Analytical and Microbiology Laboratory (GLP) Manuals

Analytical and microbiology laboratory GLP manuals provide guidelines and procedures typically needed in laboratory operations.

Microbiological retesting is an investigational tool that may be used as part of the Investigational Measurements Protocol. The number of retests performed on a sample should be specified in advance, and a point should be prescribed when to end testing and begin evaluating the product/material.

According to the FDA OOS Draft Guidance, repeat testing until a passing result is obtained (i.e., testing into compliance) is objectionable under the cGMPs.

If the compendia does not define retest criteria, the decision to retest shall be made by quality assurance management and must be based on a sound scientific rationale. A retesting protocol should be created and approved by laboratory supervision before any retesting.

The retest protocol must be based on the specific problem identified, the product history, the method, the batch/lot used, and any applicable compendial requirements.

Microbial Attributes of Non Sterile Solid Materials Testing

Microbial attribute testing estimates the number of and/or types of microorganisms present in a sample of material or product by utilising the total aerobic bacterial count test, the total yeast and mould count test, or the presence of specified microorganisms test. Because of the wide diversity of materials and finished solid oral drug products, not all products are equally susceptible to microbial contamination. Therefore, microbial attributes testing may not be the same for all products. This guidance has been developed to help determine the need for microbiological testing.

Microbiological Testing Practices in Cleaning Validation for Medicinal Products

If it is determined that microbiological testing is needed, where possible, it is recommended that bioburden samples be collected via rinse sampling. A known quantity of Purified Water or Water for Injection (dependent on the final rinsing type prescribed by the cleaning procedure) sufficient to completely rinse the product contact surfaces of the equipment is used. A sample of the effluent rinse is collected in a sterile container and microbiologically evaluated.

Sampling should be conducted according to a sampling plan that does not contribute to the potential contamination of the samples or impact the integrity of subsequent samples. If surface sampling is needed, sterile swabs saturated with a sterile diluent such as Sterile Water for Injection or Sterile Saline Solution are used.

Alternatively, RODAC® plates may be employed for sample sites where the entire surface of the RODAC® plate can be in contact with the equipment surface. If RODAC® plates are used, the sampled surface should be cleaned by a qualified cleaning procedure before use of the equipment for production (e.g. wiped with a sterile saturated 70% isopropyl alcohol wipe).

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Stability Study Considerations for Planned or Unplanned Deviations and Process Changes

ICH Q7A states, “Changes can be classified (e.g. as minor or major) depending on the nature and extent of the changes and the effects these changes may impart on the process.” A minor process change may be defined as a change that has no impact on registration or validation, and little or no impact on product quality. A minor process change should not require major testing or development efforts such as the generation of supportive stability data. If stability data are needed, the change should be categorised as major.

A major process change may be defined as a change that potentially impacts registration, validation, product quality or GMP compliance. The evaluation of a major process change may require development support, including the collection of stability data.

In addition to determining whether a change is major or minor, the site Validation Team is responsible for determining whether stability data are needed to support a process change.

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Sterility Testing Practices

The following items or issues should be addressed in approved methods and/or procedures:

– Validation and Revalidation of Sterility Test Methods.

– Sample Collection, Transport and Storage.

– Pooling of Sterility Test Samples.

– Sterility Test Methods.

– Negative Controls.

– Incubation of Sterility Test Samples.

– Cleaning and Sanitation of Sterility Test Area.

– Preparation, Testing, Approval and Storage of Sterility Test Media, Reagents and Supplies.

– Growth promotion tests.

– Maintenance of Sterility Test Area Equipment and Systems.

– Environmental Monitoring Program for the Sterility Test Area.

– Identification of Micro-organisms from Positive Sterility Test Samples.

– Preparation and Decontamination of Containers for Sterility Testing.

– Gowning (if applicable).

– Sterility Test Failure Investigation and Response.

– Decontamination and Disposal of Sterility Testing Waste.

– Interpretation of Sterility Test Results.

– Trending of Sterility Test Data.

– Change Control.

Endotoxin Testing

Recognised LAL Test Methods:

– Gel-clot

– Turbidimetric kinetic

– Turbidimetric endpoint

– Chromogenic kinetic

– Chromogenic endpoint

The gel-clot techniques detect or quantify endotoxins based on clotting of the LAL Reagent in the presence of endotoxin. The turbidimetric method measures increases in turbidity. Depending on the test principle used, the turbidimetric technique is classified as end-point or kinetic.

The chromogenic method measures the chromophore released from a suitable chromogenic peptide by reacting endotoxins with the LAL Reagent. Depending on the test principle employed, the chromogenic technique is classified as endpoint or kinetic chromogenic.

Guideline for Stability Testing for R&D

This section of the Stability Guideline provides recommendations on stability testing requirements but provides the flexibility to use alternative approaches with scientifically justifiable reasons.

Stability testing will provide evidence of the quality of the material tested under the influence of various environmental factors such as temperature, humidity, and light and how the material’s quality varies with time. Stability data are used to establish the re-test period for a drug substance or shelf life for a drug product and the recommended storage conditions.

Stability studies within R&D can be classified as:

– Investigational stability studies (preliminary stress/compatibility)

– Development stability studies (to support non-clinical/clinical studies and additional stress/compatibility)

– Formal stability studies on primary batches (for MAA)

Protocols should be prepared at all stages to clarify what should be done. The level of detail will increase during development.

Investigational stability studies:

Within R&D, when a new drug substance enters development, a quick assessment of the stability characteristics is required. Compatibility studies to support formulation development may also be required, and drug products should be evaluated under appropriate conditions to understand degradation pathways.

Significant levels of physical or chemical degradation can help to understand degradation pathways and develop stability-indicating methods. At a minimum, one batch of drug substance and product should be tested.

Investigational studies should involve conditions that evaluate thermolytic, hydrolytic, oxidative and photolytic degradation pathways.

Stability data generated for investigational studies are not normally included in any type of clinical application.

Water Quality Standard

Water is the most widely used material in manufacturing Active Pharmaceutical Ingredients (API) and medicinal products and in laboratory testing. Its presence is everywhere in the production environment, being used not only as a starting material but also as a cleaning agent and as the source water for steam sterilisation processes. It is used in laboratories to produce various reagents and solutions.

The control of its chemical and microbiological quality is critical in all these areas.

Sampling:

A procedure(s) must be available describing sampling requirements and cover items such as:

– Preparation of sampling containers, e.g. containers for microbiological testing need to be sterile

– Frequency of sampling

– Location of sampling points

– Quantity of sample required

– Sampling instructions

– Sample storage, including any time limitations to perform the analysis

– Training of samplers

Samples should be collected from points of use in the same way as or in a manner that simulates

how the water is used during routine production. Where possible, samples will be taken on a rotational basis to ensure that points are not always sampled on the same day.

Samples that are not taken from points of use and which may be taken from a sample point that is exposed to the elements may be flushed for a defined time to ensure the sample reflects the water in the system.

Testing:

Written, approved and validated test methods are required. Test methods per the EP or United States Pharmacopeia (USP) are validated.

Potable Water:

Testing for the full analytical requirements for Potable Water is usually outside the scope of the typical pharmaceutical laboratory. The recommended practice is to receive Analytical Reports and/or Certificates of Analysis from the municipality/supply company providing the water and supporting local test data.

If alternative water sources are used, e.g. borehole/healthy water, it may be possible to arrange for the local municipality/supply company or a contract water-testing laboratory to sample and test the water against the appropriate standard.

The local site should perform a limited number of analytical monitoring tests that have been pre-established in a local procedure or standard.

Purified Water:

The tests, methodology and specifications for Purified Water are described in the relevant Pharmacopoeias.

Endotoxin-Controlled Purified Water:

The tests, methodology and specifications for Endotoxin-Controlled Purified Water are, as minimum, the same as for Purified Water with the addition of the Bacterial Endotoxin Test with a limit of not more than 0.25 IU/ml.

Storage and Expiry Dating of Analytical Reagents

Where no conditions are specified, the material must be stored in sealed containers at room temperature in an area preventing exposure to excessive heat and humidity and commensurate with the potential hazard of the material.

Example: Flammable reagents and solvents must be stored in fire-resistant cupboards. Large packages and hazardous materials must be stored in fire-resistant cupboards and near the ground. Material producing toxic fumes must be stored in a fume cupboard or ventilated area.

Preparation & Maintenance of Stability Protocols and Stability Master Plans

The first step is to create a Stability Protocol and Stability Master Plan.

The protocol shall be based upon the ICH Stability Guidelines for new products, WHO Guidelines for stability testing of pharmaceutical products containing well-established drug substances in conventional dosage forms and other local guidelines that may be relevant to specific studies.

The Stability Protocol shall include the first or early commercial batches and/or the Annual Maintenance stability protocols. These protocols may be used for special studies when they match the requirements. For new products, it may be sufficient to only cover the ICH batches in the first version of the protocol. The first versions shall be reviewed to include Annual Maintenance protocols before Annual Maintenance studies are initiated.

If there are regulatory commitments, they may supersede the recommended ICH stability guidelines. Where different formulation types exist (for example, creams and transdermal patches), separate Integrated Protocols or Protocols should be considered.

If bracketing or matrixing is considered, the ICH Guidelines “Bracketing and Matrixing Designs for Stability Testing of New Drug Substances and Products” shall be used as guidance.

The Stability Master Plan provides a five-year forward visibility of the commercial stability set-down program by detailing scheduled new and active studies. Additional special studies shall be added to the SMP when the need for the studies is identified.

Commercial Stability Testing of API (Pure Bulk Drug)

Ongoing surveillance of the stability profiles of commercially available active pharmaceutical ingredients is an integral part of the Company’s quality assurance program. It is essential that stability studies are conducted at Stability Sites as detailed in the SMP issued by the Dossier Management Group (DMG). It is recognised that minor variations may be required due to special requirements by national authorities. In these cases, the deviations shall be embodied within the stability study protocols and noted in the SMP.

Commercial Stability Studies at Contractors

It is the contractor’s responsibility to conduct studies according to the study plan and protocol agreed upon with the sponsor company and in compliance with cGMP codes relevant to the markets being supplied.

It is the responsibility of the contractor to carry out a laboratory investigation into any ‘Out of Specification’ (OOS) results or adverse trend likely to lead to an OOS result within or at the shelf life of the product, to confirm or otherwise, their validity and to report confirmed OOS results, and/or adverse trend likely to lead to an OOS result within or at the shelf life of the product, to the Lead team/site within 3 working days (within 1 working day for products marketed in USA).

It is the contractor’s responsibility to report all data from ongoing studies to the Lead team/site at least annually or more frequently if required by the Lead Site.

Microbiological Testing for Non Sterile Drug Product

The appropriate operations management is responsible for establishing and ensuring compliance with this International Guideline for microbiological testing of non-sterile products.

It is the responsibility of each operation unit using third-party testing labs to ensure that this International Guideline is adhered to.

It is the responsibility of R&D management to initiate a formal microbiological risk assessment at the appropriate time in development and for it to be completed during Phase III.

Each receiving site is responsible for reviewing the R&D risk assessment and producing its own risk assessment based on its manufacturing process and plant. Operation sites are responsible for controlling changes that could impact the outcome of the microbiological risk assessment during the product lifecycle.

Reference & Retention Samples

Reference samples of each API batch, excipient, packaging material and bulk IMP must be retained.

The holding of reference samples is for the purpose of potential future evaluation of the quality of batches, and not for future stability testing. Reference samples are for the purpose of analysis and should be conveniently available to a laboratory with validated methodology. All necessary analytical materials and equipment are expected to be available, or are readily obtainable, to carry out all tests in the specification during the retention period.

Reference samples are also available for identification purposes.

There is no requirement to retain reference samples of both bulk IMP and packaged IMP if they are packaged in the same CCS.

Bulk IMP reference samples should be collected in standard walled HDPE bottles for oral solid dosage forms. Just so you know, additional samples are only required when packaged the first time in a different CCS.

For dosage forms other than oral solids, samples must be pulled for each CCS.

Reference samples that must be placed in foil pouches or similar enclosures to assure sterility, protection from moisture, etc., must be retained in that enclosure.

There is no mandatory requirement to take retention samples (as a physical specimen); when accurately documented, robust processes supported by, e.g. bar code technology are in place to provide assurance of segregation of products and labels.

Laboratory Equipment Qualification

Analytical equipment qualification is the collection of documented evidence demonstrating that specific equipment performs suitably for its intended purpose.

Qualification is a logical step-wise process, which generally consists of the following stages:

– User Requirement Specifications and/or Design Qualification

– Installation Qualification

– Operational Qualification

– Performance Qualification

The amount of documentation and level of detail may vary depending upon the criticality to GMP and complexity of the new or modified equipment.

Manufacture and Microbiological Testing of Sterile API & Drug Product Within R&D

The manufacture of sterile products (Investigational Medicinal Products and Active Pharmaceutical Ingredients for the purposes of this Guideline) requires facilities that are suitably designed and qualified. Design features alone do not assure the quality of sterile products, especially those prepared aseptically, which additionally relies largely upon robust processes and procedures and the training and experience of personnel.

Process qualification, considering related items such as operator gowning, sterile filtration, environmental controls, etc., is essential for sterile product manufacture.

I think specific consideration should be given depending on the type of process (e.g., aseptic processing, isolator, or blow/fill/seal technology).

Facility design philosophies, air classifications, environmental monitoring limits and other operational requirements are specified in Annex 1 of ‘The Rules Governing Medicinal Products in the European Union” and the FDA “Guideline on Sterile Drug Products Produced by Aseptic Processing”.

Additional guidance regarding specific engineering for facilities that manufacture sterile products is to be found in the ISPE Baseline Pharmaceutical Engineering Guide Volume 3.

The ICH Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients indicates that applicable GMP guidelines for drug products, which are reflected in this guideline, generally would apply to the manufacture of sterile API.

The following guidance is provided only as a summary of requirements defined in the various regulations and technical papers on sterile manufacturing and gives some additional points of clarification. The reference section includes a list of documents, which include detailed information on each of the areas discussed below, and these should be consulted for more detailed guidance.

Commercial Stability Testing For Formulated Products

In principle, all formulations, dosage strengths and primary package variations shall be tested. A set of three distinct production scale batches of each dosage strength shall be taken from each manufacturing (formulation) site that will produce the commercial product.

Each of the three batches shall be packaged in all primary packaging variants, i.e., different container closure systems, proposed for marketing, and these primary packages shall be included in the stability program.

When samples of all primary package variants cannot be obtained from the first or early three production batches, subsequent batches shall be utilised to provide stability coverage of all package variants, i.e. different container/closure systems.

Where possible, dosage form batches from at least two different lots of drug substances and key excipients shall be incorporated into the stability program.

Where bracketing is permitted by regulatory authorities in the markets affected, three batches of each dosage strength/primary package variant combination may not be required.

When primary packages marketed in the USA are identical to those marketed in the rest of the world (ROW), every effort shall be made to integrate the requirements of the USA with those of ROW so that the number of studies is minimised.

However, such integrated studies must be designed to meet the USA’s requirements with respect to supply chain specificity. When the FDA permits bracketing, three batches of each dosage strength/primary package variant combination may not be required.

However, such integrated studies must be designed to meet the USA’s requirements with respect to supply chain specificity.

Environmental Monitoring

An environmental monitoring program provides information on the non-viable particulate and microbiological quality of the environment being monitored. Such data provides an insight into the effectiveness of the control programs (e.g. cleaning, housekeeping, gowning) that are in place.

Classified environments (sometimes referred to as controlled environments) must be monitored using the appropriate regulations, guidelines, and industry practices. Areas defined as classified environments must be monitored as appropriate for that environment. Monitoring programs must be designed based on the processing requirements and risk assessment of the area.

In non-classified areas, a level of microbiological monitoring is necessary as part of an ongoing risk assessment to maintain awareness of the microbiological environmental conditions during manufacturing activities.

The monitoring programs must be performed and analysed by appropriately trained personnel.

A program must be established for sampling equipment and instrument calibration.

Samples should be collected from areas where products or components are exposed to the environment, such as critical processing zones and filling lines, where such sampling does not interfere with the process being monitored or increases the risk of product contamination.

Intensified environmental monitoring should be performed during the initial area start-up and following periods of extended shut-down or considered after area, system or equipment modifications or if significant atypical results or trends are observed in the data. Factors that may impact the environment’s integrity must be evaluated and controlled (e.g. Sterile-filtered gases that come in contact with products, primary containers, direct product contact surfaces and sanitising agents). The frequency should be based on an assessment of the risk of product failure.

Trending of Stability Data

The registered retest period/shelf life of a drug substance/product will have been set considering the specification to be registered and the trends seen in stability studies completed or ongoing at the time of the new product and/or new primary pack registration. However, these studies will have been conducted on relatively few batches, some made only at the pilot scale.

Out of Specification Results Investigation

A result is considered to be reportable and available for compliance with specifications, compendial limits or trends when it has been checked by a second analyst.

When a laboratory test produces an unexpected OOS result, repeating the test and ignoring the original result is not sufficient. An investigation must be conducted whenever an unexpected OOS test result is obtained.

An investigation must be done to determine whether the test result is due to a laboratory error, a true measure of the batch or a sampling error.

However, the OOS result may be expected and accepted (stressed and/or accelerated stability samples), and no investigation or retesting may be required. In such cases, the OOS should be documented, and a review and justification should be undertaken.

It is recommended, however, that the first occurrence of an OOS during these studies is registered and investigated using the OOS procedure and subsequent results referenced to it.

However, if the OOS result is unexpected, the investigation aims to determine the root cause of the OOS.

I think an initial assessment should be based within the laboratory and, where appropriate, should be referenced in an extended investigation, should this be required.

The presence of an OOS result does not necessarily require the batch to be rejected but indicates that an immediate investigation should be conducted for batch disposition purposes.

Therefore, the Commercial Stability Site must use trending stability data to support the retest period/shelf-life of a drug substance/product and indicate when a change to retest period/shelf-life and/or cautionary labelling statement is required. Trending must also be used as a tool to identify significant trends and recommend release alert limits when necessary.

The formulation site must take appropriate action to ensure that drug substance/product release procedures are updated to consider the latest stability trend analysis reports issued by the Commercial Stability Site.

This includes establishing or revising release alert limits if recommended by the Commercial Stability Site. Please take a look at section 5.5 for further guidance.

During ongoing studies, Commercial Stability Sites may identify OOT results. In that case, the formulation site(s) must be involved to investigate and, whenever possible, reverse the cause of the deterioration in stability characteristics of the product. If a trend is identified as adverse, the Commercial Stability Site must report this.

Analytical Procedures and Validation

The following characteristics are listed and defined by ICH (Refs: 6.1 & 6.2). They would be expected to form part of the analytical procedure and/or validation studies (but not necessarily submitted) at the time of submission of the marketing authorisation dossier (NDA, MAA, JNDA).

(a) Accuracy

(b) Precision

(d) Detection Limit

(e) Quantitation Limit

(f) Linearity

(g) Range

(h) System Suitability

(i) Robustness

Analytical procedure validation is usually performed as part of a development program. Validation information will be included in regulatory submissions, with increasing levels of detail as projects progress through development to the marketing phase.

As a guide, each of the validation characteristics listed above should be considered. In the early phases of development, a reduced number of validation characteristics may be assessed according to local procedures. When a new validated analytical procedure replaces an existing one, appropriate change control should be implemented according to local procedure.

I think it should be given to a period of ongoing monitoring to determine whether the change has resulted in a step change in results.

Note that biological testing, for example, sterility, endotoxin testing and the microbiological examination of non-sterile products, will be validated in line with Pharmacopoeial requirements.