Auditing the Microbiology Laboratory

Introduction

This training will provide a basic understanding of an audit of a typical Microbiology Laboratory. We have divided this training into two sections:

I.

Major Areas of Audit in the Micro Lab

II. Useful, Often Relevant Audit Questions

I. Major Areas of Audit in the Microbiology Laboratory

As a QC lab, the Micro dept. must comply with the same basic GMP regulations promulgated for the Analytical Chemistry Lab.
With this in mind, we have adopted the standard 8 major areas in a lab audit described ~ a year ago in GQAC training on Auditing the Analytical Chem Lab.
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Major areas of audit in the microbiological laboratory

As a reminder, the major areas of audit in a QC lab are: 1. 2. 3. 4. 5. 6. 7. 8. Sample Control Employee Training Reference Standards Equipment and Instruments Reagents and Media Recordkeeping and Documentation Laboratory Controls Method Validation

1. Sample Control

Just like with samples coming into the Analytical Chem Lab, samples coming into the Micro Lab:

Must have sample records that show chain-ofcustody. Records can be manual, e.g., forms or logbooks, or electronic, e.g., LIMS (laboratory information management system). Must be stored in an area that maintains their quality. This includes areas that are properly identified, clean and orderly, and is adequate to prevent mix-up and contamination from other samples, from chemicals and reagents, and from spillage.
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Sample Control Audit Questions

1. 2.

Ask how Micro samples are logged in and stored.

Does the sample log book (or other record) provide spaces for who delivered the sample and who then took it for testing (chain of custody)?
What type of samples might be temporarily stored while awaiting testing? What method validation or compendial ref supports the sample storage conditions (e.g., water)? What site SOP governs what happens when a water or an EM sample time point is missed? (should be a deviation). If LIMS is used for tracking all samples and activities, check if pen raw data precedes computer and whether the former is properly retained.
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3.

4.

5.

6.

(Water) Sample Control Audit Questions

1. 2.

Ask how water samples are drawn; compare to site SOP. Is the chemistry sample collected before the Micro (i.e., if a spray disinfectant is used on the sample port)? If any sampling sites are also points of use (POU) for Mfg, how comparable are the Micro sampling and Mfg cleaning/rinsing/use of the same POUs? Does the sample record contain time sample taken, samplers name, sample site, time sample delivered to Lab? Are water-sampling bottles reused with a validated sterilization cycle and a proper storage/protection in between? Is the expiration date on those stored water sampling bottles supported by a study (or any data)? If there is a maximum number of hours that may elapse between sampling and bacterial enumeration testing, is the actual time or hour of sampling recorded along with the date?
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3.

4.

5.

6.

7.

(Water) Sample Control Audit Questions

8.

Ask for the most recent enumeration data of a specific critical water POU (should contain or refer to date/time sample taken, date/time plated and put in incubator & date/time out of incubator + incubator ID+ lot # of media used). What media is used to test water for counts? (If that water goes into a European-marketed product, then R2A Media). Ask to see growth promotion testing of the media (some companies require a water system isolate to be included). When is a colony identified? Ask for the SOP covering this decision (should be obtainable from within the Lab itself workers need proximity to their SOPs). Ask to see total viable count & the organisms identified in the WFI loop (for all POUs) for past 18 mos.

9.

10.

11.

12.

2. Employee Training

Employee training includes the basics needed for every pharmaceutical employee, i.e., a combination of education, training, and experience to do his or her job. The core educational background of the microbiology staff must be in microbiology or a closely related biological science. cGMP training and task training are provided before performing the task, and with experience comes more complex testing, and specific organism identifications.

In the Microbiology Lab, much of the task training is by Technique as opposed to being by Product.
The training program should include an established training curricula specific to job functions, and periodic performance assessments to verify ongoing competency in core activities, e.g., hygiene, plating, aseptic technique, handling test failures.

Analyst Training Audit Questions

1. 2.

Ask to see the Labs org. chart. Has cross-training created sufficient designated back-ups for critical activities in the Lab (or even for reviewing analysts results)? Ask to see the training curricula for each unique Lab position. Determine the most recent effective date for a change to a Micro SOP, then examine training records for updated training on the new revision. Ask to see the Microbiology OOS procedure. Ask to see the OOSs in Micro for the past 18 mos. Can patterns be seen in tests or analysts associated w/ OOSs, and if so, what CAPAs have followed?
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3.

4.

5. 6. 7.

3. Reference Standards

The microbiology laboratory shares the same requirements that apply to the Analytical laboratory; it must also ascertain that the reference standards used are of the highest quality for the required tests Noteworthy standard requirements include those for:

- Standards which need to be frozen at extremely low temperatures, including microbiological cultures. Check procedure to handle these standards (e.g., use of liq N2). - The USP Endotoxin RSE has a defined potency of 10,000 USP Endotoxin Units (EU) per vial. The first dilution of the concentrate may not be stored in a refrigerator for more than 14 days for use in subsequent dilutions. Further dilutions may not be stored w/o supporting data (risk of lost activity via adsorption to container)

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3. Reference Standards

Biological Indicator (BI): is a challenge organism, usually as a spore, which can be characterized by its population and D-value. The D-value is the number of minutes (at defined conditions of steam, gas, or radiation) required to achieve a 1 log reduction on the total number of defined organisms. Each BI is tested by the Micro lab or an approved contract lab to confirm the vendors labeled bacterial population and, less frequently, D-value. BIs are placed in selected locations in sterilizing equipment during respective qualifications. To pass qualification, the sterilizer must show a 106 reduction in number of organisms after exposure at worst case (less time than in routine operations) and at prescribed pressure, BI spore population and resistance (D-value). See later section on audit questions, which explains importance of D-value.

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3. Reference Standards

Microbial cultures are delicate standards. Procedures should specify careful handling instructions. Preparation and resuscitation of cultures should follow the instructions of the supplier or a validated, established method. USP recommends using the "SeedLot" technique for storage of stock cultures, i.e., using working cultures and never returning unused passages back to original stock. In addition, there should be an established maximum number of passages (5 or less), and maximum storage time for working cultures. Cultures for use in compendial tests should be acquired from a national culture collection, in frozen, freeze-dried, on slants, or in ready-to-use forms. Confirmation of the purity and the identity should be performed prior to its use in quality control testing. Ready-to-use cultures may require additional confirmation of inoculum size.

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Biological Indicator Audit Questions

1.

What BIs are purchased by the site?

2.
3.

Ask to see the incoming QC test SOPs for all of them.

Is the bacterial population and purity confirmed for every supplier lot of BI?

4.

Is the D-value >= 1.5 min for 121oC steam sterilization? (required by AAMI, ISO, and USP <1035>)
Has the D-value claimed by the supplier ever been verified by a qualified lab with a BIER (biological indicator evaluator resistometer) Vessel per ANSI/AAMI ST44 or USP <55>? If no, ask how the site knows D >= 1.5 min
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5.

Biological Indicator Audit Questions (contd)

6. If accepted on certificate of analysis, what BI supplier quality assurance has been established? Ask to see file. Supplier should be approved. Read the BI vendors use instructions, then look for data on elapsed time between cycle exposure and incubation.

7.

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Purchased Cell Cultures

1.

When purchasing microorganisms from a national culture collection, what incoming QC tests are run for identity and purity? Is the ID done via genotypic analysis? How are the # of passages of working cultures tracked, and what is the maximum # permitted (PIC/S says NMT 5 passages for cutures used in pos. ctls of sterility tests)? Ask to see the records of subculturing a purchased organism. How long can a working culture be used (<= 1 wk)?

2.

3.

4.

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4. Equipment and Instruments

The same Analytical Chem Lab equipment and instrument calibration, maintenance, and qualification requirements apply to the Micro Lab. There, the most common equipment are incubators, autoclaves, refrigerators, water baths, laminar flow hoods, and automatic pipettors. Often overlooked, the glassware washer is critical in the Micro Lab. Prepn of media in poorly cleaned glassware can allow inhibitory substances to enter the media from residual cleaning detergent or from matls previously used. The washer must be qualified.
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4. Equipment and Instruments

Storage equipment the Micro Lab uses a number of storage equipment that require qualification: incubators, refrigerators, freezers, thermostated water baths, etc. Some inspectors expect thermostated water baths to have undergone OQ of the temperature consistency throughout bath. Qualifications are conducted through temperature mapping, and the number of locations depend on size. Small incubators and refrigerators would not require mapping qualification, but instead are verified with a calibrated thermometer during use. After initial qualifications, critical storage equipment are monitored through daily verifications. Temperature mapping is periodically repeated, to confirm uniformity and monitoring probe placements at worst case, hot and cold, locations. We have found that some labs fall short on requirements, e.g., 2-8 C range is monitored for cold rooms even though some stored matls need range of 2-6 C.
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4. Equipment and Instruments

If monitoring occurs via review of charts from equipment chart recorder, then challenge this review process. Verify that charts from a given time-period, typically 6 months, are appropriately reviewed, and deviations, including temporary spikes, are explained/justified or defined within the equipment SOP as an allowable excursion. In addition to mapping and routine monitoring, other controls are needed for critical storage equipment:

Procedure to capture extended temperature excursion and to assess impact on the contents. Probe and recorder calibrations, challenge of temp. alarms (all the way to the monitoring site or external company), and equipment PM. A disaster recovery plan.

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4. Equipment and Instruments

The autoclave is specialized equipment with unique requirements. When used to sterilize media, the growthpromotability of the media is part of the acceptance criteria in addition to sterility. The cycle in the autoclave/sterilizer must be validated to ensure proper heat distribution for selected media loads and volumes. Typically, manufacturers recommend using an autoclave cycle of 121 C for 15 minutes, which apply to time at temperature of the media. The sterilization time is dependent on the media volume and autoclave load. Sterilization cycles in which the autoclave is slow to come up to temperature may result in overheating of the media. Therefore, care must be taken to balance the need for a sterile media against the tendency of the media to degrade under excessive heating.
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4. Equipment and Instruments

Storage of the media in the autoclave after the liquid cycle is completed is not recommended after cooling, as it may damage the media. Improper heating or sterilizing conditionsfor commercially prepared or internally prepared mediamay result in a difference in color change, loss of clarity, altered gel strength, or pH drift from the manufacturer's recommended range. Autoclaves used for media sterilization may lack the ability to displace steam with sterile filtered air (for sealed bottles of media, this would not present a problem). For non-sealed bottles or flasks of media, non-sterile air has led to the contamination of media. Autoclaving less than the required time will also allow mediaassociated 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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4. Equipment and Instruments

The most common micro instruments are the microscopes, balances, pH meters, thermometers, spectrophotometers. Lab instruments should be calibrated on a regular schedule and verified routinely. Review pH records, we originally found very few Micro Labs standardizing pH correctly. From the simplified major tests introduced in earlier sections, pH is critical for their results. Other unique instrument/equipment calibration requirements in the Micro Lab include calibration of the lens vernier scale in the microscope and the air sampler unit that is used for environmental air sampling. Micro must ensure tight environment control when conducting Sterility tests.
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4. Equipment and Instruments

Computerized Laboratory computer-controlled systems may be used, e.g., to control and maintain environmental data. As with the QC Chemistry Lab, the microbiology lab must have a policy up front on what constitutes the instrument generated raw data the printed or the electronic data. The FDA basic guidance on security and authenticity issues, to address data integrity concerns, apply here. There must be a provision so that only authorized personnel can make data entries. Data entries may not be deleted; changes may be captured by the audit trail or by a manual process.

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4. Equipment and Instruments

One of the most common computer systems found in a Micro lab is the automated system for the genus and species 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 use of automated systems for the identification of microorganisms is relatively common in parenteral manufacturing where isolates from the environment, water systems, validation, and people are routinely identified.

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Environmentally-Controlled Chambers/Rooms Audit Questions

1. . 2.

Show how refrigerators, freezers, & liquid N2 tanks are monitored. Do these have a logbook or form for contents and is it accurate and timely updated? What, if any, disaster recovery plan exists for some of these environmentally-controlled rooms/chambers? What actions would be taken if a large walk-in refrigerator or freezer failed? What procedure exists to capture an extended temperature excursion and to assess impact on contents of affected chamber? Does that procedure allow for a 10 min (or similar) temp. excursion due to door opening? Ask to see the most recent calibration record and SOP of the temperature probe for a refrigerator (does it have an equipment #. If not, why not?). Ask to see the last PM report and its SOP (check if theres a test for the alarm) for the refrigerator, freezer, incubators, stability chamber
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3.

4.

5.

6.

7.

Env-Controlled Chambers/Rooms Audit Questions

8. 9.

Are multi-shelved incubators temp.-mapped? Ask to see temp.-mapping studies for one or some of the above chambers. If no mapping ever done, are there temp. probes for monitoring multiple locations? Ask to see the site SOP(s) for qualifying and monitoring environmentally-controlled chambers. For one of the chambers or incubators that has a chart recorder, ask for last month of archived charts. Do the charts have initials/signature and date for its installation and for its removal and review?

10.

11.

12.

13.

If the monitoring and alarm system for all or some of the chambers is computerized, ask to see its computerized system validation protocol and executed protocol.

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5. Reagents and Media

Reagents must be controlled the same way as they are controlled in the Analytical Chem Lab.

Reagent and media preparation must be traceable, preferably documented in a laboratory notebook. The minimum information required for traceability:
Dates Type and lot of material used Ingredient quantities Preparers identification

In addition, the method of reagent or media preparation is detailed/referenced.

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5. Reagents and Media

Due to the variety and number of containers produced, frequency of use, and criticality, media may require a greater level of control than ordinary reagents. Verify that containers of media are properly identified to prevent against mix-up, and are properly segregated by status (release or quarantine) and for use based on expiration date (FEFO).

The capability of the media to promote the growth of organisms may be affected by the media preparation process, sterilization (overheating), and storage.
Review analyses being conducted and inspect the plates and tubes of media being incubated.

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5. Reagents and Media

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.

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5. Reagents and Media

The LAL reagent is critical in Endotoxin testing, and must be well controlled. When the LAL reagent (lot) changes, the Micro Lab must confirm the labeled LAL reagent sensitivity (EU/ml). This is to re-calculate the Maximum Valid Dilution (MVD), which is the maximum allowable dilution of a sample to determine the endotoxin limit. MVD = (Endotoxin limit Concentration of sample soln)/ where concentration of sample in solution is known either from product testing in Anal Chem or, for a raw matl, by a precisely weighed sample and dissolution, and the endotoxin limit concentration is specified in the individual monograph (in EU per mg).
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5. Reagents and Media

Sanitizers are reagents that also need to be well controlled in the Micro Lab. There should be a lab sanitization and sanitizer preparation SOP. SOPs should specify the grade and temperature of water used to prepare sanitizing solution, method of applying sanitizer, contact time, post-contact drying method and time. Sanitizers should be qualified (effectiveness testing) with intended application quantity and contact time as well as surface. Data should be available to support expiration period of the sanitizer at its use dilution.

Data should also show that a non-sporocidal sanitizer working solution remains sterile through expiration (called monitoring for a Grade A or B area.

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Sanitizer Preparation & Use

1.

What sanitizers are used in the Micro Lab? Ask to see Micro Lab sanitization SOP and sanitizer preparation SOP. Is each lot of sanitizer released by the site QC? Does sanitization SOP specify: a) type & temp. of water used to prepare sanitizing solution; b) the method of applying sanitizer; c) contact time; d) post-contact drying method & time? Are all Micro Lab sanitizers purchased sterile or rendered sterile (if to be used in Grade A or B areas)? What qualification (i.e., sanitizer effectiveness testing) exists for each Micro Lab sanitizer and do they result in prescribed application quantity & contact time? What records show that specified contact time is achieved? What data supports exp period of the sanitizer at its use dilution? What evidence shows that a non-sporocidal sanitizer working solution, or even hand antiseptic sprays and wipes, remain sterile through expiration, and are they monitored?
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2. 3.

4.

5.

6. 7.

8.

Media Preparation, Testing & Storage Audit Questions

1.

Ask to see the most recent settle plate (e.g., TSA) media growth promotion record and its SOP.

2.
3. 4.

Is a relevant, local air isolate among the organisms tested?

What media is prepared by the Lab? Do the following appear in the media growth promotion record: a) confirmed temp., b) time-into and time-out of the incubator, c) incubator ID, d) control (lot) no. of the organism(s) plated? Does a USP-governed site trend the results of all media growth promotions lot-to-lot? (new <61> requirement). Do media preparation records refer to the autoclave cycle used for media sterilized by Micro?

5.

6.

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Media Preparation, Testing & Storage Media Preparation, Testing & Audit Questions Storage Audit Questions (contd)
7.

Is the prepared media sufficiently labeled for traceability to the starting matls, date, and analyst? Was the autoclave cycle and the expiration dating period of in-house-prepared media validated (e.g., w/ growth-promotion testing through expiration)? Check several in-house media. When the autoclave is used to sterilize media, is a printout of cycle parameters printed and attached to a media preparation worksheet or notebook page? Ask to see SOP(s) with description, picture, or both of validated media load configurations in the autoclave. What is the frequency of autoclave cycle revalidation (annual)?

8.

9.

10. 11.

When media pH is checked after autoclaving and cool-down, how is this done (should be a completely aseptic technique)?
Does analyst 100% check the plates or tubes of in-house-prepared media for a) unequal filling, b) dehydration, c) excessive bubbles?
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12.

Media Preparation, Testing Media Preparation, Testing && Storage Audit Questions Storage Audit Questions (contd)
13. Are all media stored according to mfr instructions and protected against dehydration?

14.

Are in-house-prepared media slated for EM of critical areas (e.g., Grade A) double-wrapped and terminally sterilized (or preincubated & inspected for contamination)?
How does the Micro Lab clean its glassware and/or procure media plates so that no inhibitory residue could get into prepared media? How does the Micro Lab inventory its various media and ensure disposal at expiration? Ask to see the growth promotion testing SOP for both purchased media and in-house-prepared media. For environmental monitoring media, is the media on quarantine pending the acceptable results of growth promotion? This is also S-P policy with regards to water-testing media.
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15.

16. 17. 18.

6.

Recordkeeping and Documentation

Proper recordkeeping is critical for the Micro Lab. A test should be performed as per SOP, and the laboratory notebook should provide a record of all critical details needed to confirm the integrity of the data. At a minimum, the laboratory write-up should include the following: Date Material tested Microbiologist's name (unless a personal bound notebook) Procedure number Test results and any logical operators fully defined (e.g., <) or explained Deviations (if any) Documented parameters (equipment used, microbial stock culture nos. used, media or reagent lot nos. used) Management/Second review signature

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6.

Recordkeeping and Documentation

Proper recordkeeping includes good documentation practices. Every critical piece of equipment is noted. Equipment temperatures (water baths, incubators, autoclaves) are recorded and traceable. The governing test method and revision are clearly noted.

Logbooks or forms are available and supportive of the laboratory notebook records. Test results include the original plate counts, which constitute the actual raw data and are used for the calculations for final test results. Methods for data analysis are detailed in SOPs.
All laboratory records are archived and protected against catastrophic loss. A formal record retention and retrieval program is in place, practiced at the local level.
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6.

Recordkeeping and Documentation

Documentation in the microbiology laboratory includes procedures and test methods, work instructions (i.e., calibration and maintenance), protocols, guidelines, manuals, etc. If used for the operation of the lab and are critical for validity of results, they must be approved by the Quality Unit. Furthermore, assurance must exist that testing histories are accurate and complete by having a defined system for issuance, monitoring, and reconciliation of worksheets printed/used (i.e., an audit trail. When we introduced Micro test methods, there were only three major tests. Then why do we need product specific test methods? Test methods capture unique sample requirements, e.g., validated preparatory steps prior to moving into the common steps. SOP must reflect actual practices and test methods and must be in conformance with application commitments and/or compendium requirements.
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6.

Recordkeeping and Documentation

For compendial test methods, there must be a process in place to ensure that changes are captured timely so that they may be implemented when the changes go into effect. There should be a

process in place to evaluate, comment and resolve issues related to proposed changes to the compendium before revised monographs become final.

Documentation is sufficient to show that testing was done in a laboratory and by methods that were under control. Equipment performance during test (e.g., 24-hour/7-day chart recorders) Media preparation, sterility checks, and growth-promotion and selectivity capabilities Media inventory and control testing Critical components of test conducted as specified by SOP Data and calculations verified Reports reviewed by site quality unit or qualified responsible mgr Investigation of data deviations, if any

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7. Laboratory Controls

The management ensures that the laboratory is operating in a state of control Through review of key metrics, e.g., lab related [major] deviations and OOSs due to analyst or instrument error, stability testing performed as scheduled, etc. Through self-inspection -- immediate correction of high risk issues and continuous improvement in all areas where gaps are identified Changes in the laboratory should be approved by an independent quality unit and as per a formalized change control process
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7. Laboratory Controls

Sterility Testing: the regulatory requirements follow from the USP statement, "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." This includes environmental monitoring and gowning equivalent to that used for manufacturing product, e.g., gowning area, pass-through airlock. (see sterility test audit questions section). Review records of initial positive sterility test results, particularly for all high risk aseptically filled products. Firms have difficulty justifying release of a product filled aseptically that fails an initial sterility test, without having identified specific problems associated with the controls used for the sterility test.

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7. Laboratory Controls

When the manufacturer has never found an initial positive sterility test, confirm that the absence of initial positives is not due to inappropriate control on retesting, or lack of validation to demonstrate that there is no carryover of inhibition from the product or preservative. Evaluate the time period used for sterility test sample incubation. As per USP, samples are to be incubated for at least 14 days at 2 sets of incubation temperatures.

Media fill, environmental, sterility test results and other data should be reviewed to assure the absence of slow growing organisms. Compare the methods being used for incubation to determine if they conform to those listed in approved or pending applications.

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7. Laboratory Controls

Laboratory Operations/Housekeeping In the Micro Lab, areas where EM, water, or product samples are handled/incubated must be adequately separated from areas where there are tests that involve live cultures or subculturing, microbial ID, or investigations. This is found in <1117> Best Micro Lab Practices, and recently enforced by FDA at an multinational pharma biologics API mfg site.

The Micro Lab should practice aseptic techniques during testing in general, to avoid microbial contamination and false positives.
Housekeeping must be properly maintained to prevent use of expired or contaminated testing mats. Verify cleanliness of work stations, cleared of extraneous or previous test matls, prompt removal of refuse, and clean utensils and equipment.

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7. Laboratory Controls

Stability the stability program is likely managed by the analytical laboratory or by others with only the testing performed by the Micro laboratory Often, stability Micro testing is missed or is late because of poor adherence to the stability protocol, specially for a test that is infrequent and is not included in finished product test monographs, e.g., MLT at defined intervals and shelflife expiry. The analytical laboratory may conduct only the tests specified in the finished product test monographs, without the Micro laboratory knowing that it missed required testing.

Therefore, review controls around infrequent Micro testing.

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Environmental Monitoring (EM)

1.

Ask to see the mfg suite active air and passive air viable monitoring SOPs (either classified, unclassified, or both). Is the description of where settle plates or hand-held active air samplers sufficiently specific (e.g., supplemented w/ exactly mapped sampling locations inside the suite(s)). If its possible to observe a surface EM sampling, a) see when a disinfectant spray is applied to sampling location (should only be done immediately afterward). b) check what disinfectant or cleaner was used, whether its within expiry and whether it was ever validated (mainly for a disinfectant). For settle plate monitoring, check the procedures exposure time versus actual practice. Also, If a laminar flow hood is monitored on an EM program, does Micro use a side-by-side pos. control plate or did it validate exposure time (to guarantee against agar desiccation)?

2.

3.

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Environmental Monitoring (contd)

4.

Ask whether settle plate exposure times have ever gotten shorter in the procedure(s). If they have, did the specs change downward as well? For classified suites, ask to see EM trends as well as how alert and action limits were initially calculated according to a procedure, and where these limits are officially recorded/stated.

5.

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Microbiological Isolates
1.

Explain how local manufacturing area air, non-productcontact surface, gown/glove, and water microbiological isolates are obtained and cultured? Ask to see the library list of environmental isolates. Is there an SOP on the culturing, subculturing, and librarying of local environmental isolates? Ask to see. Does the procedure explain when the library is updated or revised with new organisms (especially for growth promotion tests)? When sampling from non-product-contact surfaces, are inactivators such as Tween or lecithin used/ formulated within certain media (because of residual disinfectant)? What data can demonstrate recovery from a non-productcontact surface of a spiked test organism, using the above-mentioned inactivator in the media?
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2. 3.

4.

5.

6.

Unknown Organism ID Testing

1. 2.

What method(s) are used for microbiological isolate ID? Does Micro use genotype (i.e., DNA) ID for sterility test failures? Have the ID methods been developed/validated here or tech transferred to this Micro Lab from another company site? If fatty acid analysis via gas chromatography is used, describe the procedures for GC system suitability and calibration. Is a database used for recording and trending organism ID results? If yes, ask to see this demonstrated. Is the database validated plus Part 11- compliant? What viable particle EM is performed in the Micro Labs?

3.

4.

5.

6. 7.

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Unknown Organism ID Testing (contd)

8.

Ask to see the trends of identified organisms in the air and on the surfaces of Micro Lab & inside sterility test suite or isolator.

9.

How is trending performed. For example, are all points in a room averaged to get a result?
When do water testing counts require ID and when is a water microorganism objectionable and/or requiring full speciation? See Attachment 3, WGD 10,509 Evaluation of Microbial Isolates Found in Water or Steam Systems

10.

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Physical Micro Lab Audit Questions

1. 2.

How is the cleanliness/neatness of benches & hoods? Examine purchased reagents for exp dates (e.g., LAL CSE, Gram Stain reagents). Examine site-prepared media or reagents labeling of expiration date and internal lot number. Does Micro maintain an inventory list (w/ exp dates) of all media and other reagents. For an instrument calibrated by a contract Lab, examine a recent calibration report. Has a site mgr or supervisor signed the report to signify having reviewed it?

3.

4.

5.

6.

Does site calibrate all mechanical pipets, including maxitip pipets, repipets, and repeat dispensers (i.e., hand pump)?
Where are malfunctioning instruments/equipment stored? How are they Labeled?
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7.

Physical Micro Lab Audit Questions (contd)

8.
9.

Does the Micro Lab or other qualified site persons conduct some form of self inspection of the Micro Lab operations (EU required)? Ask if any equipment is undergoing qualification or is part of method validation, is it labeled out of service or validation in progress? Are areas in which environmental or product samples are handled/incubated adequately separated from live cultures/subculturing, microbial ID/staining, or investigations? How are staff separated (or processed as in regowning and hand scrubbing) based on whether they routinely work in sampling versus live culture areas?

10.

11.

12.

Ask to see the suite where API or excipient sampling is done for MLT. Are aseptic sampling practices and room design evident? Ask to see the aseptic sampling suite cleaning log book as well as the log book for recording HEPA start-up time, monitored pressure differentials, temp., etc.
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Physical Micro Lab Audit Questions (contd)

13.

What is the Micro Labs source of water for preparing media & other reagents? Does the lab use a Mfg area WFI or USP Purified Water drop or is the lab water a different purification system from Mfg? Does Micro Lab uses a water storage vessel to hold drawn water from some Mfg water drop outside the lab? If yes to #14, does Micro label it or claim it as WFI in the tank? Does Micro monitor their water source (even from a holding tank) just like Mfg POUs? Are any uncontrolled instructions or copies of compendia, etc. taped to the wall or the benches? Is the expiration dating of sterile utensils, sample containers, etc. supported by sterility testing or by LAL testing (or both if both apply)?
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14.

15. 16.

17.

18.

8. Method Validation

Microbiological Examination of Nonsterile Products and Sterility Test Validation

The validity of the results rests on the demonstration that the samples do not inhibit the growth of microorganisms that may be present

Preparatory to conducting the tests on a regular basis and as circumstances require subsequently, diluted samples (in relevant culture medium) are inoculated with separate viable cultures of the compendial organisms and tested.

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8. Method Validation

LAL Test Validation

-Preparatory tests are conducted to verify that the sample solution

doesnt inhibit or enhance reaction.

-Revalidation of the test method is required when conditions change that are likely to influence the test result.

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Microbiological Examination of Nonsterile Products--Audit Questions

1.

What are the Microbial Enumeration Test (MET) specs for one or two nonsterile products, and how arrived at? Ask to see the SOP for MET and the tests for specified organisms of one or two nonsterile products and compare to the to methods provided in USP <61>, <62>, and the drugs monographs (if specified). For 1 or 2 nonsterile products, when/how are plate count organisms identified?

2.

3.

4.

Does the product in question have preservatives? How are they neutralized?

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Microbiological Examination of Nonsterile Products Audit Questions

Ask to see the method validation for the microbial enumeration test (MET), and see what is the % recovery of organisms. Also, see how exhaustively the site tried to neutralize a bactericidal or preserved product in order to be able to conduct MET on it. 6. Verify that Lactose Monohydrate, NF; Gelatin, NF; Corn Starch, NF; and Talc, USP undergo the required compendial MLT if they are excipients. Do any product ongoing stability protocols require MET? Ask to see the most recent stability failure due to MLT and the resultant investigation (for US market, was a field alert issued upon initial OOS?).

7.

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Sterility Test Audit Questions

1.

For a sample of site sterile products, ask for the original and most recent bacteriostasis/fungistasis studies. For question #1, were USP <71> recommended organisms used in the bacteriostais/fungistasis based on the media used? What is the frequency of revalidating the product bacteriostasis/fungistasis (PIC/S recommends every 12 mo)?

2.

3.

4.

Is the EM during sterility testing identical to that used during sterile product mfg? Compare SOPs for these two EMs.
Are all sterility test media pre-incubated for 14 days to prove sterility prior to use or is this control test done concurrently?

5.

6.

Are negative controls/manipulation controls used in sterility tests? If not, why not?
Other than during growth promotion, how well are anaerobic conditions maintained for fluid thioglycollate incubation?
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7.

Sterility Test Audit Questions (contd)

8. In aseptic filling, are samples drawn from beginning, middle, and end as well as immediately after interruptions and operator interventions. Does the local SOP call for these intervention samples? Ask to see a list of OOSs for the sterility test. Choose at least one OOS (or called lab investigation report) to audit for agreement with applicable SOP and with scientific method/logic.

9.

10. If terminal sterilization is used, do sterility test samples include some from validation-identified coolest load location? 11. If parametric release is used for a terminally sterilized product, ask for two different sterilization batch records and compare the measured sterilization parameters to those given in the product dossier and/or NDA (e.g., fan speeds in cascading water/steam terminal sterilizer used by Baxter for Garenoxicin LVP). 12. If media fills are performed, does Micro Lab record its daily checks on the incubated vials, bottles, etc. (whether or not turbidity is observed)?

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Sterility Test Audit Questions (contd)

12. Sterility suite is designed like a mfg Grade A clean room, e.g.: a) aseptic gowning area/airlock w/ step-over bench division, full-length wall mirror, gowning instructions, hand washing, drying, and antiseptic application; b) annual cleanroom certification per ISO stds; c) NLT 10-15 Pa press. differential with adjacent rooms, the reading taken at least prior to entering suite; d) flush-mounting of power outlets, light fixtures, hands-free intercom, etc., and no extraneous equipment; e) outer surfaces of samples & equipment entering test suite is treated w/ sterile sanitizer (in EU, latter must be monitored); f) environmental monitoring similar to Aseptic Core of Mfg.

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Sterility Test Audit Questions (contd)

13. Additionally: a) janitorial/cleaning supplies must be sterilized before use; b) ultraviolet lights, if used, are kept on at all times except when testing is in progress or when viable particle EM is occurring; c) UV lights must be on an intensity measuring schedule; d) where there is more than one parallel UV tube, they should be shielded from each other; e) the LAF hood must be on at least 30 min prior to any use; f) LAF hood is annually certified (i.e., magnehelic gauge, calibration, HEPA filter scan for leaks and an Emory challenge.

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Sterility Test Isolators Audit Questions

1. Ask for isolator validation & compare to USP <1208> criteria: a) Set point of overpressure of interior can be maintained and controlled during operation? Is there a press. hold test? b) a computational fluid dynamics analysis (CFD) and/or smoke studies performed to determine the worst case airflow locations in the isolator? c) a six-log sterilization kill is confirmed in 3 consecutive validation studies, & is BI resistance to the sterilization process estimated? d) containers, media, filter sets, tubing, and other supplies kept inside the isolator are known or proved unaffected by sterilant penetration? e) frequency of re-sterilization justified with data. This includes proof of a maintained aseptic environment throughout a defined operational period.
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Sterility Test Isolators Audit Questions (contd)

2.

Ask for isolator use SOP and any gowning requirements (e.g., no rings, watches or other sharp objects, including long nails). Is the isolator directly in the flow path of an air supply grille (latter could cool sections of isolators walls to cause condensation during vapor sterilization). Ask to see isolator envir. monitoring SOP & the (viable and nonviable) specs or action/alert levels for air, surfaces, and gloves. If any isolator EM plates have shown growth, were the organisms identified? Ask to see these records. Does PM SOP include a glove (and half-suit) integrity test, preemptive replacement, and possibly submersion testing of gloves in a 0.1% peptone water followed by filtration of diluent and plating? How about transfer system gaskets and seals? How is the identity and composition of the gassing agent (sterilant) assured (e.g., incoming inspection or supplier certification)? Is the gas generator in the PM program?
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3.

4.

5.

6.

7.

8.

Endotoxin Testing Audit Questions

1.

What LAL method does the site use? If its only gel clot, can trends ever be detected? This is admittedly a key shortcoming of gel clot. Does this gel clot method use the sites own WFI as LAL diluent (in place of LAL water)? If yes, how does the site know the WFI isnt just barely below threshold for a clot? Ask for the sites SOP for preparing standards using reference std endotoxin (RSE). Compare the reconstitution and dilution instructions with that of USP <85> instructions, esp. the duration of vortexing.

2.

3.

4.

Does the site use Control Standard Endotoxin (CSE) to prepare standard curves or RSE? If CSE, has the CSE been standardized against the RSE? (see 1987 FDA Guideline on Validation of the LAL Test)
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Endotoxin Testing Audit Questions

5.

When LAL reagent (lot) changes, does the Lab confirm the Labeled LAL reagent sensitivity (EU/ml) and use the Lab value (vs the supplier-labeled value) in calculating MVD? Has the Lab ever changed suppliers for LAL reagent? If yes, did this result in a revalidation of the method (Per USP: Revalidation for the test method is required when conditions that are likely to influence the test result change.)? What disposable plasticware, pipets, or tips are used in LAL assays and how does the site know they are pyrogen-free? If pH is measured and adjusted on an LAL sample, how is this accomplished w/o adding pyrogens from pH probe or titrant? If the site depyrogenates glassware, what is the time limit for storage and use and what data is that based on?
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6.

7.

8.

9.

Micro Contract Services Audit Qs

1.

What external service providers (ESPs) does the site use for micro-related work?

2.

What documented qualification data (e.g., audit reports, CVs, completed questionnaires) can be shown for these ESPs?
What indicates that an ESP is site-quality-unit-approved? Ask to see quality/technical agreements between the site and the respective ESPs, especially when the ESP performs routine micro tests w/ product release implications. Do reports provided by ESPs contain a review signature by an appropriate manager or supervisor within the site?

3. 4.

5.

6.

Can the site request and receive copies (or review at the ESP) test result or other raw data residing at the ESP?
Do changes to any procedures or specs of contract tests require prior approval by the site? Stated in quality/tech agreement? How does site track & reassess an ESPs continuing performance?
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7.

8.

Bibliography
1.
2.

USP <55> Biological Indicators

USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms USP <71> Sterility Tests USP <85> Bacterial Endotoxins Test USP <1117> Microbiological Best Laboratory Practices USP <1208> Sterility TestingValidtion of Isolator Systems FDA Jan, 1994 Q&A on LAL Test FDA Dec, 1987 Guideline on Validation of the LAL Test FDA 1993 Guide to Inspections of Microbio Pharm. QC Labs

3.

4. 5. 6. 7. 8. 9. 10. 11. 12.

PIC/S Recommendation on Sterility Testing July, 2004

PIC/S Recommendation on Isolators Used for Aseptic Processing and Sterility Testing July, 2004 Cundell, Anthony; Managing the Microbio Quality of Pharm. Excipients.
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13.

Please ask any questions

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If you dont have any more questions, then let me ask a few:
1.

Give some requirements for a sterility test suite that resemble those for a sterile core? Give at least 3 questions that can be asked about a sterility test isolator. For a purchased biological indicator (BI), what would be on its C of A? What must the site lab test and what can it farm out? What matls and treated labware in the micro lab must have exp dating? What activities should be and what matls storage areas should be well separated in Micro? Give requirements for surface sanitizers used in Grade A or B suites. What is asked about the Micro lab glassware/labware washer and why?

2. 3.

4. 5.

6. 7. 8.

What are some questions related to bacterial cultures purchased and utilized in the Micro lab?
What can you say about the control of Micro Lab worksheets?

9.

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