Microbiological Pharmaceutical Quality Control Labs

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GUIDE TO INSPECTIONS OF MICROBIOLOGICAL PHARMACEUTICAL
QUALITY CONTROL LABORATORIES
Note: This document is reference material for investigators and other FDA
personnel. The document does not bind FDA, and does not confer any rights,
privileges, benefits, or immunities for or on any person(s).
I. INTRODUCTION
 
The Guide to the Inspection of Pharmaceutical Quality Control Laboratories provided very
limited guidance on the matter of inspection of microbiological laboratories. While that guide
addresses many of the issues associated with the chemical aspect of laboratory analysis of
pharmaceuticals, this document will serve as a guide to the inspection of the microbiology
analytical process. As with any laboratory inspection, it is recommended that an analyst
(microbiologist) who is familiar with the tests being inspected participate in these
inspections.
 
II. MICROBIOLOGICAL TESTING OF NON-STERILE PRODUCTS
 
For a variety of reasons, we have seen a number of problems associated with the
microbiological contamination of topical drug products, nasal solutions and inhalation
products. The USP Microbiological Attributes Chapter <1111> provides little specific
guidance other than "The significance of microorganisms in non-sterile pharmaceutical
products should be evaluated in terms of the use of the product, the nature of the product,
and the potential hazard to the user." The USP recommends that certain categories be
routinely tested for total counts and specified indicator microbial contaminants. For example
natural plant, animal and some mineral products for Salmonella, oral liquids for E. Coli,
topicals for P. aeruginosa and S. Aureus, and articles intended for rectal, urethral, or vaginal
administration for yeasts and molds. A number of specific monographs also include
definitive microbial limits.
 
As a general guide for acceptable levels and types of microbiological contamination in
products, Dr. Dunnigan of the Bureau of Medicine of the FDA commented on the health
hazard. In 1970, he said that topical preparations contaminated with gram negative
organisms are a probable moderate to serious health hazard. Through the literature and
through our investigations, it has been shown that a variety of infections have been traced
to the gram negative contamination of topical products. The classical example being
the Pseudomonas cepacia contamination of Povidone Iodine products reported by a hospital
in Massachusetts several years ago.
 
Therefore, each company is expected to develop microbial specifications for their non-sterile
products. Likewise, the USP Microbial Limits Chapter <61> provides methodology for
selected indicator organisms, but not all objectionable organisms. For example, it is widely
recognized thatPseudomonas cepacia is objectionable if found in a topical product or nasal
solution in high numbers; yet, there are no test methods provided in the USP that will
enable the identification of the presence of this microorganism.
 
A relevant example of this problem is the recall of Metaproterenol Sulfate Inhalation
Solution. The USP XXII monograph requires no microbial testing for this product. The
agency classified this as a Class I recall because the product was contaminated
with Pseudomonas gladioli/cepacia. The health hazard evaluation commented that the risk
of pulmonary infection is especially serious and potentially life-threatening to patients with
chronic obstructive airway disease, cystic fibrosis, and immuno-compromised patients.
Additionally, these organisms would not have been identified by testing procedures
delineated in the general Microbial Limits section of the Compendia.
 
The USP currently provides for retests in the Microbial Limits section <61> however there is
a current proposal to remove the retest provision. As with any other test, the results of
initial test should be reviewed and investigated. Microbiological contamination is not evenly
dispersed throughout a lot or sample of product and finding a contaminant in one sample
and not in another does not discount the findings of the initial sample results. Retest results
should be reviewed and evaluated, and particular emphasis should be placed on the logic
and rationale for conducting the retest.
 
In order to isolate specific microbial contaminants, FDA laboratories, as well as many in the
industry, employ some type of enrichment media containing inactivators, such as Tween or
lecithin. This is essential to inactivate preservatives usually present in these types of
product and provides a better medium for damaged or slow growing cells. Other growth
parameters include a lower temperature and longer incubation time (at least 5 days) that
provide a better survival condition for damaged or slow-growing cells.
 
For example, FDA laboratories use the test procedures for cosmetics in the Bacteriological
Analytical Manual (BAM), 6th Edition, to identify contamination in non-sterile drug products.
This testing includes an enrichment of a sample in modified letheen broth. After incubation,
further identification is carried out on Blood Agar Plates and MacConkey Agar Plates.
Isolated colonies are then identified. This procedure allows FDA microbiologists to optimize
the recovery of all potential pathogens and to quantitate and speciate all recovered
organisms. Another important aspect of procedures used by FDA analysts is to determine
growth promotion characteristics for all of the media used.
 
The selection of the appropriate neutralizing agents are largely dependent upon the
preservative and formulation of the product under evaluation. If there is growth in the
enrichment broth, transfer to more selective agar media or suitable enrichment agar may be
necessary for subsequent identification.
 
Microbiological testing may include an identification of colonies found during the Total
Aerobic Plate Count test. Again, the identification should not merely be limited to the USP
indicator organisms.
 
The importance of identifying all isolates from either or both Total Plate Count testing and
enrichment testing will depend upon the product and its intended use. Obviously, if an oral
solid dosage form such as a tablet is tested, it may be acceptable to identify isolates when
testing shows high levels. However, for other products such as topicals, inhalants or nasal
solutions where there is a major concern for microbiological contamination, isolates from
plate counts, as well as enrichment testing, should be identified.
 
III. FACILITIES, EQUIPMENT, AND
 
MEDIA
 
Begin the inspection with a review of analyses being conducted and inspect the plates and
tubes of media being incubated (caution should be exercised not to inadvertently
contaminate plates or tubes of media on test). Be particularly alert for retests that have not
been documented and "special projects" in which investigations of contamination problems
have been identified. This can be evaluated by reviewing the ongoing analyses (product or
environmental) for positive test results. Request to review the previous day's plates and
media, if available and compare your observations to the recorded entries in the logs.
Inspect the autoclaves used for the sterilization of media. Autoclaves may lack the ability to
displace steam with sterile filtered air. For sealed bottles of media, this would not present a
problem. However, for non-sealed bottles or flasks of media, non-sterile air has led to the
contamination of media. In addition, autoclaving less than the required time will also allow
media associated contaminants to grow and cause a false positive result. These problems
may be more prevalent in laboratories with a heavy workload.
 
Check the temperature of the autoclave since overheating can denature and even char
necessary nutrients. This allows for a less than optimal recovery of already stressed
microorganisms. The obvious problem with potential false positives is the inability to
differentiate between inadvertent medium contamination and true contamination directly
associated with the sample tested.
 
IV. STERILITY TESTING
 
On 10/11/91, the Agency published a proposed rule regarding the manufacture of drug
products by aseptic processing and terminal sterilization. A list of contaminated or
potentially contaminated drug products made by aseptic processing and later recalled was
also made available. Many of the investigations/inspections of the recalled products started
with a list of initial sterility test failures. FDA review of the manufacturer's production,
controls, investigations and their inadequacies, coupled with the evidence of product failure
(initial sterility test failure) ultimately led to the action.
 
The USP points out that the facilities used to conduct sterility tests should be similar to
those used for manufacturing product. The USP states, "The facility for sterility testing
should be such as to offer no greater a microbial challenge to the articles being tested than
that of an aseptic processing production facility". Proper design would, therefore, include a
gowning area and pass-through airlock. Environmental monitoring and gowning should be
equivalent to that used for manufacturing product.
 
Since a number of product and media manipulations are involved in conducting a sterility
test, it is recommended that the inspection include actual observation of the sterility test
even though some companies have tried to discourage inspection on the grounds that it
may make the firm's analyst nervous. The inspection team is expected to be sensitive to
this concern and make the observations in a manner that will create the least amount of
disruption in the normal operating environment. Nevertheless, such concerns are not
sufficient cause for you to suspend this portion of the inspection.
 
One of the most important aspects of the inspection of a sterility analytical program is to
review records of initial positive sterility test results. Request lists of test failures to facilitate
review of production and control records and investigation reports. Particularly, for the high
risk aseptically filled product, initial positive sterility test results and investigations should
be reviewed. It is difficult for the manufacturer to justify the release of a product filled
aseptically that fails an initial sterility test without identifying specific problems associated
with the controls used for the sterility test.
 
Examine the use of negative controls. They are particularly important to a high quality
sterility test. Good practice for such testing includes the use of known terminally sterilized
or irradiated samples as a system control. Alternatively, vials or ampules filled during media
fills have also been used.
 
Be especially concerned about the case where a manufacturer of aseptically filled products
has never found an initial positive sterility test. While such situations may occur, they are
rare. In one case, a manufacturer's records showed that they had never found a positive
result; their records had been falsified. Also, the absence of initial positives may indicate
that the test has not been validated to demonstrate that there is no carryover of inhibition
from the product or preservative.
 
Inspect robotic systems or isolation technology, such as La Calhene units used for sterility
testing. These units allow product withdrawal in the absence of people. If an initial test
failure is noted in a sample tested in such a system, it could be very difficult to justify
release based on a retest, particularly if test controls are negative.
 
Evaluate the time period used for sterility test sample incubation. This issue has been
recently clarified. The USP states that samples are to be incubated for at least 7 days, and a
proposal has been made to change the USP to require a period of 14 days incubation. You
are expected to evaluate the specific analytical procedure and the product for the proper
incubation period. Seven days may be insufficient, particularly when slow growing
organisms have been identified. Media fill, environmental, sterility test results and other
data should be reviewed to assure the absence of slow growing organisms. Also, you should
compare the methods being used for incubation to determine if they conform to those listed
in approved or pending applications.
 
V. METHODOLOGY AND
 
VALIDATION OF TEST
 
PROCEDURES
 
Determine the source of test procedures. Manufacturers derive test procedures from several
sources, including the USP, BAM and other microbiological references. It would be virtually
impossible to completely validate test procedures for every organism that may be
objectionable. However, it is a good practice to assure that inhibitory substances in samples
are neutralized.
 
During inspections, including pre-approval inspections, evaluate the methodology for
microbiological testing. For example, we expect test methods to identify the presence of
organisms such as Pseudomonas cepacia or other Pseudomonas species that may be
objectional or present a hazard to the user. Where pre-approval inspections are being
conducted, compare the method being used against the one submitted in the application.
Also verify that the laboratory has the equipment necessary to perform the tests and that
the equipment was available and in good operating condition on the dates of critical testing.
 
The USP states that an alternate method may be substituted for compendial tests, provided
it has been properly validated as giving equivalent or better results.
 
You may find that dehydrated media are being used for the preparation of media. Good
practice includes the periodic challenge of prepared media with low levels of organisms. This
includes USP indicator organisms as well as normal flora. The capability of the media to
promote the growth of organisms may be affected by the media preparation process,
sterilization (overheating) and storage. These represent important considerations in any
inspection and in the good management of a microbiology laboratory.
 
VI. DATA STORAGE
 
Evaluate the test results that have been entered in either logbooks or on loose analytical
sheets. While some manufacturers may be reluctant to provide tabulations, summaries, or
printouts of microbiological test results, this data should be reviewed for the identification of
potential microbial problems in processing. When summaries of this data are not available
the inspection team is expected to review enough data to construct their own summary of
the laboratory test results and quality control program.
 
Some laboratories utilize preprinted forms only for recording test data. Some laboratories
have also pointed out that the only way microbiological test data could be reviewed during
inspections would be to review individual batch records. However, in most cases, preprinted
forms are in multiple copies with a second or third copy in a central file. Some companies
use log-books for recording data. These logbooks should also be reviewed.
 
Additionally, many manufacturers are equipped with an automated microbial system for the
identification of microorganisms. Logs of such testing, along with the identification of the
source of the sample, are also of value in the identification of potential microbial problems
in processing.
 
The utilization of automated systems for the identification of microorganisms is relatively
common in the parenteral manufacturer where isolates from the environment, water
systems, validation and people are routinely identified.
 
Microbiologists in our Baltimore District are expert on the use of automated microbic
analytical systems. They were the first FDA laboratory to use such equipment and have
considerable experience in validating methods for these pieces of equipment. Contact the
Baltimore District laboratory for information or questions about these systems. Plants with
heavy utilization of these pieces of equipment should be inspected by individuals from the
Baltimore District laboratory.
 
VII. MANAGEMENT REVIEW
 
Microbiological test results represent one of the more difficult areas for the evaluation and
interpretation of data. These evaluations require extensive training and experience in
microbiology. Understanding the methodology, and more importantly, understanding the
limitations of the test present the more difficult issues. For example, a manufacturer found
high counts of Enterobacter cloacae in their oral dosage form product derived from a natural
substance. Since they did not isolate E. coli, they released the product. FDA analysis
found E.cloacae in most samples from the batch and even E. coli in one sample. In this case
management failed to recognize that microbiological contamination might not be uniform,
that other organisms may mask the presence of certain organisms when identification
procedures are performed, and that microbiological testing is far from absolute. The
inspection must consider the relationship between the organisms found in the samples and
the potential for the existence of other objectionable conditions. For example, it is logical to
assume that if the process would allow E.cloacae to be present, it could also allow the
presence of the objectionable indicator organism. The microbiologist should evaluate this
potential by considering such factors as methodology, and the growth conditions of the
sample as well as other fundamental factors associated with microbiological analysis.
 
Evaluate management's program to audit the quality of the laboratory work performed by
outside contractors.
 
VIII. CONTRACT TESTING
 
LABORATORIES
 
Many manufacturers contract with private or independent testing laboratories to analyze
their products. Since, these laboratories will conduct only the tests that the manufacturer
requests, determine the specific instructions given to the contractor. Evaluate these
instructions to assure that necessary testing will be completed. For example, in a recent
inspection of a topical manufacturer, total plate count and testing for the USP indicator
organisms were requested. The control laboratory performed this testing only and did not
look for other organisms that would be objectionable based on the product's intended use.
 
Analytical results, particularly for those articles in which additional or retesting is conducted,
should be reviewed. Test reports should be provided to the manufacturer for tests
conducted. It is not unusual to see contract laboratories fail to provide complete results,
with both failing as well as passing results.
 
Bacteriostasis/fungiostasis testing must be performed either by the contract lab or the
manufacturer. These test results must be negative otherwise any sterility test results
obtained by the contractor on the product may not be valid.