Auditing the Microbiology Laboratory

Auditing the Microbiology Auditing the Microbiology LaboratoryLaboratory

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IntroductionIntroduction

This training will provide a basic understanding This training will provide a basic understanding of an audit of a typical Microbiology of an audit of a typical Microbiology Laboratory.Laboratory.

We have divided this training into two sections:We have divided this training into two sections:

I. Major Areas of Audit in the Micro LabI. Major Areas of Audit in the Micro Lab

II. Useful, Often Relevant Audit Questions II. Useful, Often Relevant Audit Questions

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I. Major Areas of Audit in the I. Major Areas of Audit in the Microbiology LaboratoryMicrobiology Laboratory

As a QC lab, the Micro dept. must As a QC lab, the Micro dept. must comply with the same basic GMP comply with the same basic GMP regulations promulgated for the regulations promulgated for the Analytical Chemistry Lab. Analytical Chemistry Lab.

With this in mind, we have adopted With this in mind, we have adopted the standard – 8 major areas in a lab the standard – 8 major areas in a lab audit – described ~ a year ago in audit – described ~ a year ago in GQAC training on Auditing the GQAC training on Auditing the Analytical Chem Lab. Analytical Chem Lab.

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Major areas of audit in the Major areas of audit in the microbiological laboratorymicrobiological laboratory

As a reminder, the major areas of audit in a QC As a reminder, the major areas of audit in a QC lab are:lab are:

1. Sample Control 1. Sample Control 2. Employee Training2. Employee Training3. Reference Standards3. Reference Standards4. Equipment and Instruments4. Equipment and Instruments5. Reagents and Media5. Reagents and Media6. Recordkeeping and Documentation6. Recordkeeping and Documentation7. Laboratory Controls7. Laboratory Controls8. Method Validation8. Method Validation

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1. Sample Control1. Sample Control

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

Must have sample records that show chain-of-Must have sample records that show chain-of-custody. Records can be manual, e.g., forms custody. Records can be manual, e.g., forms or logbooks, or electronic, e.g., LIMS or logbooks, or electronic, e.g., LIMS (laboratory information management system). (laboratory information management system).

Must be stored in an area that maintains their Must be stored in an area that maintains their quality. This includes areas that are properly quality. This includes areas that are properly identified, clean and orderly, and is adequate identified, clean and orderly, and is adequate to prevent mix-up andto prevent mix-up and contamination from contamination from other samples, from chemicals and reagents, other samples, from chemicals and reagents, and from spillage.and from spillage.

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Sample Control Audit Questions

1.1. Ask how Micro samples are logged in and stored.Ask how Micro samples are logged in and stored.

2.2. Does the sample log book (or other record) provide Does the sample log book (or other record) provide spaces for who delivered the sample and who then spaces for who delivered the sample and who then took it for testing (chain of custody)?took it for testing (chain of custody)?

3.3. What type of samples might be temporarily stored What type of samples might be temporarily stored while awaiting testing? while awaiting testing?

4.4. What method validation or compendial ref supports What method validation or compendial ref supports the sample storage conditions (e.g., water)?the sample storage conditions (e.g., water)?

5.5. What site SOP governs what happens when a water What site SOP governs what happens when a water or an EM sample time point is missed? (should be a or an EM sample time point is missed? (should be a deviation).deviation).

6.6. If LIMS is used for tracking all samples and If LIMS is used for tracking all samples and activities, check if pen raw data precedes computer activities, check if pen raw data precedes computer and whether the former is properly retained.and whether the former is properly retained.

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1.1. Ask how water samples are drawn; compare to site SOP.Ask how water samples are drawn; compare to site SOP.

2.2. Is the chemistry sample collected before the Micro (i.e., if a Is the chemistry sample collected before the Micro (i.e., if a spray disinfectant is used on the sample port)?spray disinfectant is used on the sample port)?

3.3. If any sampling sites are also points of use (POU) for Mf’g, If any sampling sites are also points of use (POU) for Mf’g, how comparable are the Micro sampling and Mf’g how comparable are the Micro sampling and Mf’g cleaning/rinsing/use of the same POU’s? cleaning/rinsing/use of the same POU’s?

4.4. Does the sample record contain time sample taken, Does the sample record contain time sample taken, sampler’s name, sample site, time sample delivered to Lab?sampler’s name, sample site, time sample delivered to Lab?

5.5. Are water-sampling bottles reused with a validated Are water-sampling bottles reused with a validated sterilization cycle and a proper storage/protection in sterilization cycle and a proper storage/protection in between? between?

6.6. Is the expiration date on those stored water sampling Is the expiration date on those stored water sampling bottles supported by a study (or any data)?bottles supported by a study (or any data)?

7.7. If there is a maximum number of hours that may elapse If there is a maximum number of hours that may elapse between sampling and bacterial enumeration testing, is the between sampling and bacterial enumeration testing, is the actual time or hour of sampling recorded along with the actual time or hour of sampling recorded along with the date? date?

(Water) Sample Control Audit Questions

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(Water) Sample Control Audit Questions

8.8. Ask for the most recent enumeration data of a specific Ask for the most recent enumeration data of a specific critical water POU (should contain or refer to date/time critical water POU (should contain or refer to date/time sample taken, date/time plated and put in incubator & sample taken, date/time plated and put in incubator & date/time out of incubator + incubator ID+ lot # of media date/time out of incubator + incubator ID+ lot # of media used).used).

9.9. What media is used to test water for counts? (If that What media is used to test water for counts? (If that water goes into a water goes into a European-marketed productEuropean-marketed product, then R2A , then R2A Media).Media).

10.10. Ask to see growth promotion testing of the media (some Ask to see growth promotion testing of the media (some companies require a water system isolate to be included).companies require a water system isolate to be included).

11.11. When is a colony identified? Ask for the SOP covering this When is a colony identified? Ask for the SOP covering this decision (should be obtainable from within the Lab itself—decision (should be obtainable from within the Lab itself—workers need proximity to their SOPs).workers need proximity to their SOPs).

12.12. Ask to see total viable count & the organisms identified in Ask to see total viable count & the organisms identified in the WFI loop (for all POUs) for past 18 mos.the WFI loop (for all POUs) for past 18 mos.

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2.2. Employee TrainingEmployee Training Employee training includes the basics needed for every Employee training includes the basics needed for every

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

In the Microbiology Lab, much of the task training is by In the Microbiology Lab, much of the task training is by Technique as opposed to being by Product.Technique as opposed to being by Product.

The training program should include an established The training program should include an established training curricula specific to job functions, and periodic training curricula specific to job functions, and periodic performance assessments to verify ongoing competency in performance assessments to verify ongoing competency in core activities, e.g., hygiene, plating, aseptic technique, core activities, e.g., hygiene, plating, aseptic technique, handling test failures.handling test failures.

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1.1. Ask to see the Lab’s org. chart. Ask to see the Lab’s org. chart.

2.2. Has cross-training created sufficient Has cross-training created sufficient designated back-ups for critical activities in the Lab designated back-ups for critical activities in the Lab (or even for reviewing analysts’ results)?(or even for reviewing analysts’ results)?

3.3. Ask to see the training curricula for each unique Lab Ask to see the training curricula for each unique Lab position.position.

4.4. Determine the most recent effective date for a change Determine the most recent effective date for a change to a Micro SOP, then examine training records for to a Micro SOP, then examine training records for updated training on the new revision.updated training on the new revision.

5.5. Ask to see the Microbiology OOS procedure. Ask to see the Microbiology OOS procedure.

6.6. Ask to see the OOSs in Micro for the past 18 mos. Ask to see the OOSs in Micro for the past 18 mos.

7.7. Can patterns be seen in tests or analysts associated w/ Can patterns be seen in tests or analysts associated w/ OOSs, and if so, what CAPAs have followed?OOSs, and if so, what CAPAs have followed?

Analyst Training Audit Questions

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3.3. Reference StandardsReference Standards The microbiology laboratory shares the same requirements The microbiology laboratory shares the same requirements

that apply to the Analytical laboratory; it must also that apply to the Analytical laboratory; it must also ascertain that the reference standards used are of the ascertain that the reference standards used are of the highest quality for the required testshighest quality for the required tests

Noteworthy standard requirements include those for:Noteworthy standard requirements include those for:

- Standards which need to be frozen at extremely low - Standards which need to be frozen at extremely low temperatures, including microbiological cultures. Check temperatures, including microbiological cultures. Check procedure to handle these standards (e.g., use of liq N2).procedure to handle these standards (e.g., use of liq N2).

- The USP Endotoxin RSE has a defined potency of 10,000 - The USP Endotoxin RSE has a defined potency of 10,000 USP Endotoxin Units (EU) per vial. The first dilution of the USP Endotoxin Units (EU) per vial. The first dilution of the concentrate may not be stored in a refrigerator for more concentrate may not be stored in a refrigerator for more than 14 days for use in subsequent dilutions. Further than 14 days for use in subsequent dilutions. Further dilutions may not be stored w/o supporting data (risk of dilutions may not be stored w/o supporting data (risk of lost activity via adsorption to container)lost activity via adsorption to container)

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3.3. Reference StandardsReference Standards Biological Indicator (BI)Biological Indicator (BI): is a challenge organism, : is a challenge organism,

usually as a spore, which can be characterized by its usually as a spore, which can be characterized by its population and population and D-value. The D-value is the number of minutes (at D-value. The D-value is the number of minutes (at defined conditions of steam, gas, or radiation) defined conditions of steam, gas, or radiation) required to achieve a 1 log reduction on the total required to achieve a 1 log reduction on the total number of defined organisms.number of defined organisms.

Each BI is tested by the Micro lab or an approved Each BI is tested by the Micro lab or an approved contract lab to confirm the vendor’s labeled bacterial contract lab to confirm the vendor’s labeled bacterial population and, less frequently, D-value.population and, less frequently, D-value.

BIs are placed in selected locations in sterilizing BIs are placed in selected locations in sterilizing equipment during respective qualifications. To pass equipment during respective qualifications. To pass qualification, the sterilizer must show a 10qualification, the sterilizer must show a 106 6 reduction reduction in number of organisms after exposure at worst case in number of organisms after exposure at worst case (less time than in routine operations) and at (less time than in routine operations) and at prescribed pressure, BI spore population and prescribed pressure, BI spore population and resistance (D-value). See later section on audit resistance (D-value). See later section on audit questions, which explains importance of D-value.questions, which explains importance of D-value.

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3.3. Reference StandardsReference Standards Microbial cultures are delicate standards. Procedures Microbial cultures are delicate standards. Procedures

should specify careful handling instructions. Preparation should specify careful handling instructions. Preparation and resuscitation of cultures should follow the instructions and resuscitation of cultures should follow the instructions of the supplier or a validated, established method. USP of the supplier or a validated, established method. USP recommends using the "Seed-Lot" technique for storage of recommends using the "Seed-Lot" technique for storage of stock cultures, i.e., using working cultures and stock cultures, i.e., using working cultures and nevernever returning unused passages back to original stock. In returning unused passages back to original stock. In addition, there should be an established maximum number addition, there should be an established maximum number of passages (5 or less), and maximum storage time for of passages (5 or less), and maximum storage time for working cultures. working cultures.

Cultures for use in compendial tests should be acquired Cultures for use in compendial tests should be acquired from a national culture collection, in frozen, freeze-dried, from a national culture collection, in frozen, freeze-dried, on slants, or in ready-to-use forms. Confirmation of the on slants, or in ready-to-use forms. Confirmation of the purity and the identity should be performed prior to its use purity and the identity should be performed prior to its use in quality control testing. Ready-to-use cultures may in quality control testing. Ready-to-use cultures may require additional confirmation of inoculum size. require additional confirmation of inoculum size.

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1.1. What BI’s are purchased by the site?What BI’s are purchased by the site?

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

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

4.4. Is the D-value >= 1.5 min for 121oC steam Is the D-value >= 1.5 min for 121oC steam sterilization? (required by AAMI, ISO, and USP sterilization? (required by AAMI, ISO, and USP <1035>)<1035>)

5.5. Has the D-value claimed by the supplier ever been Has the D-value claimed by the supplier ever been verified by a qualified lab with a BIER (biological verified by a qualified lab with a BIER (biological indicator evaluator resistometer) Vessel per indicator evaluator resistometer) Vessel per ANSI/AAMI ST44 or USP <55>? If “no”, ask how the ANSI/AAMI ST44 or USP <55>? If “no”, ask how the site knows D >= 1.5 minsite knows D >= 1.5 min

Biological Indicator Audit Questions

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6.6. If accepted on certificate of analysis, what BI If accepted on certificate of analysis, what BI supplier quality assurance has been established? supplier quality assurance has been established? Ask to see file. Supplier should be “approved.”Ask to see file. Supplier should be “approved.”

7.7. Read the BI vendor’s use instructions, then look Read the BI vendor’s use instructions, then look for data on elapsed time between cycle exposure for data on elapsed time between cycle exposure and incubation. and incubation.

Biological Indicator Audit Questions (cont’d)

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1.1. When purchasing microorganisms from a When purchasing microorganisms from a national culture collection, what incoming QC national culture collection, what incoming QC tests are run for identity and purity? Is the ID tests are run for identity and purity? Is the ID done via genotypic analysis?done via genotypic analysis?

2.2. How are the # of passages of working cultures How are the # of passages of working cultures tracked, and what is the maximum # tracked, and what is the maximum # permitted (PIC/S says NMT 5 passages for permitted (PIC/S says NMT 5 passages for cutures used in pos. ctls of sterility tests)? cutures used in pos. ctls of sterility tests)?

3.3. Ask to see the records of subculturing a Ask to see the records of subculturing a purchased organism.purchased organism.

4.4. How long can a working culture be used How long can a working culture be used (<= 1 wk)?(<= 1 wk)?

Purchased Cell Cultures

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4. Equipment and Instruments4. Equipment and Instruments

The same Analytical Chem Lab equipment and The same Analytical Chem Lab equipment and instrument calibration, maintenance, and instrument calibration, maintenance, and qualification requirements apply to the Micro qualification requirements apply to the Micro Lab.Lab.

There, the most common equipment are There, the most common equipment are incubators, autoclaves, refrigerators, water incubators, autoclaves, refrigerators, water baths, laminar flow hoods, and automatic baths, laminar flow hoods, and automatic pipettors. pipettors.

Often overlooked, the glassware washer is Often overlooked, the glassware washer is critical in the Micro Lab. Prep’n of media in critical in the Micro Lab. Prep’n of media in poorly cleaned glassware can allow inhibitory poorly cleaned glassware can allow inhibitory substances to enter the media from residual substances to enter the media from residual cleaning detergent or from mat’ls previously cleaning detergent or from mat’ls previously used. The washer must be qualified. used. The washer must be qualified.

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Storage equipment – the Micro Lab uses a number of storage Storage equipment – the Micro Lab uses a number of storage equipment that require qualification: incubators, refrigerators, equipment that require qualification: incubators, refrigerators, freezers, thermostated water baths, etc. Some inspectors freezers, thermostated water baths, etc. Some inspectors expect thermostated water baths to have undergone OQ of the expect thermostated water baths to have undergone OQ of the temperature consistency throughout bath. temperature consistency throughout bath.

Qualifications are conducted through temperature mapping, Qualifications are conducted through temperature mapping, and the number of locations depend on size. Small incubators and the number of locations depend on size. Small incubators and refrigerators would not require mapping qualification, but and refrigerators would not require mapping qualification, but instead are verified with a calibrated thermometer during use. instead are verified with a calibrated thermometer during use.

After initial qualifications, critical storage equipment are After initial qualifications, critical storage equipment are monitored through daily verifications. Temperature mapping monitored through daily verifications. Temperature mapping is periodically repeated, to confirm uniformity and monitoring is periodically repeated, to confirm uniformity and monitoring probe placements at worst case, hot and cold, locations. We probe placements at worst case, hot and cold, locations. We have found that some labs fall short on requirements, e.g., 2-have found that some labs fall short on requirements, e.g., 2-8º C range is monitored for cold rooms even though some 8º C range is monitored for cold rooms even though some stored mat’ls need range of 2-6º C.stored mat’ls need range of 2-6º C.

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4. Equipment and Instruments4. Equipment and InstrumentsIf monitoring occurs via review of charts from If monitoring occurs via review of charts from equipment chart recorder, then challenge this review equipment chart recorder, then challenge this review process. Verify that charts from a given time-period, process. Verify that charts from a given time-period, typically 6 months, are appropriately reviewed, and typically 6 months, are appropriately reviewed, and deviations, including temporary spikes, are deviations, including temporary spikes, are explained/justified or defined within the equipment explained/justified or defined within the equipment SOP as an allowable excursion. SOP as an allowable excursion.

In addition to mapping and routine monitoring, other In addition to mapping and routine monitoring, other controls are needed for critical storage equipment:controls are needed for critical storage equipment:

Procedure to capture extended temperature Procedure to capture extended temperature excursion and to assess impact on the contents. excursion and to assess impact on the contents.

Probe and recorder calibrations, challenge of Probe and recorder calibrations, challenge of temp. alarms (all the way to the monitoring site or temp. alarms (all the way to the monitoring site or external company), and equipment PM.external company), and equipment PM.

A disaster recovery plan.A disaster recovery plan.

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The autoclave is specialized equipment with unique The autoclave is specialized equipment with unique requirements. When used to sterilize media, the requirements. When used to sterilize media, the growth-promotability of the media is part of the growth-promotability of the media is part of the acceptance criteria in addition to sterility.acceptance criteria in addition to sterility.

The cycle in the The cycle in the autoclave/sterilizerautoclave/sterilizer must be must be validated to ensure proper heat distribution for validated to ensure proper heat distribution for selected media loads and volumes. Typically, selected media loads and volumes. Typically, manufacturers recommend using an autoclave cycle manufacturers recommend using an autoclave cycle of 121º C for 15 minutes, which apply to time at of 121º C for 15 minutes, which apply to time at temperature of the media. The sterilization time is temperature of the media. The sterilization time is dependent on the media volume and autoclave load. dependent on the media volume and autoclave load.

Sterilization cycles in which the autoclave is slow to Sterilization cycles in which the autoclave is slow to come up to temperature may result in overheating of come up to temperature may result in overheating of the media. Therefore, care must be taken to balance the media. Therefore, care must be taken to balance the need for a sterile media against the tendency of the need for a sterile media against the tendency of the media to degrade under excessive heating. the media to degrade under excessive heating.

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4. Equipment and Instruments4. Equipment and Instruments Storage of the media in the autoclave after the liquid cycle is Storage of the media in the autoclave after the liquid cycle is

completed is not recommended after cooling, as it may damage completed is not recommended after cooling, as it may damage the media. Improper heating or sterilizing conditions—for the media. Improper heating or sterilizing conditions—for commercially prepared or internally prepared media—may result in commercially prepared or internally prepared media—may result in a difference in color change, loss of clarity, altered gel strength, or a difference in color change, loss of clarity, altered gel strength, or pH drift from the manufacturer's recommended range.pH drift from the manufacturer's recommended range.

Autoclaves used for media sterilization may lack the ability to Autoclaves used for media sterilization may lack the ability to displace steam with sterile filtered air (for sealed bottles of media, displace steam with sterile filtered air (for sealed bottles of media, this would not present a problem). For non-sealed bottles or flasks this would not present a problem). For non-sealed bottles or flasks of media, non-sterile air has led to the contamination of media. of media, non-sterile air has led to the contamination of media.

Autoclaving less than the required time will also allow media- Autoclaving less than the required time will also allow media- associated contaminants to grow and cause a false positive result. associated contaminants to grow and cause a false positive result. These problems may be more prevalent in laboratories with a These problems may be more prevalent in laboratories with a heavy workload.heavy workload.

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4. Equipment and Instruments4. Equipment and Instruments The most common micro instruments are the microscopes, The most common micro instruments are the microscopes,

balances, pH meters, thermometers, spectrophotometers. balances, pH meters, thermometers, spectrophotometers. Lab instruments should be calibrated on a regular schedule Lab instruments should be calibrated on a regular schedule and verified routinely. and verified routinely.

Review pH records, we originally found very few Micro Labs Review pH records, we originally found very few Micro Labs standardizing pH correctly. From the simplified major tests standardizing pH correctly. From the simplified major tests introduced in earlier sections, pH is critical for their introduced in earlier sections, pH is critical for their results.results.

Other unique instrument/equipment calibration Other unique instrument/equipment calibration requirements in the Micro Lab include calibration of the requirements in the Micro Lab include calibration of the lens vernier scale in the microscope and the air sampler lens vernier scale in the microscope and the air sampler unit that is used for environmental air sampling. Micro unit that is used for environmental air sampling. Micro must ensure tight environment control when conducting must ensure tight environment control when conducting Sterility tests. Sterility tests.

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4. Equipment and Instruments4. Equipment and Instruments Computerized LaboratoryComputerized Laboratory – computer-controlled – computer-controlled

systems may be used, e.g., to control and systems may be used, e.g., to control and maintain environmental data. As with the QC maintain environmental data. As with the QC Chemistry Lab, the microbiology lab must have a Chemistry Lab, the microbiology lab must have a policy up front on what constitutes the instrument policy up front on what constitutes the instrument generated raw data generated raw data the printed or the the printed or the electronic data. electronic data.

The FDA basic guidance on security and The FDA basic guidance on security and authenticity issues, to address data integrity authenticity issues, to address data integrity concerns, apply here.concerns, apply here.

There must be a provision so that only authorized There must be a provision so that only authorized personnel can make data entries. Data entries personnel can make data entries. Data entries may not be deleted; changes may be captured by may not be deleted; changes may be captured by the audit trail or by a manual process. the audit trail or by a manual process.

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One of the most common computer systems found in One of the most common computer systems found in a Micro lab is the automated system for the genus a Micro lab is the automated system for the genus and species identification of microorganisms.and species identification of microorganisms.

Logs of such testing, along with the identification of Logs of such testing, along with the identification of the source of the sample, are also of value in the the source of the sample, are also of value in the identification of potential microbial problems in identification of potential microbial problems in processing.processing.

The use of automated systems for the identification The use of automated systems for the identification of microorganisms is relatively common in parenteral of microorganisms is relatively common in parenteral manufacturing where isolates from the environment, manufacturing where isolates from the environment, water systems, validation, and people are routinely water systems, validation, and people are routinely identified. identified.

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Environmentally-Controlled Chambers/Rooms Audit Questions

..1.1. Show how refrigerators, freezers, & liquid N2 tanks are Show how refrigerators, freezers, & liquid N2 tanks are monitored. monitored.

2.2. Do these have a logbook or form for contents and is it Do these have a logbook or form for contents and is it accurate and timely updated?accurate and timely updated?

3.3. What, if any, disaster recovery plan exists for some of these What, if any, disaster recovery plan exists for some of these environmentally-controlled rooms/chambers?environmentally-controlled rooms/chambers?

4.4. What actions would be taken if a large walk-in refrigerator or What actions would be taken if a large walk-in refrigerator or freezer failed?freezer failed?

5.5. What procedure exists to capture an extended temperature What procedure exists to capture an extended temperature excursion and to assess impact on contents of affected excursion and to assess impact on contents of affected chamber? Does that procedure allow for a 10 min (or similar) chamber? Does that procedure allow for a 10 min (or similar) temp. excursion due to door opening? temp. excursion due to door opening?

6.6. Ask to see the most recent calibration record and SOP of the Ask to see the most recent calibration record and SOP of the temperature probe for a refrigerator (does it have an temperature probe for a refrigerator (does it have an equipment #. If not, why not?).equipment #. If not, why not?).

7.7. Ask to see the last PM report and its SOP (check if there’s a Ask to see the last PM report and its SOP (check if there’s a test for the alarm) for the refrigerator, freezer, incubators, test for the alarm) for the refrigerator, freezer, incubators, stability chamberstability chamber

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Env-Controlled Chambers/Rooms Audit Questions

8.8. Are multi-shelved incubators temp.-mapped?Are multi-shelved incubators temp.-mapped?

9.9. Ask to see temp.-mapping studies for one or some of the Ask to see temp.-mapping studies for one or some of the above chambers. If no mapping ever done, are there above chambers. If no mapping ever done, are there temp. probes for monitoring multiple locations?temp. probes for monitoring multiple locations?

10.10. Ask to see the site SOP(s) for qualifying and monitoring Ask to see the site SOP(s) for qualifying and monitoring environmentally-controlled chambers.environmentally-controlled chambers.

11.11. For one of the chambers or incubators that has a chart For one of the chambers or incubators that has a chart recorder, ask for last month of archived charts. recorder, ask for last month of archived charts.

12.12. Do the charts have initials/signature and date for its Do the charts have initials/signature and date for its installation and for its removal and review?installation and for its removal and review?

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

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5. Reagents and Media5. Reagents and Media Reagents must be controlled the same way as they Reagents must be controlled the same way as they

are controlled in the Analytical Chem Lab.are controlled in the Analytical Chem Lab.

Reagent and media preparation must be traceable, Reagent and media preparation must be traceable, preferably documented in a laboratory notebook. preferably documented in a laboratory notebook. The minimum information required for traceability: The minimum information required for traceability:

●● DatesDates●● Type and lot of material usedType and lot of material used●● Ingredient quantities Ingredient quantities ●● Preparer’s identificationPreparer’s identification

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

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5. Reagents and Media5. Reagents and Media

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

The capability of the media to promote the growth of The capability of the media to promote the growth of organisms may be affected by the media preparation organisms may be affected by the media preparation process, sterilization (overheating), and storage.process, sterilization (overheating), and storage.

Review analyses being conducted and inspect the plates Review analyses being conducted and inspect the plates and tubes of media being incubated. and tubes of media being incubated.

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Be particularly alert for retests that have not Be particularly alert for retests that have not been documented and "special projects" in been documented and "special projects" in which investigations of contamination which investigations of contamination problems have been identified. problems have been identified.

This can be evaluated by reviewing the This can be evaluated by reviewing the ongoing analyses (product or environmental) ongoing analyses (product or environmental) for positive test results. Request to review for positive test results. Request to review the previous day's plates and media, if the previous day's plates and media, if available and compare your observations to available and compare your observations to the recorded entries in the logs. the recorded entries in the logs.

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The The LAL reagentLAL reagent is critical in Endotoxin testing, and is critical in Endotoxin testing, and must be well controlled.must be well controlled.

When the LAL reagent (lot) changes, the Micro Lab When the LAL reagent (lot) changes, the Micro Lab must confirm the labeled LAL reagent sensitivity λ must confirm the labeled LAL reagent sensitivity λ (EU/ml). This is to re-calculate the Maximum Valid (EU/ml). This is to re-calculate the Maximum Valid Dilution (MVD), which is the maximum allowable Dilution (MVD), which is the maximum allowable dilution of a sample to determine the endotoxin limit. dilution of a sample to determine the endotoxin limit.

MVD = (Endotoxin limit × Concentration of sample sol’n)/λ MVD = (Endotoxin limit × Concentration of sample sol’n)/λ

where concentration of sample in solution is known where concentration of sample in solution is known either from product testing in Anal Chem or, for a raw either from product testing in Anal Chem or, for a raw mat’l, by a precisely weighed sample and dissolution, mat’l, by a precisely weighed sample and dissolution, and the endotoxin limit concentration is specified in and the endotoxin limit concentration is specified in the individual monograph (in EU per mg).the individual monograph (in EU per mg).

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SanitizersSanitizers are reagents that also need to be well are reagents that also need to be well controlled in the Micro Lab. controlled in the Micro Lab.

There should be a lab sanitization and sanitizer There should be a lab sanitization and sanitizer preparation SOP. SOPs should specify the grade and preparation SOP. SOPs should specify the grade and temperature of water used to prepare sanitizing temperature of water used to prepare sanitizing solution, method of applying sanitizer, contact time, solution, method of applying sanitizer, contact time, post-contact drying method and time. post-contact drying method and time.

Sanitizers should be qualified (effectiveness testing) Sanitizers should be qualified (effectiveness testing) with intended application quantity and contact time with intended application quantity and contact time as well as surface. Data should be available to as well as surface. Data should be available to support expiration period of the sanitizer at its use support expiration period of the sanitizer at its use dilution. dilution.

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

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1.1. What sanitizers are used in the Micro Lab? Ask to see What sanitizers are used in the Micro Lab? Ask to see Micro Lab sanitization SOP and sanitizer preparation SOP.Micro Lab sanitization SOP and sanitizer preparation SOP.

2.2. Is each lot of sanitizer released by the site QC?Is each lot of sanitizer released by the site QC?

3.3. Does sanitization SOP specify: a) type & temp. of water Does sanitization SOP specify: a) type & temp. of water used to prepare sanitizing solution; b) the method of used to prepare sanitizing solution; b) the method of applying sanitizer; c) contact time; d) post-contact drying applying sanitizer; c) contact time; d) post-contact drying method & time?method & time?

4.4. Are all Micro Lab sanitizers purchased sterile or rendered Are all Micro Lab sanitizers purchased sterile or rendered sterile (if to be used in Grade A or B areas)?sterile (if to be used in Grade A or B areas)?

5.5. What qualification (i.e., sanitizer effectiveness testing) What qualification (i.e., sanitizer effectiveness testing) exists for each Micro Lab sanitizer and do they result in exists for each Micro Lab sanitizer and do they result in prescribed application quantity & contact time?prescribed application quantity & contact time?

6.6. What records show that specified contact time is achieved?What records show that specified contact time is achieved?

7.7. What data supports exp period of the sanitizer at its use What data supports exp period of the sanitizer at its use dilution?dilution?

8.8. What evidence shows that a non-sporocidal sanitizer What evidence shows that a non-sporocidal sanitizer working solution, or even hand antiseptic sprays and working solution, or even hand antiseptic sprays and wipes, remain sterile through expiration, and are they wipes, remain sterile through expiration, and are they monitored? monitored?

Sanitizer Preparation & Use

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

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

3.3. What media is prepared by the Lab? What media is prepared by the Lab?

4.4. Do the following appear in the media growth promotion Do the following appear in the media growth promotion record: record: a) confirmed temp., b) time-into and time-out of the a) confirmed temp., b) time-into and time-out of the incubator, incubator, c) incubator ID, d) control (lot) no. of the organism(s) c) incubator ID, d) control (lot) no. of the organism(s) plated?plated?

5.5. Does a USP-governed site trend the results of all media Does a USP-governed site trend the results of all media growth promotions lot-to-lot? (new <61> requirement).growth promotions lot-to-lot? (new <61> requirement).

6.6. Do media preparation records refer to the autoclave cycle Do media preparation records refer to the autoclave cycle used for media sterilized by Micro? used for media sterilized by Micro?

Media Preparation, Testing & Storage Audit Questions

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7.7. Is the prepared media sufficiently labeled for traceability to Is the prepared media sufficiently labeled for traceability to the starting mat’ls, date, and analyst? Was the autoclave the starting mat’ls, date, and analyst? Was the autoclave cycle and the expiration dating period of in-house-prepared cycle and the expiration dating period of in-house-prepared media validated (e.g., w/ growth-promotion testing through media validated (e.g., w/ growth-promotion testing through expiration)? Check several in-house media.expiration)? Check several in-house media.

8.8. When the autoclave is used to sterilize media, is a printout of When the autoclave is used to sterilize media, is a printout of cycle parameters printed and attached to a media preparation cycle parameters printed and attached to a media preparation worksheet or notebook page?worksheet or notebook page?

9.9. Ask to see SOP(s) with description, picture, or both of Ask to see SOP(s) with description, picture, or both of validated media load configurations in the autoclave. validated media load configurations in the autoclave.

10.10. What is the frequency of autoclave cycle revalidation What is the frequency of autoclave cycle revalidation (annual)? (annual)?

11.11. When media pH is checked after autoclaving and cool-down, When media pH is checked after autoclaving and cool-down, how is this done (should be a completely aseptic technique)?how is this done (should be a completely aseptic technique)?

12.12. Does analyst 100% check the plates or tubes of in-house-Does analyst 100% check the plates or tubes of in-house-prepared media for a) unequal filling, b) dehydration, c) prepared media for a) unequal filling, b) dehydration, c) excessive bubbles?excessive bubbles?

Media Preparation, Testing & Storage Media Preparation, Testing & Storage Audit Questions Audit Questions Media Preparation, Testing & Storage Audit Questions (cont’d)

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Media Preparation, Testing & Storage Media Preparation, Testing & Storage Audit Questions Audit Questions

13. Are all media stored according to mf’r instructions and 13. Are all media stored according to mf’r instructions and protected against dehydration?protected against dehydration?

14.14. Are in-house-prepared media slated for EM of critical areas Are in-house-prepared media slated for EM of critical areas (e.g., Grade A) double-wrapped and terminally sterilized ((e.g., Grade A) double-wrapped and terminally sterilized (oror preincubated & inspected for contamination)? preincubated & inspected for contamination)?

15.15. How does the Micro Lab clean its glassware and/or procure How does the Micro Lab clean its glassware and/or procure media plates so that no inhibitory residue could get into media plates so that no inhibitory residue could get into prepared media?prepared media?

16.16. How does the Micro Lab inventory its various media and How does the Micro Lab inventory its various media and ensure disposal at expiration? ensure disposal at expiration?

17.17. Ask to see the growth promotion testing SOP for both Ask to see the growth promotion testing SOP for both purchased media and in-house-prepared media. purchased media and in-house-prepared media.

18.18. For environmental monitoring media, is the media on For environmental monitoring media, is the media on quarantine pending the acceptable results of growth quarantine pending the acceptable results of growth promotion? This is also S-P policy with regards to water-promotion? This is also S-P policy with regards to water-testing media.testing media.

Media Preparation, Testing & Storage Audit Questions (cont’d)

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6.6. Recordkeeping and DocumentationRecordkeeping and Documentation Proper Proper recordkeepingrecordkeeping is critical for the Micro Lab. A is critical for the Micro Lab. A

test should be performed as per SOP, and the test should be performed as per SOP, and the laboratory notebook should provide a record of all laboratory notebook should provide a record of all critical details needed to confirm the integrity of the critical details needed to confirm the integrity of the data. At a minimum, the laboratory write-up should data. At a minimum, the laboratory write-up should include the following:include the following:

Date Date Material tested Material tested Microbiologist's name (unless a personal bound Microbiologist's name (unless a personal bound

notebook)notebook) Procedure number Procedure number Test results and any logical operators fully defined Test results and any logical operators fully defined

(e.g., <) or explained (e.g., <) or explained Deviations (if any) Deviations (if any) Documented parameters (equipment used, microbial Documented parameters (equipment used, microbial

stock culture nos. used, media or reagent lot nos. stock culture nos. used, media or reagent lot nos. used) used)

Management/Second review signature Management/Second review signature

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6.6. Recordkeeping and DocumentationRecordkeeping and Documentation Proper recordkeeping includes good documentation practices. Proper recordkeeping includes good documentation practices.

Every critical piece of equipment is noted. Equipment Every critical piece of equipment is noted. Equipment temperatures (water baths, incubators, autoclaves) are temperatures (water baths, incubators, autoclaves) are recorded and traceable. recorded and traceable.

The governing test method and revision are clearly noted. The governing test method and revision are clearly noted.

Logbooks or forms are available and supportive of the Logbooks or forms are available and supportive of the laboratory notebook records. Test results include the original laboratory notebook records. Test results include the original plate counts, which constitute the actual raw data and are plate counts, which constitute the actual raw data and are used for the calculations for final test results. Methods for used for the calculations for final test results. Methods for data analysis are detailed in SOPs.data analysis are detailed in SOPs.

All laboratory records are archived and protected against All laboratory records are archived and protected against catastrophic loss. A formal record retention and retrieval catastrophic loss. A formal record retention and retrieval program is in place, practiced at the local level.program is in place, practiced at the local level.

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6.6. Recordkeeping and DocumentationRecordkeeping and Documentation

DocumentationDocumentation in the microbiology laboratory includes in the microbiology laboratory includes procedures and test methods, work instructions (i.e., calibration procedures and test methods, work instructions (i.e., calibration and maintenance), protocols, guidelines, manuals, etc. If used and maintenance), protocols, guidelines, manuals, etc. If used for the operation of the lab and are critical for validity of for the operation of the lab and are critical for validity of results, they must be approved by the Quality Unit. results, they must be approved by the Quality Unit. Furthermore, assurance must exist that testing histories are Furthermore, assurance must exist that testing histories are accurate and complete by having a defined system for accurate and complete by having a defined system for issuance, monitoring, and reconciliation of worksheets issuance, monitoring, and reconciliation of worksheets printed/used (i.e., an audit trailprinted/used (i.e., an audit trail..

When we introduced Micro test methods, there were only three When we introduced Micro test methods, there were only three major tests. Then why do we need product specific test major tests. Then why do we need product specific test methods? Test methods capture unique sample requirements, methods? Test methods capture unique sample requirements, e.g., validated preparatory steps prior to moving into the e.g., validated preparatory steps prior to moving into the common steps. common steps.

SOP must reflect actual practices and test methods and must SOP must reflect actual practices and test methods and must be in conformance with application commitments and/or be in conformance with application commitments and/or compendium requirements.compendium requirements.

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6.6. Recordkeeping and DocumentationRecordkeeping and Documentation For compendial test methods, there For compendial test methods, there mustmust be a process in place be a process in place

to ensure that changes are captured timely so that they may to ensure that changes are captured timely so that they may be implemented when the changes go into effect. be implemented when the changes go into effect. There There shouldshould be a process in place to evaluate, comment and resolve issues be a process in place to evaluate, comment and resolve issues related to proposed changes to the compendium before related to proposed changes to the compendium before revised monographs become final.revised monographs become final.

Documentation is sufficient to show that testing was done in a Documentation is sufficient to show that testing was done in a laboratory and by methods that were under control. laboratory and by methods that were under control. Equipment performance during test (e.g., 24-hour/7-day Equipment performance during test (e.g., 24-hour/7-day

chart recorders) chart recorders) Media preparation, sterility checks, and growth-promotion Media preparation, sterility checks, and growth-promotion

and selectivity capabilities and selectivity capabilities Media inventory and control testing Media inventory and control testing Critical components of test conducted as specified by SOP Critical components of test conducted as specified by SOP Data and calculations verified Data and calculations verified Reports reviewed by site quality unit or qualified Reports reviewed by site quality unit or qualified

responsible mg’rresponsible mg’r Investigation of data deviations, if any Investigation of data deviations, if any

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7.7. Laboratory ControlsLaboratory Controls

The management ensures that the laboratory is The management ensures that the laboratory is operating in a state of control operating in a state of control

Through review of key metrics, e.g., lab related Through review of key metrics, e.g., lab related [major] deviations and OOSs due to analyst or [major] deviations and OOSs due to analyst or instrument error, stability testing performed as instrument error, stability testing performed as scheduled, etc.scheduled, etc.

Through self-inspection -- immediate correction of high Through self-inspection -- immediate correction of high risk issues and continuous improvement in all areas risk issues and continuous improvement in all areas where gaps are identifiedwhere gaps are identified

Changes in the laboratory should be approved by an Changes in the laboratory should be approved by an independent quality unit and as per a formalized independent quality unit and as per a formalized change control process change control process

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7.7. Laboratory ControlsLaboratory Controls Sterility TestingSterility Testing: the regulatory requirements follow from : the regulatory requirements follow from

the USP statement, "The facility for sterility testing should the USP statement, "The facility for sterility testing should be such as to offer no greater a microbial challenge to the be such as to offer no greater a microbial challenge to the articles being tested than that of an aseptic processing articles being tested than that of an aseptic processing production facility." This includes environmental production facility." This includes environmental monitoring and gowning equivalent to that used for monitoring and gowning equivalent to that used for manufacturing product, e.g., gowning area, pass-through manufacturing product, e.g., gowning area, pass-through airlock. (see sterility test audit questions section).airlock. (see sterility test audit questions section).

Review records of initial positive sterility test results, Review records of initial positive sterility test results, particularly for all high risk aseptically filled products. particularly for all high risk aseptically filled products. Firms have difficulty justifying release of a product filled Firms have difficulty justifying release of a product filled aseptically that fails an initial sterility test, without having aseptically that fails an initial sterility test, without having identified specific problems associated with the controls identified specific problems associated with the controls used for the sterility test.used for the sterility test.

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7.7. Laboratory ControlsLaboratory Controls When the manufacturer has never found an initial positive When the manufacturer has never found an initial positive

sterility test, confirm that the absence of initial positives is sterility test, confirm that the absence of initial positives is not due to inappropriate control on retesting, or lack of not due to inappropriate control on retesting, or lack of validation to demonstrate that there is no carryover of validation to demonstrate that there is no carryover of inhibition from the product or preservative.inhibition from the product or preservative.

Evaluate the time period used for sterility test sample Evaluate the time period used for sterility test sample incubation. As per USP, samples are to be incubated for at incubation. As per USP, samples are to be incubated for at least 14 days at 2 sets of incubation temperatures. least 14 days at 2 sets of incubation temperatures.

Media fill, environmental, sterility test results and other Media fill, environmental, sterility test results and other data should be reviewed to assure the absence of slow data should be reviewed to assure the absence of slow growing organisms. growing organisms.

Compare the methods being used for incubation to Compare the methods being used for incubation to determine if they conform to those listed in approved or determine if they conform to those listed in approved or pending applications.pending applications.

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7.7. Laboratory ControlsLaboratory Controls Laboratory Operations/HousekeepingLaboratory Operations/Housekeeping

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

The Micro Lab should practice aseptic techniques during The Micro Lab should practice aseptic techniques during testing in general, to avoid microbial contamination and testing in general, to avoid microbial contamination and false positives.false positives.

Housekeeping must be properly maintained to prevent Housekeeping must be properly maintained to prevent use of expired or contaminated testing mat’s. Verify use of expired or contaminated testing mat’s. Verify cleanliness of work stations, cleared of extraneous or cleanliness of work stations, cleared of extraneous or previous test mat’ls, prompt removal of refuse, and clean previous test mat’ls, prompt removal of refuse, and clean utensils and equipment.utensils and equipment.

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7.7. Laboratory ControlsLaboratory Controls

StabilityStability – the stability program is likely managed by – the stability program is likely managed by the analytical laboratory or by others with only the the analytical laboratory or by others with only the testing performed by the Micro laboratorytesting performed by the Micro laboratory

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

Therefore, review controls around infrequent Micro Therefore, review controls around infrequent Micro testing. testing.

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1.1. Ask to see the mf’g suite active air and passive air viable Ask to see the mf’g suite active air and passive air viable monitoring SOPs (either classified, unclassified, or both). monitoring SOPs (either classified, unclassified, or both). Is the description of where settle plates or hand-held Is the description of where settle plates or hand-held active air samplers sufficiently specific (e.g., active air samplers sufficiently specific (e.g., supplemented w/ exactly mapped sampling locations supplemented w/ exactly mapped sampling locations inside the suite(s)). inside the suite(s)).

2.2. If it’s possible to observe a surface EM sampling, If it’s possible to observe a surface EM sampling, a) see when a disinfectant spray is applied to sampling a) see when a disinfectant spray is applied to sampling location (should only be done immediately afterward). location (should only be done immediately afterward). b) check what disinfectant or cleaner was used, whether b) check what disinfectant or cleaner was used, whether it’s it’s within expiry and whether it was ever validated (mainly within expiry and whether it was ever validated (mainly for a for a disinfectant). disinfectant).

3.3. For settle plate monitoring, check the procedure’s For settle plate monitoring, check the procedure’s exposure time versus actual practice. Also, If a laminar exposure time versus actual practice. Also, If a laminar flow hood is monitored on an EM program, does Micro use flow hood is monitored on an EM program, does Micro use a side-by-side pos. control plate or did it validate a side-by-side pos. control plate or did it validate exposure time (to guarantee against agar desiccation)?exposure time (to guarantee against agar desiccation)?

Environmental Monitoring (EM)Environmental Monitoring (EM)

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Environmental Monitoring (cont’d)Environmental Monitoring (cont’d)

4.4. Ask whether settle plate exposure times have ever Ask whether settle plate exposure times have ever gotten shorter in the procedure(s). If they have, did gotten shorter in the procedure(s). If they have, did the specs change downward as well?the specs change downward as well?

5.5. For classified suites, ask to see EM trends as well as For classified suites, ask to see EM trends as well as how alert and action limits were initially calculated how alert and action limits were initially calculated according to a procedure, and where these limits are according to a procedure, and where these limits are officially recorded/stated.officially recorded/stated.

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Microbiological IsolatesMicrobiological Isolates

1.1. Explain how local manufacturing area air, non-product-Explain how local manufacturing area air, non-product-contact surface, gown/glove, and water microbiological contact surface, gown/glove, and water microbiological isolates are obtained and cultured?isolates are obtained and cultured?

2.2. Ask to see the “library” list of environmental isolates.Ask to see the “library” list of environmental isolates.

3.3. Is there an SOP on the culturing, subculturing, and Is there an SOP on the culturing, subculturing, and “librarying” of local environmental isolates? Ask to see. “librarying” of local environmental isolates? Ask to see.

4.4. Does the procedure explain when the “library” is Does the procedure explain when the “library” is updated or revised with new organisms (especially for updated or revised with new organisms (especially for growth promotion tests)? growth promotion tests)?

5.5. When sampling from non-product-contact surfaces, are When sampling from non-product-contact surfaces, are inactivators such as Tween or lecithin used/ formulated inactivators such as Tween or lecithin used/ formulated within certain media (because of residual disinfectant)? within certain media (because of residual disinfectant)?

6.6. What data can demonstrate recovery from a non-What data can demonstrate recovery from a non-product-contact surface of a spiked test organism, using product-contact surface of a spiked test organism, using the above-mentioned inactivator in the media?the above-mentioned inactivator in the media?

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qq Unknown Organism ID TestingUnknown Organism ID Testing

1.1. What method(s) are used for microbiological isolate What method(s) are used for microbiological isolate ID?ID?

2.2. Does Micro use genotype (i.e., DNA) ID for sterility test Does Micro use genotype (i.e., DNA) ID for sterility test failures?failures?

3.3. Have the ID methods been developed/validated here or Have the ID methods been developed/validated here or tech transferred to this Micro Lab from another tech transferred to this Micro Lab from another company site?company site?

4.4. If fatty acid analysis via gas chromatography is used, If fatty acid analysis via gas chromatography is used, describe the procedures for GC system suitability and describe the procedures for GC system suitability and calibration.calibration.

5.5. Is a database used for recording and trending Is a database used for recording and trending organism ID results? If yes, ask to see this organism ID results? If yes, ask to see this demonstrated.demonstrated.

6.6. Is the database validated plus Part 11- compliant?Is the database validated plus Part 11- compliant?

7.7. What viable particle EM is performed in the Micro What viable particle EM is performed in the Micro Labs?Labs?

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Unknown Organism ID TestingUnknown Organism ID Testing(cont’d)(cont’d)

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

9.9. How is trending performed. For example, are all points How is trending performed. For example, are all points in a room averaged to get a result?in a room averaged to get a result?

10.10. When do water testing counts require ID and when is a When do water testing counts require ID and when is a water microorganism objectionable and/or requiring full water microorganism objectionable and/or requiring full speciation?speciation?See Attachment 3, WGD 10,509 “Evaluation of Microbial See Attachment 3, WGD 10,509 “Evaluation of Microbial Isolates Found in Water or Steam Systems”Isolates Found in Water or Steam Systems”

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Physical Micro Lab Audit Questions

1.1. How is the cleanliness/neatness of benches & hoods?How is the cleanliness/neatness of benches & hoods?

2.2. Examine purchased reagents for exp dates (e.g., LAL CSE, Examine purchased reagents for exp dates (e.g., LAL CSE, Gram Stain reagents).Gram Stain reagents).

3.3. Examine site-prepared media or reagents’ labeling of Examine site-prepared media or reagents’ labeling of expiration date and internal lot number. expiration date and internal lot number.

4.4. Does Micro maintain an inventory list (w/ exp dates) of all Does Micro maintain an inventory list (w/ exp dates) of all media and other reagents.media and other reagents.

5.5. For an instrument calibrated by a contract Lab, examine For an instrument calibrated by a contract Lab, examine a recent calibration report. Has a site mg’r or supervisor a recent calibration report. Has a site mg’r or supervisor signed the report to signify having reviewed it?signed the report to signify having reviewed it?

6.6. Does site calibrate all mechanical pipets, including Does site calibrate all mechanical pipets, including maxitip pipets, repipets, and repeat dispensers (i.e., maxitip pipets, repipets, and repeat dispensers (i.e., hand pump)?hand pump)?

7.7. Where are Where are malfunctioningmalfunctioning instruments/equipment instruments/equipment stored? How are they Labeled? stored? How are they Labeled?

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8. Does the Micro Lab or other qualified site persons conduct 8. Does the Micro Lab or other qualified site persons conduct some form of self inspection of the Micro Lab operations (EU some form of self inspection of the Micro Lab operations (EU required)?required)?

9.9. Ask if any equipment is undergoing qualification or is part of Ask if any equipment is undergoing qualification or is part of method validation, is it labeled “out of service” or “validation method validation, is it labeled “out of service” or “validation in progress”? in progress”?

10.10. Are areas in which environmental or product samples are Are areas in which environmental or product samples are handled/incubated adequately separated from live handled/incubated adequately separated from live cultures/subculturing, microbial ID/staining, or investigations?cultures/subculturing, microbial ID/staining, or investigations?

11.11. How are staff separated (or processed as in regowning and How are staff separated (or processed as in regowning and hand scrubbing) based on whether they routinely work in hand scrubbing) based on whether they routinely work in sampling versus live culture areas?sampling versus live culture areas?

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

Physical Micro Lab Audit Questions (cont’d)

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Physical Micro Lab Audit Questions (cont’d)

13.13. What is the Micro Lab’s source of water for preparing media & What is the Micro Lab’s source of water for preparing media & other reagents? Does the lab use a Mf’g area WFI or USP other reagents? Does the lab use a Mf’g area WFI or USP Purified Water drop or is the lab water a different purification Purified Water drop or is the lab water a different purification system from Mf’g?system from Mf’g?

14.14. Does Micro Lab uses a water storage vessel to hold drawn Does Micro Lab uses a water storage vessel to hold drawn water from some Mf’g water drop outside the lab? water from some Mf’g water drop outside the lab?

15.15. If yes to #14, does Micro label it or claim it as WFI in the tank?If yes to #14, does Micro label it or claim it as WFI in the tank?

16.16. Does Micro monitor their water source (even from a holding Does Micro monitor their water source (even from a holding tank) just like Mf’g POU’s?tank) just like Mf’g POU’s?

17.17. Are any uncontrolled instructions or copies of compendia, etc. Are any uncontrolled instructions or copies of compendia, etc. taped to the wall or the benches? taped to the wall or the benches?

18.18. Is the expiration dating of sterile utensils, sample containers, Is the expiration dating of sterile utensils, sample containers, etc. supported by sterility testing or by LAL testing (or both if etc. supported by sterility testing or by LAL testing (or both if both apply)? both apply)?

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8.8. Method ValidationMethod Validation

Microbiological Examination of Nonsterile Products and Microbiological Examination of Nonsterile Products and Sterility Test ValidationSterility Test Validation

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

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

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8.8. Method ValidationMethod Validation LAL Test ValidationLAL Test Validation

--Preparatory tests are conducted to verify that the sample Preparatory tests are conducted to verify that the sample solution doesn’t inhibit or enhance reaction. solution doesn’t inhibit or enhance reaction.

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

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Microbiological Examination of Microbiological Examination of Nonsterile Products--Audit QuestionsNonsterile Products--Audit Questions

1.1. What are the Microbial Enumeration Test (MET) What are the Microbial Enumeration Test (MET) specs for one or two nonsterile products, and specs for one or two nonsterile products, and how arrived at?how arrived at?

2.2. Ask to see the SOP for MET and the tests for Ask to see the SOP for MET and the tests for specified organisms of one or two nonsterile specified organisms of one or two nonsterile products and compare to the to methods products and compare to the to methods provided in USP <61>, <62>, and the drugs’ provided in USP <61>, <62>, and the drugs’ monographs (if specified). monographs (if specified).

3.3. For 1 or 2 nonsterile products, when/how are For 1 or 2 nonsterile products, when/how are plate count organisms identified? plate count organisms identified?

4.4. Does the product in question have Does the product in question have preservatives? How are they neutralized?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 Ask to see the method validation for the microbial enumeration test (MET), and see what is the % recovery of organisms. test (MET), and see what is the % recovery of organisms. Also, see how exhaustively the site tried to neutralize a Also, see how exhaustively the site tried to neutralize a bactericidal or preserved product in order to be able to bactericidal or preserved product in order to be able to conduct MET on it.conduct MET on it.

6.6. Verify that Lactose Monohydrate, NF; Gelatin, NF; Corn Verify that Lactose Monohydrate, NF; Gelatin, NF; Corn Starch, NF; and Talc, USP undergo the required compendial Starch, NF; and Talc, USP undergo the required compendial MLT if they are excipients.MLT if they are excipients.

7.7. Do any product ongoing stability protocols require MET? Ask Do any product ongoing stability protocols require MET? Ask to see the most recent stability failure due to MLT and the to see the most recent stability failure due to MLT and the resultant investigation (for US market, was a field alert issued resultant investigation (for US market, was a field alert issued upon initial OOS?).upon initial OOS?).

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Sterility Test Audit Questions

1.1. For a sample of site sterile products, ask for the original and For a sample of site sterile products, ask for the original and most recent bacteriostasis/fungistasis studies.most recent bacteriostasis/fungistasis studies.

2.2. For question #1, were USP <71> recommended organisms For question #1, were USP <71> recommended organisms used in the bacteriostais/fungistasis based on the media used in the bacteriostais/fungistasis based on the media used?used?

3.3. What is the frequency of revalidating the product What is the frequency of revalidating the product bacteriostasis/fungistasis (PIC/S recommends every 12 mo)?bacteriostasis/fungistasis (PIC/S recommends every 12 mo)?

4.4. Is the EM during sterility testing identical to that used during Is the EM during sterility testing identical to that used during sterile product mf’g? Compare SOPs for these two EMs.sterile product mf’g? Compare SOPs for these two EMs.

5.5. Are all sterility test media pre-incubated for 14 days to prove Are all sterility test media pre-incubated for 14 days to prove sterility prior to use or is this control test done concurrently?sterility prior to use or is this control test done concurrently?

6.6. Are negative controls/manipulation controls used in sterility Are negative controls/manipulation controls used in sterility tests? If not, why not?tests? If not, why not?

7.7. Other than during growth promotion, how well are anaerobic Other than during growth promotion, how well are anaerobic conditions maintained for fluid thioglycollate incubation?conditions maintained for fluid thioglycollate incubation?

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8.8. In aseptic filling, are samples drawn from beginning, middle, In aseptic filling, are samples drawn from beginning, middle, and end as well as immediately after interruptions and and end as well as immediately after interruptions and operator interventions. Does the local SOP call for these operator interventions. Does the local SOP call for these intervention samples?intervention samples?

9.9. Ask to see a list of OOSs for the sterility test. Choose at Ask to see a list of OOSs for the sterility test. Choose at least one OOS (or called lab investigation report) to audit for least one OOS (or called lab investigation report) to audit for agreement with applicable SOP and with scientific agreement with applicable SOP and with scientific method/logic.method/logic.

10.10. If terminal sterilization is used, do sterility test samples If terminal sterilization is used, do sterility test samples include some from validation-identified coolest load location?include some from validation-identified coolest load location?

11.11. If parametric release is used for a terminally sterilized If parametric release is used for a terminally sterilized product, ask for two different sterilization batch records and product, ask for two different sterilization batch records and compare the measured sterilization parameters to those compare the measured sterilization parameters to those given in the product dossier and/or NDA (e.g., fan speeds in given in the product dossier and/or NDA (e.g., fan speeds in cascading water/steam terminal sterilizer used by Baxter for cascading water/steam terminal sterilizer used by Baxter for Garenoxicin LVP).Garenoxicin LVP).

12.12. If media fills are performed, does Micro Lab record its daily If media fills are performed, does Micro Lab record its daily checks on the incubated vials, bottles, etc. (whether or not checks on the incubated vials, bottles, etc. (whether or not turbidity is observed)? turbidity is observed)?

Sterility Test Audit Questions (cont’d)

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12.12. Sterility suite is designed like a mf’g Grade A clean room, e.g.: Sterility suite is designed like a mf’g Grade A clean room, e.g.:

a) aseptic gowning area/airlock w/ step-over bench division, a) aseptic gowning area/airlock w/ step-over bench division, full-length wall mirror, gowning instructions, hand washing, full-length wall mirror, gowning instructions, hand washing, drying, and antiseptic application; drying, and antiseptic application;

b) annual cleanroom certification per ISO stds;b) annual cleanroom certification per ISO stds;

c) NLT 10-15 Pa press. differential with adjacent rooms, thec) NLT 10-15 Pa press. differential with adjacent rooms, the reading taken at least prior to entering suite; reading taken at least prior to entering suite;

d) flush-mounting of power outlets, light fixtures, hands-free d) flush-mounting of power outlets, light fixtures, hands-free intercom, etc., and no extraneous equipment; intercom, etc., and no extraneous equipment;

e) outer surfaces of samples & equipment entering test suite ise) outer surfaces of samples & equipment entering test suite is treated w/ sterile sanitizer (in EU, latter must be monitored); treated w/ sterile sanitizer (in EU, latter must be monitored);

f) environmental monitoring similar to Aseptic Core of Mf’g.f) environmental monitoring similar to Aseptic Core of Mf’g.


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13.13. Additionally: Additionally:

a) janitorial/cleaning supplies must be sterilized before use;a) janitorial/cleaning supplies must be sterilized before use;

b) ultraviolet lights, if used, are kept on at all times except b) ultraviolet lights, if used, are kept on at all times except when when testing is in progress or when viable particle EM is occurring; testing is in progress or when viable particle EM is occurring;

c) UV lights must be on an intensity measuring schedule;c) UV lights must be on an intensity measuring schedule;

d) where there is more than one parallel UV tube, they should d) where there is more than one parallel UV tube, they should bebe shielded from each other; shielded from each other;

e) the LAF hood must be on at least 30 min prior to any use;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,f) LAF hood is annually certified (i.e., magnehelic gauge, calibration, HEPA filter scan for leaks and an Emory challenge. 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:1. Ask for isolator validation & compare to USP <1208> criteria:

a) Set point of overpressure of interior can be maintained anda) Set point of overpressure of interior can be maintained and controlled during operation? Is there a press. hold test? controlled during operation? Is there a press. hold test?

b) a computational fluid dynamics analysis (CFD) and/orb) a computational fluid dynamics analysis (CFD) and/or smoke studies performed to determine the worst case airflow smoke studies performed to determine the worst case airflow locations in the isolator? locations in the isolator?

c) a six-log sterilization kill is confirmed in 3 consecutivec) a six-log sterilization kill is confirmed in 3 consecutive validation studies, & is BI resistance to the sterilization validation studies, & is BI resistance to the sterilization process estimated? process estimated?

d) containers, media, filter sets, tubing, and other supplies keptd) containers, media, filter sets, tubing, and other supplies kept inside the isolator are known or proved unaffected by inside the isolator are known or proved unaffected by sterilant penetration? sterilant penetration?

e) frequency of re-sterilization justified with data. Thise) frequency of re-sterilization justified with data. This includes proof of a maintained aseptic environment throughout includes proof of a maintained aseptic environment throughout a defined operational period. a defined operational period.

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Sterility Test Isolators Audit Questions (cont’d)

2.2. Ask for isolator use SOP and any gowning requirements (e.g., no Ask for isolator use SOP and any gowning requirements (e.g., no rings, watches or other sharp objects, including long nails).rings, watches or other sharp objects, including long nails).

3.3. Is the isolator directly in the flow path of an air supply grille (latter Is the isolator directly in the flow path of an air supply grille (latter could cool sections of isolator’s walls to cause condensation during could cool sections of isolator’s walls to cause condensation during vapor sterilization).vapor sterilization).

4.4. Ask to see isolator envir. monitoring SOP & the (viable and non-Ask to see isolator envir. monitoring SOP & the (viable and non-viable) specs or action/alert levels for air, surfaces, and gloves. viable) specs or action/alert levels for air, surfaces, and gloves.

5.5. If any isolator EM plates have shown growth, were the organisms If any isolator EM plates have shown growth, were the organisms identified? Ask to see these records. identified? Ask to see these records.

6.6. Does PM SOP include a glove (and half-suit) integrity test, Does PM SOP include a glove (and half-suit) integrity test, preemptive replacement, and possibly submersion testing of gloves preemptive replacement, and possibly submersion testing of gloves in a 0.1% peptone water followed by filtration of diluent and in a 0.1% peptone water followed by filtration of diluent and plating? How about transfer system gaskets and seals?plating? How about transfer system gaskets and seals?

7.7. How is the identity and composition of the gassing agent (sterilant) How is the identity and composition of the gassing agent (sterilant) assured (e.g., incoming inspection or supplier certification)?assured (e.g., incoming inspection or supplier certification)?

8.8. Is the gas generator in the PM program? Is the gas generator in the PM program?

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Endotoxin Testing Audit QuestionsEndotoxin Testing Audit Questions

1.1. What LAL method does the site use? If it’s only gel What LAL method does the site use? If it’s only gel clot, can trends ever be detected? This is admittedly clot, can trends ever be detected? This is admittedly a key shortcoming of gel clot.a key shortcoming of gel clot.

2.2. Does this gel clot method use the site’s own WFI as Does this gel clot method use the site’s own WFI as LAL diluent (in place of LAL water)? If yes, how does LAL diluent (in place of LAL water)? If yes, how does the site know the WFI isn’t just barely below the site know the WFI isn’t just barely below threshold for a clot?threshold for a clot?

3.3. Ask for the site’s SOP for preparing standards using Ask for the site’s SOP for preparing standards using reference std endotoxin (RSE). Compare the reference std endotoxin (RSE). Compare the reconstitution and dilution instructions with that of reconstitution and dilution instructions with that of USP <85> instructions, esp. the duration of USP <85> instructions, esp. the duration of vortexing.vortexing.

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

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Endotoxin Testing Audit Questions

5.5. When LAL reagent (lot) changes, does the Lab confirm When LAL reagent (lot) changes, does the Lab confirm the Labeled LAL reagent sensitivity (EU/ml) and use the the Labeled LAL reagent sensitivity (EU/ml) and use the Lab value (vs the supplier-labeled value) in calculating Lab value (vs the supplier-labeled value) in calculating MVD?MVD?

6.6. Has the Lab ever changed suppliers for LAL reagent? If Has the Lab ever changed suppliers for LAL reagent? If yes, did this result in a revalidation of the method (Per yes, did this result in a revalidation of the method (Per USP: “Revalidation for the test method is required USP: “Revalidation for the test method is required when conditions that are likely to influence the test when conditions that are likely to influence the test result change.”)?result change.”)?

7.7. What disposable plasticware, pipets, or tips are used in What disposable plasticware, pipets, or tips are used in LAL assays and how does the site know they are LAL assays and how does the site know they are pyrogen-free?pyrogen-free?

8.8. If pH is measured and adjusted on an LAL sample, how If pH is measured and adjusted on an LAL sample, how is this accomplished w/o adding pyrogens from pH is this accomplished w/o adding pyrogens from pH probe or titrant? probe or titrant?

9.9. If the site depyrogenates glassware, what is the time If the site depyrogenates glassware, what is the time limit for storage and use and what data is that based limit for storage and use and what data is that based on?on?

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

2.2. What documented qualification data (e.g., audit reports, CVs, What documented qualification data (e.g., audit reports, CVs, completed questionnaires) can be shown for these ESPs? completed questionnaires) can be shown for these ESPs?

3.3. What indicates that an ESP is site-quality-unit-approved?What indicates that an ESP is site-quality-unit-approved?

4.4. Ask to see quality/technical agreements between the site and Ask to see quality/technical agreements between the site and the respective ESPs, especially when the ESP performs routine the respective ESPs, especially when the ESP performs routine micro tests w/ product release implications.micro tests w/ product release implications.

5.5. Do reports provided by ESPs contain a review signature by an Do reports provided by ESPs contain a review signature by an appropriate manager or supervisor within the site?appropriate manager or supervisor within the site?

6.6. Can the site request and receive copies (or review at the ESP) Can the site request and receive copies (or review at the ESP) test result or other raw data residing at the ESP? test result or other raw data residing at the ESP?

7.7. Do changes to any procedures or specs of contract tests require Do changes to any procedures or specs of contract tests require prior approval by the site? Stated in quality/tech agreement? prior approval by the site? Stated in quality/tech agreement?

8.8. How does site track & reassess an ESP’s continuing How does site track & reassess an ESP’s continuing performance?performance?

Micro Contract Services Audit Q’s

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BibliographyBibliography1.1. USP <55> Biological IndicatorsUSP <55> Biological Indicators

2.2. USP <61> Microbiological Examination of Nonsterile Products: USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests Microbial Enumeration Tests

3.3. USP <62> Microbiological Examination of Nonsterile Products: USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified MicroorganismsTests for Specified Microorganisms

4.4. USP <71> Sterility TestsUSP <71> Sterility Tests

5.5. USP <85> Bacterial Endotoxins TestUSP <85> Bacterial Endotoxins Test

6.6. USP <1117> Microbiological Best Laboratory PracticesUSP <1117> Microbiological Best Laboratory Practices

7.7. USP <1208> Sterility Testing—Validtion of Isolator SystemsUSP <1208> Sterility Testing—Validtion of Isolator Systems

8.8. FDA Jan, 1994 Q&A on LAL TestFDA Jan, 1994 Q&A on LAL Test

9.9. FDA Dec, 1987 Guideline on Validation of the LAL TestFDA Dec, 1987 Guideline on Validation of the LAL Test

10.10. FDA 1993 Guide to Inspections of Microbio Pharm. QC LabsFDA 1993 Guide to Inspections of Microbio Pharm. QC Labs

11.11. PIC/S “Recommendation on Sterility Testing” July, 2004PIC/S “Recommendation on Sterility Testing” July, 2004

12.12. PIC/S “Recommendation on Isolators Used for Aseptic Processing PIC/S “Recommendation on Isolators Used for Aseptic Processing and Sterility Testing” July, 2004and Sterility Testing” July, 2004

13.13. Cundell, Anthony; “Managing the Microbio Quality of Pharm. Cundell, Anthony; “Managing the Microbio Quality of Pharm. Excipients.”Excipients.”

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Please ask any questionsPlease ask any questions

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If you don’t have any more questions, then let me ask a few:If you don’t have any more questions, then let me ask a few:

1.1. Give some requirements for a sterility test suite that resemble those Give some requirements for a sterility test suite that resemble those for a sterile core?for a sterile core?

2.2. Give at least 3 questions that can be asked about a sterility test Give at least 3 questions that can be asked about a sterility test isolator.isolator.

3.3. For a purchased biological indicator (BI), what would be on its C of A? 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 must the site lab test and what can it farm out?

4.4. What mat’ls and treated labware in the micro lab must have exp What mat’ls and treated labware in the micro lab must have exp dating? dating?

5.5. What activities should be and what mat’ls storage areas should be well What activities should be and what mat’ls storage areas should be well separated in Micro?separated in Micro?

6.6. Give requirements for surface sanitizers used in Grade A or B suites.Give requirements for surface sanitizers used in Grade A or B suites.

7.7. What is asked about the Micro lab glassware/labware washer and why?What is asked about the Micro lab glassware/labware washer and why?

8.8. What are some questions related to bacterial cultures purchased and What are some questions related to bacterial cultures purchased and utilized in the Micro lab?utilized in the Micro lab?

9.9. What can you say about the control of Micro Lab worksheets?What can you say about the control of Micro Lab worksheets?

Auditing the Microbiology Laboratory

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