly in the settling of bacterial drops. It has
been reported that a decrease in relatively humidity promote an increase
of the time of occurrence of Staphylococci in the drop and dust phases of
an aerosol. High humidity assists the settle down of bacterial drops as well
as of particles of the bacterial dust and the lowering of the concentration
of the bacteria on air. Vlodavets concluded that the concentration of
viable microorganisms on air is influenced by two facts: 1) the settling of
organisms (i.e. physical loss) and (2) the death rate (i.e. biological loss) of
the microorganisms.10 Within cleanroom environments humans are the
primary source of contamination. Typically, 80 to 90 percent of normal
microbial-flora identified in a cleanroom environment is generated from
humans.11-13 Engineering controls within cleanroom should maintain a low
relative humidity hence the settlement of air organisms in cleanroom is
decreased, facilitates the clearance of particulates.
Passive air sample by settle plate is a useful method for assessing air
contamination by microorganisms. Passive sampling consists of letting
particles settle by gravity on a flat surface that required long exposure time
(i.e. approximately 1 hour). Only particles in larger size range >10 μm are
likely to land on plates. Results are expressed in CFU/plate/time or in CFU/
m2/hour.14 Settle plates are not likely to be validated for recovery method
because there is no accurate measurement of the volume of air sampled.
Volumetric air monitoring meant for a microbiological air sampler
physically drawing a known volume of air through or over a particle
collection device which can be a liquid or a solid culture media or a
nitrocellulose membrane and the quantity of microorganism present is
measured in colony forming units (CFU) per m3 of air. Active air sampling
is ideal when monitoring a low bioburden environment. The most known
methods are impaction, centrifugal and membrane (or gelatin) samplers.
Instruments should be calibrated and the method is able to be validated.15
Several studies have attempted to compare the values of microbial loads
on air obtained through both active and passive sampling methods,
but with inconsistent results: in some cases, there was significant
correlation16-19 while in others there was none.20-24 However, current
active air can be more advantageous and effective in assessing airborne
viable contamination in cleanrooms than settle plate monitoring. There
may be no advantage in performing these two parallel methods for
the detection of airborne contamination specifically because they may
increase the number of interventions into critical areas, which may in
turn increase the risk of contamination without providing any added
benefit in terms of data collection and/or process control.24
Microorganisms Recovery
from Surfaces
Microorganisms are easy to spread within the cleanroom. They can
be transferred directly from one surface to another by touching the
surface with an object that is contaminated with microorganisms.12,25
When microorganisms are released into the manufacturing area air, they
will be deposited onto these surfaces as either aerosol particles or as
liquid droplets. There are many types of surfaces in the pharmaceutical
production areas and cGMPs equipment, all with distinct physical-
chemical properties. The type of surface greatly influences their ability to
survive and their possibility to contaminate other materials.26 The surface
14
methods that are used most is the contact-plate method is suitable
for flat, firm surfaces, (considering both recovery and repeatability),
whereas swabbing is better for flexible and uneven surfaces and for
heavily contaminated surfaces.26-28 Recovery levels of surface-monitoring
methods are typically low, due to variability in sampling procedure,
analytical methods being employed (i.e., dilution), and the use of growth-
based techniques.25
Different media are employed, and in the case of swabs, different results
have been reported for wet and dry swab methods. It is widely known
the poor correlation between the amount of microbial contamination
on surfaces and the recovery obtained. For example, a collaborative
study comparing surface monitoring methods showed that artificially
contaminated stainless steel at a theoretical level of 1.4 CFU per cm2
(about 35 CFU per sample) gave recoveries of 25 to 30% when bacterial
spores were employed.29 As reported by Kang and colleagues (2007),
recovery of Listeria monocytogenes from stainless steel inoculated test
areas, or coupons, using a sonicating brush head (contact or noncontact)
yielded a recovery level of about 60% compared with swab and direct
agar contact methods (about 20%).30 Many factors may contribute to
this poor correlation, including differences in materials used (e.g., cotton,
polyester, rayon, calcium alginate), the microorganisms targeted for
culture, variations in surface, and differences in the personnel collecting
and processing samples.30 Additional sources of error are the potential for
non-homogenous surface resulting in unequal or incomplete removal of
microorganisms from surfaces.31 Two additional variables of swab method
could be that optimum elution may vary with each organism and some
media and diluents may be inhibitory to certain microorganisms.25
The Replicate Organism Detection and Counting (RODAC) method was
described first by Hall and Hartnett (1964) as a means of direct sampling
of surfaces.32 The method is used not only for sampling of flat surfaces but
also for personnel environmental (i.e., gowning) sampling.12 A variant of
this method is the touch plate where personnel will place their gloved
finger-pads (i.e. fingertips) on the surface of the RODAC plate for getting
an estimate of the microorganisms on the tips.12,33 There are several
limitations to RODAC method. The most known is the requirement of a flat
uniform surface. A second limitation is that this method is very sensitive
to residual disinfectant that may be on the sampled surface and could be
transferred to the agar. This limitation can be overcome somehow by the
addition of neutralizing agents into the nutrient agar. The third limitation
is that the smaller size of the RODAC 50 mm limits the countable number
of colonies on the plate. Therefore, the maximum range of 250 CFU for
standard 100 mm standard Petri dishes34 would not apply to RODAC (i.e.
maximum range is 50 CFU). Surface sampling has been found to recover
<50% even with high inoculum on standardized coupons. Recovery
is also highly influenced by microorganism species and number of
microorganisms present at the time of sampling. The fourth limitation
is the residual agar transferred onto the surface being sampled; media
residue on the surface must be promptly removed as media residue
could serve as a nutrient source for microbial proliferation. Prior to their
implementation the type of media and incubation conditions must be
qualified. Such qualification may include laboratory studies using the
compendial growth promotion microorganisms and/or representative
microorganisms recovered from the facility environment.7
Swab and contact plate methods are not interchangeable because
results may vary. If you decide to use both methods in the same area
make sure the data is analyzed independently. This is because it has
been reported that RODAC plates are superior to the swab technique for
the detection of Gram-positive cocci, whereas Gram-negative rods can
be detected more often by the swab technique.35 These two techniques
were not designed to obtain full recovery but to be suitable enough to
establish trends for the assessment for environmental control.
Microbial Identification
Many factors influence the capacity of microorganism’s recovery by current
EM methods. Microorganisms are likely to be found in air and surface
forming clusters of one or distinct strains, associated to dead skin scalp,
soils or inorganic material. In addition, metabolic active microorganisms
normally are found in different stages of cell cycle. When conditions
become unfavorable for growth bacteria stop replicating and viability
starts to decrease. A high initial bacterial load increases the likelihood to
be recovered and identified.31
USP <1116> suggests that microbial recoveries should be identified
at a rate sufficient to support the EM program. Once isolation of the
microorganism is achieved, microbial characterization and identification
is performed.36-37 Typically, microbial identification systems (either
genotypic or phenotypic based) are employed after primary screening
and characterization are performed through Gram staining. Identification
platforms may vary between pharmaceuticals. Genotypic and Phenotypic
automated systems are commercially available. The system chosen must
be validated.36-37
Characterization will represent the establishment of microbial profiles,
including the evaluation of sources, routes of ingress and susceptibility
to elimination or reduction. Characterizations can reveal useful clues as to
the possible source of isolates. Routine characterization of isolates should
continue to determine whether isolates are part of the normal microflora
or represent something atypical.38
A well-established microbial EM program must identify, at least at the
genus level, microorganisms isolated from EM.38 Class ISO 5 and ISO 6
microbial recoveries must be identified at the species level. Meanwhile,
the level of identification of microorganisms from ISO 7 and ISO 8 class
could be determined based on a risk assessment analysis. For example,
15
for non-sterile class ISO 8 manufacturing or support areas it may be
sufficient to identify isolates to morphology level by gram staining on a
routine basis. Higher level of identification is recommended for microbes
isolated from aseptic processing areas and may require identification to
species level. The key concern is to determine the state of control for the
facility with some relative level of confidence. Indeed, there is regulatory
guidance that suggests the requirement to identify isolates to strain
level when investigating microbial excursions or sterility failure. FDA
2004 Aseptic Guidance document, page 35 section B. Microbiological
media and Identification establish that “Characterization of recovered
microorganisms provides vital information for the environmental
monitoring program. Environmental isolates often correlate with the
contaminants found in a media fill or product sterility testing failure,
and the overall environmental picture provides valuable information
for an investigation. Monitoring critical and immediately surrounding
clean areas as well as personnel should include routine identification of
microorganisms to the species (or, where appropriate, genus) level”.39
Characteristics such as colony morphology, cellular morphology, Gram
stain and the presence, or absence, of endospores remain important
in identification to genus level, while additional biochemical and
physiological tests may often be able to differentiate to species level. The
Gram stain method is prone to a significant level of operator error, which
has encouraged the development of alternate methods for showing the
difference in cell structures. Nowadays, automated gram staining systems
have provided some level of reproducibility.
Most pharmaceutical companies identify isolates recovered from samples
that have exceeded their alert and actions levels. Other pharmaceutical
companies have designed a grid for randomly identified isolates. Less often
others may identify the morphology of representative colonies captured.
Most aseptic pharmaceutical laboratories identify microorganisms at the
species level. Most Pharmaceutical companies identify filamentous fungi
isolates to at least the genus level if the colony counts reach action level, but
I would recommend that it be done sooner (at the alert level) to remediate
a potential fungal plum within critical control manufacturing work spaces.
Fast growing microorganisms should be identified at the species level, if
possible. Fast growing bacteria grow in such a way that they appear to
be “spread” across the plate. Such fast growing phenotype is a potential
threat to the manufacturing environment and the product. Some species
of Bacillus tend to form spreaders on moist semisolid media. Early read
of test samples before the end of incubation or test plates transfer to the
following incubation temperature is recommended to get an accurate
count if spreaders are present. A final word of advice is to be consistent
with the microbial identification platform used to identify isolates from
different sources like EM samplings or finished product because the same
strain tested using two different microbial identification platforms may
give the laboratory results two different species names. This discrepancy
may make it difficult to correlate matching microbes found in the finished
product test with microbes recovered from the manufacturing area or
specific raw materials used for product compounding.
Microorganism Prevalence
in Cleanrooms
The low density of aerosolized particulates within cleanrooms should
reduce the amount of both inorganic and biological contamination on and
within the assembled products. The nutrient-deprived (i.e. oligotrophic),
ultraclean, and desiccated conditions designed to be maintained within
cleanrooms and most manufacturing areas strives to limit the proliferation
and or survival of microbial life in these environments.40-48 Rigorous
maintenance procedures, such as regular cleaning,41 HEPA air filtration,44
and constant low humidity and temperature control, make these facilities
inhospitable to microbial life and become unlikely microbial persistence.43-44
Microorganisms within the clean room are likely to be transient;
accidental occurrences of microorganisms possibly introduced into the
cleanroom by an assortment of external sources.48 However, their path of
passage may be the same each time. With a careful study of the EM data
one may confidently predict this origin of microbial influx and ultimately
design a mitigation step to prevent it from becoming a continued source
of contamination for the controlled facilities. The identification of these
entry sources is essential to improve microbial contamination prevention.
There is no such thing as endogenous microflora for a constructed
manufacturing facility. The establishment of any microorganism within
the cleanroom shall be considered as a control breach and must be
investigated and eradicated.
Bacteria can adapt to distinct environmental conditions. These include
adaptations to changes in temperature, pH, concentrations of ions
such as sodium, and the nature of the surrounding available nutrients.
Bacteria react to a sudden change in their environment by expressing or
repressing of various sets of genetic operons that control the expression
of inducible proteins and other critical cellular components. These re-
sponses change the properties of both the interior of the microorganism
and its surface chemistry. A well-known example of this adaptation is
the so-called heat shock response (also known as stress shock response).
The name derives from the fact that the response was first observed in
bacteria suddenly shifted to a higher growth temperature.50
It is widely recognized that the isolation of numerous bacterial species
(including novel microorganisms) within cleanrooms capable of surviving
diverse, unfavorable environmental conditions is most probably due
to previous inherent resistant traits (i.e., spore formation, cytoplasmic
condensation, stress shock proteins, etc.) rather than post adaption. The
engineering controls as well as frequent cleaning and disinfection of clean
rooms is the major challenge for the minimizing the establishment of any
16
microbial entity.51 By not rotating non-sporicidal with periodic sporicidal
disinfectants in the clean room one may encourage the establishment
of one type of microbe not susceptible to the antimicrobial solution. For
rapid growth in different environments, bacteria need to adjust their
enzyme levels to rapidly benefit from the nutrient mix that is currently
available in the surrounding. If the living environment undergoes rapid
changes, the bacterium’s own production of proteins may need to be
altered to adapt to these changes in an effective way. The opportunity for
growth of bacteria is determined not only by the organic composition of
their surroundings but also by sudden changes in the living environment.
High rates of bacteria growth in a stable environment requires a certain
kind of physiology, but environmental changes also require rapid
adjustments of the bacteria’s inducible protein production. Therefore, this
microbial phenotype type(s) must be available prior to the environmental
change.51-53
Most Gram positive rod-shaped bacteria are capable of existing in two
forms, dormant spores and active vegetative cells. Vegetative cells form
spores under adverse conditions as a means of survival. Spore formation
protects the bacteria from starvation, drying, freezing, harsh chemicals,
and extreme heat. When conditions become favorable, the spores
germinate, allowing each spore to once again become a vegetative cell
with the ability to reproduce. Among the bacteria, sporulation is not a
means of reproduction since each cell forms a single spore which later
germinates into a single cell again. Most sporulating bacteria that grow
in the presence of air belong to the Genus Bacillus, and those microbes
that grow only in the absence of air belong to the Genus Clostridium. The
endospores of several Bacillus species isolated from spacecraft assembly
facilities have previously exhibited various levels of resistance to H2O2
treatment.45,52 Gram positive bacteria are more likely to survive indoor low
humidity conditions than Gram negative bacterium.46-48
Microorganisms also vary in their optimal growth temperatures. For
example, psychrophilic bacterium prefers colder temperatures, usually
below 15°C. Mesophiles thrive best at moderate temperatures, typically 20
to 45°C. Thermophiles have adapted well to hotter temperatures, usually
45° to 80°C. Most human source microflora are mesophilic.55 Mesophilic
bacteria grow best at or near human body temperature, but are also
capable of growth at room temperature.48
Spore-forming Bacillus species are not the only ones that shows
physiologically flexible phenotype able to persist in the inhospitable
conditions of clean room environments. La Duc et al (2007) reported
that the significant differences observed in the cultivable bacterial
populations among the certified clean rooms are more likely attributable
to the amounts of human activity and/or routine maintenance of each
facility, rather than geographic location.56-59 The isolation of numerous
bacterial species (including novel microorganisms) capable of surviving
diverse, unfavorable environmental conditions is a testament to the
remarkable distribution of physiologically diverse unicellular life. The
occurrence of thermophiles (Geobacillus spp.) in mesophilic condition,
obligate anaerobes (Paenibacillus sp.) in oxygen-rich environments,
and halotolerant alkaliphiles (Oceanobacillus sp. and Exiguobacterium
sp.) in neutral pH environments supports the adaptability and
resistance of these microorganisms to environments that are not their
first choice of growth.46
Conclusion
The range and types of microorganisms able to be recovered from
EM samples within cleanrooms is very limited. The parameters that
contribute to the ability to recover these microorganisms include
the microorganism’s physiology, environmental conditions (stress
factors), and nutrient composition of the culture medium and the
selected incubation conditions used to recover them. Due to the
described limitations of the EM methods currently available for
pharmaceutical manufacturing monitoring, it is unlikely to obtain all
the microorganisms that may occur in the clean rooms that employ the
need for human intervention. When manufacturing is performed in the
presence of people, most likely the microorganisms recovered will be
human source Gram positive, mesophilic aerobic or facultative aerobic
bacteria. The current commercial growth media allows for the recovery
and enumeration of these human and terrestrial microflora within a
7-day incubation period. Although, as stated in the introduction the
need for microbial environmental monitoring is a global regulatory and
compendia expectation and should not be taken lightly. The frustration
and caveats with the available commercial methods to perform these
tasks is that their recovery capability of natural microorganisms is
not optimal. As industrial microbiologists, it is our responsibility to
understand their limitations (with evidence available from the cited
references) and how to apply these insights on their practical usefulness
to monitor and indicate when our facilities are in or out of control
and meeting our company’s compliance requirements as defined and
documented in our validation studies and risk assessment reports.
Acknowledgments
I would like to thank Dennis E. Guilfoyle, Ph.D., Sr. Director, Microbiology
& Analytical Regulatory Compliance, Johnson & Johnson for critical review
and input of the manuscript.
17
References
1. 21 Code of Federal Regulations 211. Good Manufacturing Practice
for Finished Pharmaceuticals.
2. USP (2017) “Microbiological Control and Monitoring of Aseptic
Processing Environments” In: USP Vol 40, General Chapter 1116,
Rockville, Maryland, USA
3. European Commission (2008) “Manufacture of Sterile Medicinal
Products” In EudraLex - The Rules Governing Medicinal Products
in the European Union (EU), Volume 4 EU Guidelines to Good
Manufacturing Practice - Medicinal Products for Human and
Veterinary Use - Annex 1: Manufacture of Sterile Medicinal
Products, Brussels, Belgium.
4. International Organization for Standardization ISO. 14644-1:2015.
Cleanrooms and associated controlled environments—part I:
classification of air cleanliness. http://www.iso.org/iso/catalogue_
detail.htm?csnumber=25052.
5. ISO 11137:(2006) Sterilization of health care products – Radiation
– Part 2: Establishing the sterilization dose
6. ISO 14698-1:(2003) Cleanrooms and associated controlled
environments – Biocontamination control. Part 1: General
principles and methods. UNI; pp14698–1.
7. Parenteral Drug Association (PDA) Technical Report #13 Revised
(2014) Fundamentals of an Environmental Monitoring Program
8. Dalmaso, G and Denoya, C (2015) “Qualification of an
Environmental Monitoring Program”. Technical Bulleting,
http://www.cemag.us/article/2015/01/microbial-control-and-
monitoring-aseptic-processing-cleanrooms
9. Belloni, L (2000). “Colloidal interactions”. J Phys: Condensed
Matter 12(46); R549
10. Vlodavets, VV (1964) Dynamics of bacterial aerosol in the dust
and drop phase Mikrobiologiya 33(1) (Translation form Russian
by Charles T Ostertag Jr. [1965] United States Army Biological
Laboratories Fort Detrick, Frederik Maryland)
11. Sandle, T (2011). A Review of Cleanroom Microflora: Types, Trends,
and Patterns, J. PDA J Pharm Sci Technol, 65 (4):392-403).
12. Brandes, R. Aseptic Processing: Qualification of Personnel, Mass &
Peither AG-GMP Publishing, 2012.
13. Cundell AM (2005) Risk based approach to pharmaceutical
microbiology. In: Miller MJ, ed. Encyclopedia of Rapid
Microbiology Methods. River Grove, IL: Davis Healthcare
14. Pasquarella C, et al (2000) The index of microbial air
contamination. J. of Hosp Inf;46: 241–256
15. Blomquist G (1994) Center for Disease Control and Prevention
(CDC). Guidelines for environmental infection control in health-
care facilities, Atlanta; 2003.
16. Whyte, W (1981) Setting and impaction of particles into
containers in manufacturing pharmacies, J. PDA J Pharm Sci
Technol; 36:255-268
17. Whyte W (1986) Sterility assurance and models for assessing
airborne bacterial contamination. J. PDA J Pharm Sci Technol;
40:188–197;
18. Orpianesi C, et al. (1983) Evaluation of microbial contamination
in a hospital environment. Comparison between the Surface Air
System and the traditional method. Nuovi Ann Ig Microbiol. 34:
pp 171–185
19. Perdelli F, et al (2000) Relationship between settling microbial
load and suspended microbial load in operating rooms. J of
Occup Med and Toxi;12:373–380
20. Petti S, et al (2003) Comparison between different methods to
monitor the microbial level of indoor air contamination in the
dental office. Ann Ig; 15:725–733
21. Ljungqvist B and Reinmüller B (1993) Interaction between air
movements and the dispersion of contaminants: clean zones with
unidirectional air flow. J Parenter Sci Technol. 47(2):60–69.
22. Ljungqvist B and Reinmüller B. (2000) Airborne viable particles
and total number of airborne particles: comparative studies of
active air sampling. J. PDA J Pharm Sci Technol. 54:112–116.
23. Sayer WJ, et al (1972) Hospital airborne bacteria as estimated by
the Andersen sampler versus the gravity settling culture plate.
Am;58: 558–566
24. Andon BM (2006) Active air vs. passive air (settle plate)
monitoring in routine environmental monitoring programs. J.
PDA J Pharm Sci Technol. 60(6);350-5
25. Rose LJ, Noble-Wang J and, Arduino MJ Chapter 2.6.2 Surface
Sampling in Manual of Environmental Monitoring Yates MV,
18
Nakatsu CH, Miller RV and, Pillai SD Ed 4th (American Society of
Microbiology)
26. Salaman-Byron-Byron AL (2010) Bioburden Method Suitability
for Cleaning and Sanitation Monitoring: How Far We Have to Go?
Pharma Tech USA 34(8).
27. Niskanen A and Pohja MS (1977) Comparative studies on the
sampling and investigation of microbial contamination of
surfaces by the contact plate and swab methods. J. of Appl.
Bacteriol. 42, 53–63
28. Marshall V, et al (1998) Comparative mold and yeast recovery
analysis (the effect of differing incubation temperature ranges
and growth media). J. PDA J Pharm Sci Technol 52(4); 165–169
29. Salo SA. et al (2000) Validation of the microbiological methods
hygicult dipslide, contact plate, and swabbing in surface hygiene
control: a Nordic collaborative study. J AOAC Intl. 83, 1357–1365
30. Kang D et al (2007) Evaluation of quantitative recovery methods
for Listeria monocytogenes applied to stainless steel. J AOAC Intl.
90 (3), 810–816 (2007).
31. Frank JF (2001) Microbial attachment to food and food contact
surfaces in Advances in Food and Nutrition Research Taylor, S.L.,
Ed. (CA. Academic Press, San Diego) 43: 320–370.
32. Hall L.B and and M.J. Hartnett (1964) Measurements of Bacteria
contamination on surfaces in Hospitals Public Health Rep
79(11):1021-1024
33. USP (2017) Sterile Compounding In: USP Vol 40, General Chapter
797, Rockville, Maryland, USA
34. Breed R and Dotterrer WD (1916) The Number of Colonies
Allowable On Satisfactory Agar Plates. J Bacteriol. 1:321-331
35. Lemmen SW, et al (2001) Comparison of two sampling methods
for the detection of Gram-positive and Gram-negative bacteria
in the environment: moistened swabs versus Rodac plates. Int. J.
Hyg. Environ. Health 203;245-248
36. USP (2017) Microbial characterization, Identification and Strain
Typing In: USP Vol 40, General Chapter 1113, Rockville, Maryland,
USA
37. European Pharmacopeia Chapter 5.1.6 (2017) Alternative
methods for control of microbiological quality Suppl 9.2
38. Salaman-Byron-Byron AL (2006) Implementation of Microbial
Environmental Monitoring Program for Non-Aseptic Processes.
Am Pharma Rev 9(5):10 - 15.
39. Food and Drug Administration (FDA) (2004) “Guidance for
Industry - Sterile Drug Products Produced by Aseptic Processing -
Current Good Manufacturing Practice”, Rockville, Maryland, USA
40. Favero MS, et al (1966) Comparative levels and types of microbial
contamination detected in industrial clean rooms. Appl
Microbiol.14(4): 539-51.
41. Favero MS, et al (1968) Microbiological sampling of surfaces. J
Appl Bacteriol. 31:336–346
42. Henderson, K (2000) Contamination control plan, p. D-19494.
JPL, Pasadena, CA; NASA-KSC. 1999. Launch Site Requirement
Planning Group facilities handbook of Payload Hazardous
Servicing Facility (PHSF). Publication K-STSM-14.1.15 rev D. KSC,
Cape Canaveral, FL
43. Satomi K., et al (2001) Molecular microbial diversity of a
spacecraft assembly facility. Syst. Appl. Microbiol. 24: 311-320
44. Venkateswaran, K, et al (2001). Molecular microbial diversity of a
spacecraft assembly facility. Syst. Appl. Microbiol. 24:311-320
45. Reinmüller, B. (2001) People as a Contamination Source - Clothing
Systems. In: Dispersion and Risk Assessment of Airborne
Contaminants in Pharmaceutical Cleanrooms. Royal Institute
of Technology, Building Services Engineering, Bulletin No. 56,
Stockholm, 54-77
46. La Duc MT et al (2007) Isolation and characterization of bacteria
capable of tolerating the extreme conditions of clean room
environments. Appl Environ Microbiol 73; 2600-11
47. Gen-fu W and Xiao-hua L (2007) Characterization of predominant
bacteria isolates from clean rooms in a pharmaceutical
production unit J Zhejiang Univ Sci B.; 8(9): 666–672.
48. Sandle, T (2011) A Review of Cleanroom Microflora: Types, Trends,
and Patterns, J. PDA J Pharm Sci Technol, 65 (4):392-403.
49. Pasanen AL, et al (1991) Significance of air humidity and air
velocity for fungal spore release into the air. Atmos. Environ.
25A:459 –462.
50. Arsene F (2000) The heat shock response of Escherichia coli. J
Food Microbiol 55;3–9
19
51. Chapman, JS (2003) Disinfectant resistance mechanisms, cross-
resistance and co-resistance. Int. Biodeter. Biodegr. 51(4):271-276.
52. Pavlov, MY & Ehrenberg, M. (2013) Optimal control of gene
expression for fast proteome adaptation to environmental
change. PNAS 110 (51): 20527-20532
53. Tsai FC and Macher JM (2005) Concentrations of airborne
culturable bacteria in 100 large US office buildings from the BASE
study Indoor Air 15 (Suppl 9): 71–81
54. Kempf, MJ, et al (2005) Recurrent isolation of hydrogen peroxide-
resistant spores of Bacillus pumilus from a spacecraft assembly
facility. Astrobiology.5(3):391-405
55. Proksch E.; Brandner J.M.; Jensen J.M. (2008) The Skin: An
Indispensable Barrier, Exp. Dermatol. 17 (12):1063–72
56. Frankel et al (2013) Seasonal Variations of Indoor Microbial
Exposures and Their Relation to Temperature, Relative Humidity,
and Air Exchange Rate Applied and Environ Microbiol 78(23);
8289 – 8297
57. Hyde, W (1998) Origin of bacteria in the clean room and their
growth requirements. J. PDA J Pharm Sci Technol; 52:154–164
58. Hussong D and Madsen R. (2004) Analysis of environmental
microbiology data from clean room samples. Pharm Technol.
Aseptic Processing Suppl:10–15.
59. Pacheco FL et al (2010) The bacterial diversity of pharmaceutical
clean rooms analyzed by the Fatty Acid methyl ester technique. J.
PDA J Pharm Sci Technol; 64(2):156-66
Author Biographies
Angel L. Salaman-Byron MS, PhD is Principal Process Scientist at Janssen
Biotech Malvern, PA, USA. Dr. Salaman-Byron received his PhD degree
in Medical Microbiology from the University of Puerto Rico, Rio Piedras
Campus. Besides his scientific contribution during his dissertation research
Dr. Salaman-Byron has contributed to the pharmaceutical literature
with publications appearing in American Pharmaceutical Review and
Pharmaceutical Technology as well as a White papers contributor. Dr.
Salaman-Byron has worked with oral dosages small molecules as well as
large molecules manufacturer and aseptic filling process, such as Wyeth,
Pfizer, Amgen and AbbVie.
The Hottest Topics in
Microbiology
Karen Ginsbury
PCI Pharmaceutical Consulting Israel Ltd.
Recently I asked participants in a senior level think tank, what were
the most challenging microbiological issues on their “to do” lists. The
answers included:
• Correlation of field failures for bacteria to hygiene in the
manufacturing environment or against efficacy testing.
• People.
• Perception that you can validate manual procedures leads to
miscalculations.
• Use of contract workers and high turnover.
• Seems like a lot of us are disinfecting/sanitizing but what do we
actually intend to achieve.
• Dealing with the ambiguity of bioburden control involved with
microbiologically controlled products and the perception (by
some) that we are making sterile products.
• Need to educate about risks.
• Risk assessment of environmental organisms and the need. for
better knowledge and control. Use the risk assessment for how
significant is the organism for the consumer. How do I ID the
organism and how do I do the risk assessment –the regulators
are all over the place.
• What truly is an objectionable/specified organism–base on a risk
assessment (documented) for YOUR product type and also any
USP specified organism.
• Contract manufacturer fights us on requests for objectionable
organisms? Our spec is just specified orgs. Reply: USP <1111>
requirement to assess any organism you pick up in the
environment.
• Raw material suppliers have no idea about the market they
are supplying and refuse to do it. There is a price on CMOs and
sometimes cheaper and easier to bring the baby back home.
20
You may have to pay for your requirements.
• Non-viable particulate count room qualification per ISO
regulations –rooms are controlled and we claim class E –now
required “new class E = D” for non-sterile products?
• Everyone talks about “risk-based” EM and EM trending and yet I
have yet to see one example of how anyone is actually DOING it!
And have never seen anyone using relevant statistical analysis
of EM data.
• I was so excited by <1115> but it says nothing: product
specification is NMT 0 cfu/ml.
So…ouch. You can hear the pain and then start to filter out issues. Actually
when you sort through the list, there aren’t as many different concerns as
it first appears. Here is what I found:
1. Increased emphasis on non-sterile products. Increasing
regulatory scrutiny. Unclear requirements, but nevertheless
expectations for controlled manufacturing environment,
microbial limit testing and preservative efficacy testing if
the product is preserved. The only regulatory body who has
published guidance for non-sterile environments is the World
Health Organization (WHO). Neither the EU GMPs or the US
GMPs or FDA guidances address this topic. The EU is currently
revising Annex 1 on Sterile Products and in their concept
paper on the revision indicated that the revision might address
suitable environments for manufacturing non-sterile products.
Since the revised Annex has yet to be published, the jury is out.
What should you be doing?
Regarding non-sterile products there are two references which might
help.
The first is the USP General Chapter <1115>, Bioburden Control of Non-
Sterile Drug Substances and Products. As one of the think tank participants
pointed out, it looked promising but is actually quite confusing/confused.
On the one hand, it advocates “a pragmatic scientific approach to the
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management of the microbial bioburden in non‐ sterile drug products in
keeping with patient risk and contamination control objectives based on
risk management principles”. The chapter is advocating process control
rather than reliance on end product testing which is commendable and
the desired state.
<1115> is an informational chapter and not an enforceable regulation.
On the other hand, the chapter advocates for any environmental
monitoring program to be risk‐based in terms of sample selection and
frequency and to be used to confirm microbial control and not linked to
product release.
This too is commendable as a statement for those well versed in process
control as opposed to product control. However, in practice it often results
in a check box exercise which is far removed from true risk management
and monitoring. Environmental monitoring is a RISK MONITORING
tool and not, as commonly and mistakenly believed, a risk reduction
measure. EM samples, represent intelligence gathering, allowing ongoing
assessment and understanding of the state of control of the environment.
Increased counts, and in non-sterile production, since the baseline
counts are generally far higher than in sterile production, it is easier to
identify trends earlier and to respond. But if the EM is disconnected
from production of specific products and batches, and, is frequently
conducted during “at rest” conditions, the purpose of the monitoring is
lost, the responses don’t happen and failures in Microbial Limit Tests are
then written off as “laboratory error.” According to Tony Cundell, one of
the authors of the chapter, the intent was for improved and cost‐ effective
bioburden controls for non‐sterile drug products to reduce the number of
product recalls arising as a result of microbial contamination. No question
that this necessary – especially after the recent episode of contaminated
sunflower seeds resulted in recalls of many brands of health bars including
my personal favorite the Cliff bar!
The second essential reference is PDA’s Technical Report #67: Exclusion
of Objectionable Organisms from Non-sterile Pharmaceuticals, Medical
Devices and Cosmetics. This is a points to consider document that
addresses product considerations, risk management, how to identify
clinical considerations related to defining objectionable organisms for
a particular product as well as process design to mitigate microbial
risk – all valid and appropriate considerations in setting up a microbial
control strategy.
2. How can we make risk assessments less burdensome and more
useful/effective?
Assessments need to be simplified. The risk question needs to be sharpened.
For example, for non-sterile products what is the risk question if performing
a risk assessment for the manufacturing process? How about “what could
go wrong resulting in unacceptably high total count or presence of
objectionable organisms in the product”
22
However, from a microbial point of view, the answer is not straightforward.
It ties in with the above observation that “Seems like a lot of us are
disinfecting / sanitizing but what do we actually intend to achieve.”
The answer to that is that we need an environment, populated with
operators appropriately gowned to ensure that things do not go wrong
resulting in high total counts or objectionable organisms. Since the two
are related and since microbes multiply by doubling in short periods of
time, the answer to the disinfecting/sanitizing is the following:
The purpose of disinfecting in a non-sterile area to maintain routinely
low levels of microbial contamination with the intention of preventing
product processed in the environment from ever exceeding the Microbial
Limit specification for total count or objectionable organisms.
3. Environmental monitoring – what is needed and how do we
reply risk assessment and contamination recovery rates (per
USP <1116>).
The answer to this question is a follow on from #2 above for non-sterile
products.
What is needed is a truly risk-based program, with samples at those areas
MOST LIKELY to disseminate contamination to the process and therefore
to product.
4. Control of outsourced manufacture and testing
With respect to microbiological risk – any program we have in our own
company needs to be mirrored in some manner at our contractor to
provide the necessary assurances. We audit for that and if there are
concerns they must be remedied under the CAPA program before signing
a contract. Ongoing evaluation of the supplier for bioburden controls and
microbial test must include review of EM data.
5. How much work should we be doing with identification
of organisms and how does that tie in with cleaning and
disinfection programs and field failures, hygiene and the
people factor?
There should be an SOP defining identification policy. Firstly, sufficient
isolates must be collected to obtain a baseline flora for an area and that
is more than only identifying those isolates where the alert or action
level was exceeded. Thereafter, the policy should be based on potential
significance to product and therefore to patient and periodic across the
board identification to see if there are new isolates recovered and to
test recovered wild types for sensitivity to the disinfectants used in our
sanitization regime. A sudden increase in fungal counts is reason for a
hysterical response – yes I did choose that word – rapid and aggressive
action to identify and seal off the source (often cracks or building
movement when there is heavy digging nearby from construction) and
repeated cycles of sporicidal disinfectants. You do not want to be the
next facility shut down for fungal contamination (Google “compounding
pharmacies and fungal contamination” for more details)!
And beyond that consider the following hot topics with the regulators, all
of which have implications within the microbiological area:
• Quality Metrics and Culture
• Data Integrity –the microbiology lab has a special place in
warning letters for “throwing away plates” or “failure to record
colonies” all of which is easy to do because there is no trail
whatsoever (never mind a part 11 compliant, time and date
stamped, automated computerized trail). In the microbiology
laboratory, most companies still work with an operator manually
examining incubated test samples and recording the outcome.
The temptation to cheat is facilitated by the ease with which it
can be done – which leads into cultural issues and independent
verification checks.
• Multiplicity of regulations (IVT has a link with over SEVENTY
microbiological references)
• Revision (planned) of Annex 1 to the EU GMPs (Sterile Products
annex) and updated ISO 14644 (in effect but are contractors
ready for that?)
And how do all of the above tie in with the recent massive recall of health /
granola bars due to listeria contamination of sunflower seeds?
To conclude – new issues? Not really. More of a new focus on non-sterile
manufacture. Is non-sterile manufacture, as traditionally perceived,
less complex than aseptic processing? In fact from a microbiological
perspective it might be MORE complex – because the goal is poorly
designed. Sterile means the complete absence of viable organisms, or
at least bacteria and fungi. What does Non-Sterile mean: NMT 100 cfu /
g total, NMT 10 cfu/g fungi and no objectionable organisms. Try telling
the bugs to stop replicating at 100 or the fungi at 10. If your microbial
control strategy does not strive to keep them out, they will sooner or later
23
take over and you will have OOS Microbial Limit Tests. A control strategy is
defined in ICH Q10 as:
“a planned set of controls, derived from current product and process
understanding, that assures process performance and product quality”
A microbial control strategy is based on risk assessment, implemen-
tation of controls directly related to understanding of microbial risks
posed by product and process such that the controls assure process
performance and product quality. i.e. no MLT failures. Environmental
monitoring is one of the risk monitoring tools which allows you to
show ongoing control and while not directly related to batch release
decisions, is definitely one indicator that the batch might be fit or unfit
for release from a microbial perspective.
One final comment. No think tank or article on hot micro topics would
be complete without discussing rapid methods. There are many available.
For the most part, pharma companies seem to have implemented
these mostly in the area of microbial identification. However, there are
increasing interesting offerings in the area of real time counts of viable
and total particles where the total particles include and can distinguish
viable organisms. This is an evolving area and…watch this space!
Author Biography
Karen Ginsbury is a UK trained pharmacist with a Master of Science degree
in Microbiology from Birkbeck College University of London. Karen is CEO
of PCI Pharmaceutical Consulting Israel Ltd and has worked in and with the
pharmaceutical industry for close to 30 years. She provides Quality Systems
consultancy and her company has successfully navigated companies through
FDA, Health Canada, EU and other regulatory inspections as well as setting up
and improving effective Quality Systems which have increased the efficiency
of the company.
Biologics Production: Impact
of Bioburden Contaminations
of Non-Sterile Process
Intermediates on Patient Safety
and Product Quality
Friedrich von Wintzingerode
Roche-Genentech
Large Scale Production of
Biologics is Susceptible to
Microbial Contamination
The term “Biologics” is used for a class of therapeutics that are produced
by recombinant DNA technology and generally fall into three major
categories: (i) therapeutic proteins, (ii) monoclonal antibodies (mAbs),
and (iii) antibody-drug conjugates.
Large scale production of biologics consists of two process steps,
which often occur at different production sites: (i) Drug Substance (DS)
manufacture (manufacture of the Active Pharmaceutical Ingredient),
and (ii) Drug Product (DP) manufacture (manufacture of the Final Drug,
e.g. formulated mAbs filled in vials or syringes). In the DS manufacturing
process bacterial or mammalian cells are used to express the desired
recombinant protein. Following cell expansion and production phases
these cultures are harvested, the recombinant protein is recovered and
is purified to separate the target protein from host cells, cell debris,
and process- and host-related impurities. The purified protein is then
formulated and filtered into containers for storage and shipment to the
DP manufacturing site where the formulated DS is then filled into vials
or syringes to produce the final drug product. In the DP process most
biologics are subject to terminal sterilization as they are administered
parenterally (e.g. intravenous, subcutaneous, intravitreal). However,
for biologics manufacturing the risk of microbial contamination is not
24
limited to the final product. Many biologics manufacturing steps (e.g.
protein purification, conditioning, formulation) occur under non-sterile
conditions in aqueous systems at ambient temperature or 2-8 °C under
substantially neutral pH conditions, making the large-scale production
of biologics susceptible to microbial contamination (see Figures 1
and 2 for a typical mAb production process). Regardless of where in
the DS or DP process they occur, microbial contaminations can have
a significant impact on product quality and patient safety. Microbial
contaminations can lead to product degradation or modification due
to the introduction of microbial enzymes. In addition, the introduction
of soluble components derived from the microorganisms can adversely
impact patient safety. The investigation of microbial excursions must
adequately assess impact to both product quality and patient safety
prior to making decisions regarding lot release.
Development of a successful microbial control strategy for large-
scale production of biologics should be based on a risk assessment to
ensure end-to-end microbial control of the process through adequate
prevention and detection. The risk assessments require review of the
microbial quality of direct materials, utilities (WFI, process gases, etc.),
and individual steps within the series of unit operations that constitute
the DS and DP manufacturing process. For each step, the assessment
should evaluate process/operational conditions and existing controls
regarding the equipment, the manufacturing environment, and
personnel preventive controls for their effectiveness at controlling and
minimizing microbial contamination, with a goal of prevention of ingress,
and proliferation in the process, equipment, and product. In addition, the
existing microbial detection controls should be assessed to ensure they
provide continued verification that the microbial prevention controls
are working as intended. Detection controls consist of bioburden and
endotoxin testing against established limits on samples obtained from
defined process steps. For critical process steps (e.g. production culture
and DS) the acceptable bioburden limit is extremely low and valid
bioburden results exceeding acceptance criteria would lead to rejection
of the batch.
Microbial Risk of Biologics
Manufacturing is Not Limited to
Living Microorganisms and Intact
Microbial Cells
The active pharmaceutical ingredient in biologics products are usually
heat sensitive. Therefore, filtration using membranes with pore sizes
ranging from 0.02 to 0.2 µm size is the method of choice for bioburden
reduction and terminal sterilization. While 0.02 to 0.2 μm filtration can
effectively remove intact microbial cells from the product, this process
does not allow removal of subcellular structures of bacteria and fungi.
If co-purified with the product subcellular structures like microbial
toxins and so called Pathogen Associated Molecular Pattern (PAMPs),
i.e. endotoxins, lipopeptides, lipoproteins, flagellin, bacterial and fungal
DNA, and cell wall polysaccharides potentially lead to toxic, allergic, or
inflammatory responses in humans. In addition, co-purified extracellular
proteases or endoglycosidases potentially lead to product degradation
or modification.
In other words, bioburden contaminations of non-sterile process
intermediates represent a risk even after 0.02 to 0.2 μm filtration, and
even if both Drug Substance and Drug Product specifications are met.
This risk is also described in relevant guidelines:
USP<1229.3> “Monitoring of Bioburden”:
“Bioburden is a potential risk to the patient not only because the sterilization
process might not be completely effective, but also post-processing
because of the possible presence of residual materials such as allergens,
endotoxins, and exotoxins. It may also have adverse impact on
product quality and stability.”
FDA, Questions and Answers on cGMP (January 2011):
“What manufacturing contamination risks are presented by the different
pathogenic agents ?
… Microbial toxins can be divided into two general groups: exotoxins and
endotoxins. ….. Exotoxins, especially heat-stable exotoxins, can remain in
the ingredient throughout the manufacturing process and adversely affect
patient health…….. Some species of molds produce toxic byproducts
called mycotoxins. ….”
25
Bioburden and Endotoxin Testing Play a Key Role
in The Assessment of Microbial Risk of Non-Sterile
Process Intermediates
The microbial detection control strategy of large scale biologics
production defines bioburden and endotoxin sampling points for non-
sterile process intermediates. Endotoxin testing using the compendial
LAL assay allows universal detection of bacterial endotoxins, while
direct detection of other toxins and non-endotoxin PAMPs derived from
bioburden in process intermediates is a challenge, making it difficult
to reliably confirm their presence or absence. Bioburden analysis
performed on representative samples before 0.2 μm filtration (so called
pre-filtration samples) allows determination of the total microbial
load of the product and serves as a surrogate test for toxins and non-
endotoxin PAMPs. The potential load of toxins and non-endotoxin
PAMPs can be calculated based on the bioburden concentration (e.g.
CFU/10 mL), cell characteristics of the contaminating organism, process
step, and microbiological factors of toxins and non-endotoxin PAMPs.
The calculated load can be compared to safety limits specific for toxins
and non-endotoxin PAMPs.
Bioburden analysis can also indicate the potential presence of
degradative enzymes, e.g.; extracellular proteases, or endoglycosidases,
which could cause product degradation or modification. In some cases
stability studies can be conducted to assess the potential impact of
these microbial components on product quality.
Case-by-Case Assessment of
Bioburden (CCAB): A Comprehensive
Approach to Assess the Impact of
Bioburden Contaminations of Non-
Sterile Process Intermediates on
Product Quality and Patient Safety.
The following sections describe the Case by Case Assessment of Bioburden
(CCAB) procedure and underlying rationales. The CCAB procedure is used
to investigate bioburden action limit excursion in the Roche/Genentech
Biologics manufacturing network and is only one component of the
quality investigation. The investigation must be reviewed holistically and
all findings that may impact product quality, patient safety, equipment
fitness and process performance must be considered before determining
acceptability of the batch. The applicability of CCAB is assessed by quality
based on the criticality of the impacted process and whether there are
mechanisms for additional removal of impurities derived from bioburden
in subsequent process step(s).
Figure 4 illustrates the key aspects of CCAB, which are described in more
detail below.

Patient Safety Assessment (PSA)
General Aspects
Patient safety assessment focuses on subcellular (extracellular) com-
ponents of bacteria and fungi, which show the following characteristics:
• components potentially pass filters installed in the
manufacturing process to reduce bioburden or viral loads (0.02
– 0.2 μm pore size).
• components cannot be detected using analytical methods,
which are established in a routine GMP QC environment.
• components are PAMPs, allergens or exotoxins relevant to
human safety.
Subcellular/extracellular components of bacteria and fungi, which show
all aforementioned characteristics, are termed “critical com-ponents”.
Patient safety assessment procedure is used to determine the patient
safety impact based on hypothetical assumptions about the amount
and potency of critical components in a microbial load. The assumption
is that critical components will be co-purified into the Drug Substance
and the finished pharmaceutical product (DP).
Critical components comprise the following subcellular microbial structures:
Protein exotoxins
Bacteria are capable of producing a wide range of protein toxins
most of them are actively released from bacterial cells during growth
and therefore are considered as exotoxins (Alouf, 2000). Examples
for bacterial protein exotoxins are hemolysins and staphylococcal
enterotoxins (Dingens et al., 2000). Production of hemolysins is also
reported for fungal species like Aspergillus fumigatus (Wartenberg et al.,
2011) and Candida spp. (Luo et al., 2001). Several protein exotoxins like
staphylococcal enterotoxins and toxic shock syndrome toxin 1 can act
as classical allergens (Novak et al., 2003). Currently, over 300 different
protein exotoxins are described (Alouf, 2000). In contrast to bacterial
endotoxins, which are universally detected by the LAL assay a wide range
of different test methods exists for bacterial protein toxins (Pimbley and
Patel, 1998). Most protein exotoxins occur as single-chain holoproteins
varying from approximately 2-3 kDa to approximately 300 kDa (Alouf,
2000). As a worst case scenario it is assumed that protein exotoxins are
not excluded by 0.02 – 0.2 µm filters.
Non-protein exotoxins
Fungal species are capable of producing a wide range of non-protein
exotoxins also referred to as mycotoxins (Bennett and Klich, 2003).
No universal test method exists, which detects all mycotoxins (Turner
et al., 2009). For bacteria only a few non-protein toxins are described.
Tetrodotoxin (TTX or puffer fish toxin) is one of the most potent
neurotoxins. It is produced by different bacterial strains covering a
26
wide taxonomic range (e.g. strains of the genera Pseudomonas, Bacillus
or Microbacterium) (Lu and Yi, 2009). Some mycobacteria produce
mycolactones, which are suspected to cause Buruli ulcer, an unusual
skin disease (Hong et al., 2008). A variety of bacterial strains from
different bacterial genera (Pseudomonas, Burkholderia, Acinetobacter,
and Rhodococcus) have been shown to produce rhamnolipids as LPS-like
exotoxins (Andrä et al., 2006). As a worst case scenario it is assumed that
non-protein exotoxins are not excluded by 0.02 – 0.2 µm filters.
MALP-2 like lipopeptides/proteins
Lipopeptides/proteins are peptides or proteins modified by covalent
linkages to lipids, with various functions in the bacterial cell. They are
widespread in Gram-negative bacteria, but are also found in Gram-
positive bacteria. The overwhelming majority of lipopeptides/proteins
are anchored to the cell membrane (Narita et al., 2004; Hutchings et
al., 2008). Of relevance to CCAB are lipopeptides/proteins similar to
MALP-2 lipopeptide (a 2 kDa macrophage-activating lipopeptide) from
Mycoplasma fermentans that are found in the extracellular protein
fraction. MALP-2 has been shown to have an inflammatory potency
similar to that of LPS (Galanos et al., 2000). Since bacterial lipopeptides
can be shown to be endotoxic on account of their chemical structure
(Schromm et al., 2007), they can be assumed to have a potency
essentially comparable to that of MALP-2 or LPS. Based on the current
literature fungal lipopeptides/proteins do not act as PAMPs (Sorrell and
Chen, 2009). Lipopeptides/proteins vary greatly in size. As a worst case
scenario it is assumed that they are not excluded by 0.02 – 0.2 µm filters.
Flagellin
Flagellin is the protein component of the bacterial flagellum, the
most common organelle for locomotion of Gram-positive and Gram-
negative bacteria. Flagellar filaments and monomers are shed into the
environment (Neish, 2007). Flagellin is a potent inducer of inflammatory
responses (Neish, 2007). Approximately 20,000 flagellin subunits are
present in a single flagellar filament of 20 nm diameter and 10-15 µm
length (Hughes and Erhardt, 2011). Depending on the bacterial species,
flagellins have molecular masses ranging from 28 to 80 kDa (Winstanley
and Morgan, 1997). Given these numbers it is highly unlikely that single
flagellin subunits are size excluded by 0.02 – 0.2 µm filters.
Bacterial and fungal DNA
Release of plasmids and chromosomal DNA has been shown for Bacillus
subtilis during growth (Lorenz et al., 1991). As a worst case scenario
it is assumed that the majority of bacteria and fungi are capable of
actively releasing DNA. Bacterial DNA induces inflammatory responses
in humans because of the high frequency of unmethylated CpG motifs
(Krieg, 2002; Akira et al., 2006). Unmethylated CpG motifs has also been
found in Aspergillus fumigatus DNA (Ramirez-Ortiz et al., 2008). As a
worst case scenario it is assumed that unmethylated CpG motifs are also
abundant in other fungal species. It is highly unlikely that 0.02 – 0.2 µm
filters exclude immunostimulating microbial DNA since CpG motifs are
also present on short DNA stretches or fragmented DNA.
Cell wall polysaccharides level by ribosomal DNA sequences do not produce exotoxins relevant to
humans. It is current scientific standard that ribosomal DNA sequences
Polysaccharides are structural elements of bacterial and fungal cell
of microbial isolates including those of clinical relevance are deposited
walls. Microbial cell wall synthesis is a highly dynamic process. As a
in public databases like GenBank/EMBL or SILVA1. Therefore it can be
worst case scenario it is assumed that cell wall polysaccharides are shed
excluded that exotoxin producing microorganisms recovered from
into the environment. For Gram-positive bacteria peptidoglycan (PGN)
a contamination cannot be identified to genus or species level by
constitute 30-70% of the dry weight of the cell wall (Schlegel, 1985).
ribosomal DNA sequencing2. Additional investigations (e. g. Gram-
The cell wall of Gram-positive bacteria contains lipoteichoic acid (LTA),
staining, test for motility etc.) are needed to assess critical components
which is absent in Gram-negative bacteria. Both LTA and PGN are potent
other than exotoxins.
inducers of inflammatory responses (Rockel and Hartung, 2012). For
Gram-negative bacteria endotoxins and lipoproteins constitute up to
80% of the dry weight of the cell wall, whereas PGN constitutes only a
small fraction of less than 10% (Schlegel, 1985). The fungal cell wall is 80- Calculation of Hypothetical Loads of
90% polysaccharides with most of the remainder consisting of protein
and lipid. The most important structural elements of the fungal cell wall
Critical Components
are chitin and ß-glucan (Bartnicki-Garcia, 1968). As a worst case scenario Microbiological factors for calculating hypothetical loads of critical
it is assumed that cell wall polysaccharides or fragments of it are not components are given in Table 1.
excluded by 0.02 – 0.2 µm filters.

Calculation of Carryover of
PSA Procedure
Critical Components
Evaluation of Microorganisms For the calculation of the hypothetical carryover of critical components
into the DS and the DP, a distinction is made between downstream
The starting point for the compilation of a PSA is a quantifiable microbial
processing and upstream processing.
contamination. Identification of the representative microorganisms in
the contamination uses ribosomal DNA sequencing technique. Upstream processing

Situation A: Upstream processing comprises the process steps from fermentation or

The microorganism is identified to genus or species level. Information
about critical components and cell characteristics which might impact
patient safety is obtained through literature searches. Literature searches
should cover but are not restricted to:
• Presence of exotoxins relevant to human safety
• Presence of MALP-2 like lipopeptide/proteins
• Presence of flagellin
• Cell wall type (gram-positive, gram-negative, fungal cell wall)
• Cell size (bacteria and yeasts) or size of conidia (molds)
Exceptions include microorganisms that for methodological reasons
cannot be unambiguously assigned to a species or genus based on
ribosomal DNA sequences, but for which the presence of toxins can
be assumed due to their taxonomic affiliation (e.g. members of the
Enterobacteriaceae family).
Situation B:
The taxonomic affiliation of the recovered microorganism is unknown
as it cannot be identified to species, genus or family level. It is assumed
that microorganisms, which cannot be identified to genus or species
cell culture through harvest including cell separation and clarification,
and where applicable, inclusion body preparation/renaturation (E. coli
processes). A distinction must be made between cell culture products
and E.coli products.
Cell culture products:
The protein active substances (e.g. monoclonal antibodies) are secreted
into the cell culture medium. Any critical component present is able
to bind non-specifically to the proteins and thereby be carried over
into down stream processing. The assumption is made here that 10%
of the critical component binds (non-specifically) to the protein active
substances3.
E. coli products:
Nonspecific binding of any critical component present to the protein
active substance (in the form of inclusion bodies or as periplasmic
proteins) is possible only after cell disruption. The assumption is made
here that 10% of the critical component binds (nonspecifically) to
the inclusion bodies/to the refolded or the periplasmic protein active
substance and is thereby carried over into down stream processing
for E. coli products. Critical components bound to the E. coli cell wall/
cell surface are removed during cell disruption/clarification and cannot

27
thereby be carried over into downstream processing. Carryover of critical
components, by this route, into the DS or into the DP can therefore be
ruled out.
Downstream processing
Downstream processing comprises the process steps from loading
onto the first chromatography column up to bulk compounding and
formulation. In the event of microbial contamination in downstream
processing, complete carryover of the critical component into the bulk
active substance and the finished pharmaceutical product is assumed
for in-process samples. No distinction is made between “early” and “late”
process steps.
Calculation of Critical
Component Dose
The pharmaceutical product is assumed to be administered at a certain
dose per day. The calculation of the hypothetical dose of critical
component exposed to humans is based on the product concentration of
the corresponding drug substance solution. If the product concentration
is not available, the lowest product concentration value defined by the
specification is used. The number of doses per mL can be calculated from
the maximum dosage of the drug. This information can be obtained
from product inserts or leaflets.
Assessment of Risk Potential
Safety limits for critical components are given in Table 2. A worst case
body weight should be selected based on the description for each
product or information obtained from clinical trials. If no documentation
is available, 50 kg body weight for absolute dosing (non-weight-based)
or dosing per patient should be selected to represent worst case
scenario.
Product Quality Impact
Assessment (PQIA)
General Aspects
Co-purified extracellular proteases, endoglycosidases or other
microbial enzymes can potentially lead to product degradation or
modification, impacting product quality. To further investigate this
hypothesis microbial proteomics of a panel of microorganisms that
have been recovered from Biologics product streams and known to
produce extracellular proteases and other modifying enzymes (Bacillus
28
cereus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and
Staphylococcus aureus) was conducted (Roche/Genentech internal
study, Figure 5 illustrates the experimental setup).
Proteomic analysis of microbial cell supernatants showed that release
of those enzymes is strongly influenced by growth conditions (high and
low nutrient media, starvation and no starvation). Cell supernatants
were taken from panel microorganisms grown under optimal enzyme
production conditions, spiked into two different Biologics (one
therapeutic mAb and one key signaling protein) and incubated at 20-25
°C and 2-8 °C for up to 49 days. After incubation spiked product samples
were analyzed for product integrity. In some cases significant changes
of product integrity were seen even when spiked cell supernatants
resulted from cell counts as low as 100 CFU/mL.
The PQIA procedure considers these study results. A review and
evaluation of relevant QC data provides an indicator whether there is
impact to product quality. In certain cases, additional product stability
testing is required to determine impact.
PQIA procedure
The PQIA differentiates between process steps with potential for
subsequent reduction of modifying or degrading components derived
from bioburden, and process steps for which no further reduction of
modifying or degrading components would be expected.
Process steps with further downstream purification
For all steps of upstream processing and all downstream process steps up
to the load of the last process step with potential reduction of modifying
or degrading components, PQIA is performed as follows:
• Assess relevant CoA data (e.g., identity, potency tests) of the
impacted drug substance or drug product lot.
• Compare data against reference materials or other released
lots for potential anomalies.
Process steps with no further downstream purification
For all process steps where further reduction of critical components
derived from bioburden is not expected (e.g. final UFDF, thawed bulk,
bulk compounding, and drug product processing steps) the PQIA is
performed as follows:
• Assess relevant CoA data (e.g., identity, potency tests) of the
impacted drug substance or drug product lot.
• Compare data against reference materials or other released
lots for potential anomalies.
• Perform a product stability study under protocol using the
product stability indicating assays for the product. Appropriate
subject matter experts must assess any anomalous data for
impact to product quality.

Summary
Large scale production of biologics is susceptible to microbial
contamination. Bioburden contaminations of non-sterile process
intermediates represent a risk to patient safety and product quality.
Even after bioburden removal by 0.2 µm filtration, and even if both Drug
Substance and Drug Product specifications are met, subcellular microbial
components like toxins, lipopeptide/lipoproteins, flagellin, bacterial
and fungal DNA, cell wall polysaccharides, extracellular proteases or
endoglycosidases remain in the product. Those microbial components
potentially lead to toxic, allergic or inflammatory responses in humans
or product degradation or modification. The CCAB approach described
here enables a comprehensive assessment of these risks.
Acknowledgement
The author would like to thank Ray Arnold, Wolfgang Eder, Emabelle
Ramnarine for helpful comments on the manuscript and Sven
Deutschmann, Holger Kavermann, Michael Knight, Christian John,
Markus Haberger, Ingo Lindner, Andreas Adler, Andreas Krug, Prof. Ulrich
Zähringer (Research Center Borstel, Germany), Prof. Uwe Völker and Elke
Hammer (University of Greifswald, Germany), for their contributions to
develop the CCAB concept.
References
• Akira et al., 2006. Cell. 124:783-801.
• Alouf and Popoff. 2006. “The comprehensive Sourcebook of
Bacterial Protein Toxins”. 3rd edition. Elsevier Ltd.
• Alouf, 2000. Bacterial Protein Toxins: An Overview, in: Bacterial
Toxins. Methods and Protocols. Holst O, ed., Walker, J. M.
Humana Press, Totowa, NJ, USA, pp. 1-27.
• Andrä et al., 2006. Biol. Chem. 387:301-310.
• Bartnicki-Carcia. 1968. Annu. Rev. Microbiol. 22:87-108.
• Bennett and Klich. 2003. Clin. Microbiol. Rev. 16:497-516.
• Bhatia and Zahoor, 2007. J. Clin. Diagn. Res. 3:188-197.
• Braune et al., 2001. British J. Dermatology. 144:111-117.
• Dingens et al., 2000. Clin. Microbiol. Rev. 13:16-34.
• Galanos et al., 2000. J. Endotoxin Res. 6:471-476.
• Gottlieb and Van Etten. 1966. J. Bacteriol. 91:161-168.
• Hagen et al., 2011. Curr. Oncol. 8:e109-e116.
• Higgins et al., 2007. Expert Rev. Vaccines 6:747-759.
29
• Hong et al., 2008. Nat. Prod. Rep. 25:447-454.
• Hughes and Erhardt. 2011. eLS. John Wiley & Sons, Ltd:
Chichester.
• Huleatt et al., 2007. Vaccine. 25:763-775.
• Hutchings et al., 2008. Trends in Microbiol. 17:13-21.
• Kimbrell et al., 2008. Immunol. Lett. 118:132-141.
• Krieg. 2002. Annu. Rev. Immunol. 20:709-760.
• Lorenz et al., 1991. Arch. Microbiol. 156:319-326.
• Lu and Yi, 2009. Annals of Microbiology. 59:453-458.
• Luo et al., 2001. J. Clin. Microbiol. 39:2971-2974.
• Müller and Löffler. 1992 Mykologie, 5. Aufl., Thieme Verlag,
Stuttgart
• Narita et al., 2004. Arch. Microbiol. 182:1-6.
• Neish. 2007. Am. J. Physiol. Gastrointest. Liver Physiol. 292:462-
466.
• Novak et al., 2003. J. Allergy Clin. Immunol. 112:215-216.
• Pickett et. al., 2007. European J. Neurologia. 14:E11.
• Pimbley and Patel. 1998. J. Appl. Microbiol. Biotechnol. 84:98-
109.
• Ramirez-Ortiz et al., 2008. Infection Immunity. 76:2123-2129.
• Rockel and Hartung. 2012. Frontiers in Pharmacology. 3:1-19.
• Schlegel, H. G. (1985), Allgemeine Mikrobiologie, 6. Aufl.,
Thieme Verlag, Stuttgart.
• Schromm et al., 2007. J. Biol. Chem. 282:11030-11037.
• Sorrell and Chen. 2009. Adv. Exp. Med. Biol. 666:108-20.
• Stouthamer. 1973. Antonie van Leeuwenhoek. 39:545-565.
• Turley et al., 2011. Vaccine. 29:5145-5152.
• Turner et al., 2009. Anal. Chimica Acta. 632:168-180.
• Wartenberg et al., 2011. Int. J. med. Microbiol. 301:602-611.
• Williams et al., 1988. Int. J. Immunopharmac. 10:405-414.
• Winstanley and Morgan. 1997. Microbiology. 143:3071-3084.