GMP Compliance Auditing of Non-sterile Quality Control Microbiology Laboratories

DONALD J. ENGLISH

SENIOR MANAGER – R&D MICROBIOLOGY

AVON PRODUCTS, INC.

SUFFERN, NEW YORK 10901

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Personnel Job Description

Organization

Training and Skills

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Personnel – Job Descriptions Must contain the following:

Job title

Reporting title of the position’s immediate supervisor.

Key functional and relational responsibilities

A summary of the general nature and level of the position.

A list of duties or tasks that a position is responsible for conducting

needs to be clearly described.

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Personnel - Organization

Organizational Chart Organizational structure should be defined.

Adequate laboratory staffing should be present.

Organizational chart should show independence of each quality unit, such as quality assurance or

quality control.

Supervisors must have the education, training and experience to perform assigned

job functions.

Adequate number of properly trained personnel for the size of the organization.

Personnel – Organizational Charts

Needs to illustrate the reporting relationships within the Quality Control Microbiology Testing Laboratory, as well as, that within the Quality System.

Manager of Department

Preservative Challenge Testing Supervisor

Microbial Content Supervisor

Quality Control Director

Microbial Media and Count Diluent Preparation Supervisor

Technician TechnicianMicrobiologist MicrobiologistMicrobiologist Microbiologist

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Personnel – Training and Skills

Personnel should have skills based upon relevant training and experience acquired or combination thereof, that are appropriate to their responsibilities and activities.

Appropriate cGMP training should be provided on an annual basis.

Newly Recruited Personnel

Training should be appropriate to the duties assigned to them.

Training is an on-going process that is subject to regular updates.

Personnel – Training Records

Types of Training Conducted: GMP and GLP Training (e.g. Yearly frequency) Laboratory Training (Must be conducted at a periodic frequency). Standard Operating Procedures (e.g. Laboratory Equipment and Microbial Growth Media and

Count Diluent Preparation and Sterilization)

Microbial Test Methods

Microbial Content – Raw Ingredients and Finished Products

Preservative Challenge Testing

Environmental Sampling and Testing (e.g. Air, Compressed Air and Water).

Microbial Monitoring for Cleaning and Sanitization of Manufacturing Equipment

Performance Assessment or Proficiency Training Aseptic technique, conductance of a microbial test method, etc.

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Personnel - Responsibilities

All laboratory personnel should:

Know their position in the organizational structure.

Know their defined responsibilities and activities.

Have access to documents and comply with documents relevant to their particular responsibility.

Comply with personal hygiene standards.

Be encourage to report irregularities and other non-conformities.

Have adequate educational training and skills to perform assigned responsibilities and activities.

Microbiology Laboratory Design Prevent Accidential Microbial Contamination to Samples and Personnel:

Separate areas for conducting microbial analysis of samples and support functions such as sample login,

media preparation, sample preparation and storage of personnel belongings.

Surfaces should be nonporous, cleanable and sanitizable.

Adequate lightning.

Adequate ventilation to minimize the possibility of air currents.

Adequate electrical service for laboratory equipment.

Adequate sink areas with running hot and cold water service.

Laboratory access is restricted to minimize foot traffic by non-laboratory personnel.

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Microbiology Laboratory Equipment General Recommendations:

Have a master list of equipment requiring calibration and preventative maintenance. All equipment needs to be maintained and routinely calibrated according to manufacturers directions.

Each piece of equipment should have either a maintenance logbook or electronic file containing service reports with the following information: Date of service. What was repaired? Was re-calibration necessary?

Calibration stickers – should be present on certain pieces of equipment to aid when calibration is required and contain the following information: Equipment Type Serial or Identification Number Date of Calibration Calibrator’s name or Initials Next Scheduled date of Calibration

Malfunctioning equipment or out-of-calibration equipment needs to be labeled, removed from service until repairs have been made and re-calibration has been performed.

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Laboratory Equipment – Autoclaves/Sterilizers Initially Installed:

Installation Qualification (IQ)

Operational Qualification (PQ)

Performance Qualification: Heat distribution studies of an empty chamber

Heat distribution and penetration studies of typical loading patterns for the chamber.

Routine Annual Calibration of: Temperature Recorders

Pressure Gauges

Timers

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Laboratory Equipment – Incubators/Refrigerators/Freezers

Qualification Installation Qualification

Operational Qualification

Performance Qualification

24-hour temperature distribution mapping study of the interior chamber.

Open door test Used to determine how long the unit takes to recover back to the original operating temperature.

Simulated power failure test Used to see how long the temperature of the unit can be maintained without electrical power.

Verification of accuracy of temperature recording charts if present.

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Laboratory Equipment - Balances

Balance Calibration Each balance needs to be calibrated at least once per year. Reference weights for balance calibration should cover the operating range of the balance (e.g. 1, 10,

25, 100, 500 and 1000 grams) Each reference weight used for calibration should have been calibrated against an National Institute of

Standards and Technology (NIST) or equivalent standard. Confirm accuracy of the balance to +/- 0.1 grams

Weekly Weight Balance Calibration Check Purpose is to detect when a balance starts to be out of calibration by using a NIST or equivalent

standard balance weight.

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Laboratory Equipment – Biological SafetyCabinets/Class 100 or ISO 5 Laminar Flow Hoods

Recommended that each Biological Safety Cabinet and Laminar Flow Hood should calibrated once

per year for:

Biological Safety Cabinets, Class II, Type A2 Requirements Minimum Intake Air: 100 FPM

Downward Air Flow: 50-60 FPM Dispersed Oil Particulate (DOP) Leak Test: Less than 0.01% of DOP particles >0.3 microns in diameter Germicidal Light – verification that the UV lamp is producing the necessary wavelength

Laminar Flow Hood Requirements Airflow Test: 90 FPM +/- 20%

Dispersed Oil Particulate (DOP) Leak Test: Less than 0.01% of DOP particles >0.3 microns in diameter Particle Count Test: Not more than 100 particles of >0.5 microns per cubic foot or not more than 3520

particles of >0.5 microns per cubic meter.

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Laboratory Equipment - Centrifuges Recommended to be calibrated once per year for:

Temperature – if the temperature of the unit is controlled by refrigeration

Rotor Speed

Timer

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Laboratory Equipment – pH Meters

Recommendations: pH meters need to be calibrated and standardized on either a daily basis or when used.

Need to use at least 2 certified pH buffer solutions that is closest to the expected

pH range of the test sample.

Certified buffer solutions must be standardized against an NIST or equivalent

reference.

pH buffer solutions should not be used past their expiration date.

Taking the pH of prepared microbial growth media:

Microbial growth agar – flat bottom pH electrode

Liquid microbial growth media (e.g. broths) – standard pH electrode

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Laboratory Equipment – Microbial Identification Systems

Semi-automated and automated microbial identification systems (e.g.

phenotypic, phylogenetic and genotypic)

Installation Qualification

Operational Qualification

Performance Qualification

Use both know pedigree (ATCC) and wildtype microbial strains.

Conducted identification of microbial strains at 3 separate times by using different individuals.

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Laboratory Equipment – Temperature Monitors

The following temperature monitoring devices need to be calibrated

on at least an annual basis:

Thermometers

Temperature Recording Charts (Fixed and Portable)

Wireless Temperature Data Loggers

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Laboratory Equipment – Media Dispensers

Media dispensers (e.g. pumps, syringes) are commonly used within

Microbiology Laboratories to dispense volumes of microbial count diluents

and liquid microbial growth media into bottles, test tubes or dilution blanks.

Need to be calibrated by using appropriate sized graduate cylinders before

and after to ensure that accurate and consistent quantities are delivered

throughout the dispensing process.

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Laboratory Equipment - Spectrophotometers Spectrophometers are mostly used to prepare microbial inoculums for use in

preservative challenge, MIC/MLC and validation testing.

Calibration Recommendations: Daily or weekly usage:

Use curvettes containing 0.85% Saline Solution and Crystal Violet Gram Stain to adjust

the unit to 0 and 100% light transmittance before conducting a turbidity determination

of a microbial suspension.

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Laboratory Equipment – Water Activity Instruments

Calibration recommendations: Perform on each day of use.

Need to use 2 fresh certified standard water activity salt solutions One salt solution should have a water activity reading below the expected water activity reading of the

test sample.

The second salt solution should have a water activity reading that is above the expected water

activity reading of the test sample.

Expired salt solutions should not be used to take a water activity reading of a test sample.

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Laboratory Equipment – Pippetters

2 Types of Pippetters: Micro-pippetter (e.g. Dispenses a volume of 1 to 10 microliters).

Macro-pippetter (e.g. dispenses a volume of 0.1 to 2.0 milliliter).

Each Type of Pippetter should be calibrated for: Accuracy Percent

Precision Percent

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Laboratory Equipment - Microscopes Conducted maintenance on a microscope must be documented.

Each of the following items of a microscope should be inspected on an annual basis:

Lenses

Alignment

Mechanical Aspects

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Laboratory Equipment – Mechanical Air Samplers

Can be used to determine the microbial bioburden of air samples at various locations

within a Microbiology Laboratory and manufacturing facility.

Annual Calibration Recommendations: Timer of the unit needs to be calibrated so that the correct air volume is sampled.

If mechanical air sampler is used to determine the microbial bioburden of compressed air, the air

flow regulator will need to be calibrated.

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Laboratory Equipment – Water Treatment System

Used to produce the desired grade of water (distilled or deionized) for use in

preparing microbial media and count diluents.

Should be periodically monitored for producing the desired microbial and chemical

quality attributes:

Microbial Attributes <100 CFU/milliliter

Chemical Attributes Total Organic Carbon (TOC): <500 parts per billion (ppb) [µg/liter].

Conductivity at 25oC: <1.3 µS/cm

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Microbial Culture Maintenance

Need to Verify Laboratory Microbial Culture Maintenance Program such as:

How is the laboratory storing and maintaining microbial cultures?

Are each of the microbial cultures that are used within the laboratory passed or purchased from a

vendor (e.g. microbial suspensions, culture sticks or loops) for no more than 5 passages from the

original ATCC vials?

Is proper documentation available indicating that each microbial strain being used within the

laboratory been identified to the genus/species level by either phenotypic, phylogenetic or genotypic

means?

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Microbial Growth Media - Commercially Prepared Each received lot of prepared microbial growth media should have a Certificate of Analysis (COA) with the following

information:

Manufacturers Name and Address of Preparation Facility.

Expiration Date

pH Check

Sterility Check

Microbial Growth Promotion Data.

Each receipt of a commercially prepared lot of microbial growth media should be re-checked for the following to verify

that the shipping process did not have an adverse effect:

pH Check

Confirmation of Sterility

Microbial Growth Promotion

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Microbial Growth Media – Prepared In-HousePrepared In-House: Dehydrated Microbial Growth Media Suggestions:

Each container of dehydrated microbial growth media be labeled with the following: Date Received

Date Opened

Dehydrated microbial growth media containers within the laboratory follow FIFO practices (First In,

First Out).

Containers of dehydrated microbial growth media and supplements are stored according to

manufactures directions.

Expired dehydrated microbial growth media containers need to be discarded to prevent usage.

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Microbial Growth Media – Prepared In-House (Continue)

Prepared In-House : Rehydration and Sterilization Suggestions Proper type of water (e.g. distilled or deionized water) is used to rehydrate microbial growth media.

Each batch of microbial growth media should have a batch record: Name of Media

Date of Preparation

Manufacturers Lot Number

Expiration Date

Quantity of Each Ingredient Weighed

Quantity Prepared

Signature of Preparer

Volume Dispensed

Date of Sterilization

Sterilizer Identification and Run Number

Each batch record is reviewed for accuracy and approved by laboratory

supervisor.

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Microbial Growth Media – Prepared In-House

After sterilization before use in microbial testing, each prepared

batch of microbial growth media should be checked for the following:

pH Check

Confirmation of Sterility

Microbial Growth Promotion Testing

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Storage and Expiration Dating of Laboratory Prepared Microbial Growth Media

Batches of prepared microbial growth media should be stored under conditions

recommended by the vendor.

Microbial growth promotion studies be conducted to verify storage condition and aged

of the microbial growth medium did not affect its ability to support the growth of

microorganisms.

Growth Promotion Studies of Freshly Prepared vs. Aged Microbial Growth Media.

Weight Comparison Studies of Freshly Made to Aged Microbial Growth Agar Petri Dishes.

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Microbial Count Diluents Each batch of microbial count diluents should have an individual batch record with the following information:

Name of Microbial Count Diluent

Date of Preparation

Manufacturers Lot Number of Ingredients Used in the Diluent

Quantity of each ingredient weighed

Quantity Prepared

Signature of the Preparer

Volume Dispensed

The Number of Units Dispensed.

Each batch record is reviewed for accuracy and signed by laboratory supervisor.

After sterilization, each batch of microbial count diluent should be checked to confirm sterility.

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Chemicals and Biochemical Reagents

Includes such things as Gram stain reagents, Oxidase, Coagulase, Catalase, etc.

Checked for their expiration date prior to usage.

Expired materials should not be used in the conductance of microbial testing.

Specificity of each new lot of biochemical reagent should be checked prior to usage or at the same time

with an unknown microbial isolate by using positive and negative microbial controls:

Examples:

Gram Stain Reagents: S. aureus ATCC 6538 and Escherichia coli ATCC 8739

Coagulase Test: S. aureus ATCC 6538 and S. epidermidis ATCC 12228.

Oxidase Test: Ps. aeruginosa ATCC 9027 and Escherichia coli ATCC 8739

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Microbial Biochemical Identification Cards/Kits and Reagents

To determine as to whether shipping conditions had not or had an adverse effect

on the accuracy of an identification call for a microbial isolate, the following

practices is recommended:

Biochemical Identification Cards or Kits (Phenotypic) Each receipt should be tested by using the recommended Quality Control test microorganisms by the

manufacturer.

Extraction and Sequencing Reagents for 16S or 23S rRNA Analysis (Phylogenetic) Each receipt of reagents should be tested by using known pedigree (e.g. ATCC) strains of bacteria,

yeast and mold.

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Microbial Test Procedures Microbial Content Test Methods

Enumeration

Enrichment

Preservative Challenge Test Methods

Environmental Test Methods

Ambient Air

Compressed Air

Environmental Surfaces

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Microbial Content – Enumeration Plate

Count Test Method

GENERAL ASPECTS

Plate Count Neutralizing

Diluent

1:10 Dilution Dispense

&

Incubate

Count Number of Microorganisms/gram or ml

Sample

Microbial Content -Enrichment Test Method

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GENERAL ASPECTS

1.0 or 10.0 grams

Microbial Enrichment

Broth with

Neutralizers

Streak onto Selective/

Differential Microbial Growth Agars

Incubate

Gram-stain and Identify Recovered Microbial Isolates

Sample

Preservative Challenge Test Method

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GENERAL ASPECTS

Standardized Microbial

Suspension

Inoculated Product

Removal of 1.0 gram

Aliquots at Specified Intervals

Neutralization of Preservative Antimicrobial

Activity

Recovery of Surviving

Microorganisms

Calculation of Percent or Log10

Reduction

Microbial Content Test Methods - Validation Enumeration (Plate Count)

Test Microorganisms: Pseudomonas aeruginosa ATCC 9027 Staphylococus aureus ATCC 6538 Candida albicans ATCC 10231 Escherichia coli ATCC 8739 Burkholderia cepacia ATCC 25416 Aspergillus brasiliensis ATCC 16404

Level of Inoculation: Approximately <100 CFU/gram

Enrichment Test Microorganisms:

Ps. aeruginosa ATCC 9027 S. aureus ATCC 6538 C. albicans ATCC 10231* E. coli ATCC 8739 B. cepacia ATCC 25416 Salmonella enterica subsp. enterica servor Typhimurium ATCC 14028**

* Cosmetics, ** Ingestible Non-sterile drug products and dietary supplements

Level of Inoculation: 10 to 100 CFU/0.1 milliliter

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Preservative Challenge Test Method - Validation Test Microorganisms:

Pseudomonas aeruginosa ATCC 9027

Escherichia coli ATCC 8739

Staphylococus aureus ATCC 6538

Burkholderia cepacia ATCC 25416

Candida albicans ATCC 10231

Aspergillus brasiliensis ATCC 16404

Level of Inoculation: Approximately <100 CFU/gram

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Validation of Microbial Enumeration Plate Count and Preservative Challenge Test Method

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GENERAL ASPECTS

Prepare a 104

CFU/ml Suspension

1g + 8.9 ml Neut. Diluent

9.9 ml of

Neut. Diluent

0.1 ml

(<1000 CFU)

0.1 ml (<1000 CFU)

2 x 1.0 aliquots

2 x 1.0 aliquots

Incubate

Count # of microbial colonies

per Petri dish, average counts, convert average counts to Log10

values, and compare

Add to separate

100 x 15 mm Petri Dishes

and add 18 to 20-ml/Petri

Dish of melted

Microbial Growth Agar,

mix, and solidify.

(<100 CFU)

Example of Microbial Count/Preservative Challenge Validation Test Data for a Non-sterile Cream Product Formulation

Test Organism Microbial Counts with Sample

Microbial Counts without Sample

Log10 Value of Counts with Sample

Log10 Value of Counts with Sample

Log Difference

Counts Ave. Counts Ave.

Ps. aeruginosa ATCC 9027

122, 172 147 209, 190 199.5 2.17 2.3 (+) 0.13

S. aureus ATCC 6538

72, 90 81 92, 91 91.5 1.91 1.96 (+ ) 0.05

E. coli ATCC 8739

78, 90 84 81, 93 87 1.92 1.94 (+) 0.02

B. cepacia ATCC 25416

25, 35 30 28, 40 34 1.48 1.53 (+) 0.05

C. albicans ATCC 10231

8, 5 6.5 15, 9 12 0.81 1.08 (+) 0.27

A. brasiliensis ATCC 16404

54, 68 61 44, 47 45.5 1.78 1.65 (-) 0.13

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Validation of an -Enrichment Test Method

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GENERAL ASPECTS

1.0 or 10.0 grams

Microbial Enrichment

Broth with

Neutralizers

Streak onto or Inoculate Selective/

Differential and Non-selective

Microbial Growth Broths/Agars

Incubate

Gram-stain and Identify Recovered Microbial Isolates

Sample

10-100 CFU/0.1 ml

Inoculum

For each test microorganism

Types of Microbial Growth Broths and Agars for Validation of an Enrichment Broth Test Method

Enrichment Broths

Soybean-Casein Digest Medium with 4% Tween 20 and 0.5% Soy Lecithin (USP Section 62)

Modified Letheen Broth (FDA Bacteriological Analytical Manual – Cosmetics)

Eugon LT 100 Broth (ISO Microbial Methods for Cosmetics)

D/E Neutralizing Broth

Selective/Differential and Non-Selective Microbial Growth Broths/Agars

Cetrimide (FDA BAM, ISO and USP) or Pseudomonas Isolation Agar – Ps. aeruginosa

MacConkey Broth and Agar (USP) /MacConkey Agar (FDA BAM)/ MacConkey Agar and Levine EMB Agar (ISO) – E. coli

Vogel Johnson Agar (FDA BAM-Optional) /Mannitol Salt Agar (USP)/Baird-Parker Agar (FDA BAM and ISO) – S. aureus

MacConkey Agar and Soybean-Casein Digest Agar Medium – B. cepacia

Rappaport Vassiladis Salmonella Enrichment Broth and XLD Agar (USP) – Salmonella

Reinforced Medium for Clostridia and Columbia Agar (USP) and Modified Letheen Agar/5% SBA [Anaerobic] (FDA BAM) – Clostridia

Sabouraud Dextrose Agar (ISO) – C. albicans

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Environmental Monitoring - Air

Ambient Air – certain sites within a laboratory and manufacturing facility can pose a risk in causing microbial contamination of susceptible raw ingredients, bulk finished product and finished stock.

Is there a written program including a map of air sampling sites and frequency?

What is the microbial air sampling method? Gravity Settling Plates

Mechanical Air Sampler

Are microbial identifications conducted on recovered representative microbial colonies?

Are the microbial test results trended for each air sampling site?

Compressed Air – is susceptible to microbial contamination because it is stored under humid conditions.

If being sampled, what is the compressed air sampling method?

What is the frequency of compressed air sampling?

Are microbial identifications conducted on recovered representative microbial colonies?

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Environmental Sampling – Sanitization of Equipment Surfaces

Is there a written program including identification of sampling sites for each piece of equipment?

What is the microbial sampling method?

Swabbing (e.g. Calcium alginate swabs or cotton swabs)

Rinse water analysis

Contact microbial growth agar plates

Is there proper neutralization of sanitizer/disinfectant residues if chemical sanitization is used?

Are representative recovered microbial colonies being identified to the genus/species level?

What is the microbial test criteria for each sampling site in a piece of equipment?

Is it appropriate for the type of microbial test specifications for the product being manufactured?

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Purified Water Systems

Laboratory Purified Water System

Facility Purified Water System with an Outlet in the Microbiology

Laboratory

Is there a current engineering drawing present of the facility purified water system? Need to verify that each of the components of the system indicated on the drawing has been installed.

Has a validation been conducted? Installation Qualification

Operational Qualification

Performance Qualification

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Microbial Testing of Purified Water Systems

What is the frequency of microbial testing (e.g. once per week or several

times a week)?

Are purified water samples aseptically collected?

Microbial Test Method Is the method membrane filtration or plate count?

What type of microbial growth agars are being used (e.g. R2A or Plate Count Agar)?

What is the Incubation temperature and time of incubation?

Are recovered microbial isolates identified to the genus/species level?

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Documentation

Purpose:

Provides a written record of the activities that occurs within the laboratory such as:

Training

Standard Operating Procedures

Microbial Test Methods

Microbial Test Results

Provides an operational history of the laboratory.

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Documentation – Records and Reports

Logbook, Worksheet, or Laboratory Information Management System (LIMS) Complete microbial test data must be present to show compliance with established

specifications and standards. Description of the test ample received for testing.

Source

Quantity

Lot Number

Date sample taken

Date sample was received for testing

A statement of each test method used.

Statement of sample weight or measure used for each microbial test.

A statement of microbial test result conformance with specification (Pass/Fail).

Initials/signature of the person who conducted the microbial test for a sample with date of performance.

Initials/signature of person who conducted review for accuracy (Second person for review).

Good Documentation Practices – Microbial Test Methods/Standard Operating Procedures

Is each microbial test method scientifically based (e.g. compendia or guideline)?

Is each Standard Operating Procedure/microbial test method written in a clear and

concise manner?

Is there a secondary ‘qualified” reviewer for each issued Standard Operating

Procedure/microbial test method?

Are written Standard Operating procedures and microbial test methods periodically

review and updated as necessary to document any changes?

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Good Documentation Practices – Laboratory Forms

Are laboratory forms for Standard Operating Procedures/microbial test methods standardized and

controlled?

Are errors/cross-outs on laboratory forms properly documented (e.g. initials of person with date

[mmddyy])?

Do not contain extraneous marks or strange comments?

Are laboratory forms periodically updated to reflect any changes that have occurred in a Standard

Operating Procedure or microbial test method?

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Good Documentation Practices - Logbooks

Are errors/cross-outs initialed and dated?

Are logbook reviews conducted in a timely manner?

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Documentation - Discrepancies

Any discrepancies in equipment and microbial test results for raw

ingredients, finished products and environmental test samples

should be:

Investigated

Documented

Evaluated by appropriate personnel

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Documentation - Deviations

A minor change could invalidate a test method.

Any deviation must be recorded and justified

Written specifications

Standards

Sampling plan

Microbial test methods or other laboratory control measures.

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Documentation - Investigations

Is there a formalized out-of-specification investigation procedure for microbial

test results?

Purpose of this procedure:

To establish a root cause for each microbial test result failure.

Helps to institute corrective actions to prevent a future identical microbial test result failure.

Does this investigation procedure contain the following steps?

Process defined

Detail the findings of the investigation

Reporting of results

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Conclusions

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Thank You

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