FDA QC微生物实验室检查指南 英文原版

GUIDE TO INSPECTIONS OF MICROBIOLOGICAL

CONTROL

PHARMACEUTICAL LABORATORIES

QUALITY

Note: This document is reference material for investigators and other FDA

personnel. The document does not bind FDA, and does no 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>

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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

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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 that

Pseudomonas 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

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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

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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

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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.

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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

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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

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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.

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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

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

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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

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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

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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, such as a Vitek, for the identification of microorganisms. Logs of

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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

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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

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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.

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There are no references from this document.