Laboratory Investigations of Aberrant Results
The International Conference on Harmonization (ICH) defines specifications as a “list of tests, references to analytical procedures, and appropriate acceptance criteria that are numerical limits, ranges, or other criteria for the test described.” Specifications establish the set of criteria to which a material should conform to be considered acceptable for its intended use. “Conformance to specification” means that the material (for example, raw material, finished drug product), when tested according to the listed analytical procedures, will meet the listed acceptance criteria. The term “out-of- specification (OOS) results” includes all test results that fall outside the specifications or acceptance criteria established in drug applications, drug master files (DMF), official compendia, or by the manufacturer. The term also applies to all in-process laboratory tests that are outside of established specifications.
An OOS result requires a concerted effort to determine whether it is due to a product failure or a laboratory error. In either case, an investigation is required to determine the source of the OOS result, the disposition of the product, and the impact on associated products and processes.
REGULATORY GUIDANCE
ICH Q7 Guidance for Industry, Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients, states that OOS results must be immediately reported to laboratory supervisors and/or managers and must be fully investigated. The guidance further states that any OOS result obtained should be investigated and documented according to a procedure. This procedure should include analysis of the data, assessment of whether a significant problem exists, allocation of the tasks for corrective actions, and conclusions. Any resampling and/or retesting after OOS results should be performed according to a documented procedure.
The United States Food and Drug Administration (FDA) OOS guidance further elaborates how to conduct an OOS investigation: To be meaningful, the investigation should be thorough, timely, unbiased, well documented, and scientifically sound. The first phase of such an investigation should include an initial assessment of the accuracy of the laboratory’s data. Whenever possible, this should be done before test preparations (including the composite or the homogenous source of the aliquot tested) are discarded. If this initial assessment indicates that no meaningful errors were made in the analytical method used to arrive at the data, a full-scale OOS investigation should be conducted. For contract laboratories, the laboratory should convey its data, findings, and supporting documentation to the manufacturing firm’s quality control unit (QCU), who should then initiate the full-scale OOS investigation.
A microbiological OOS, referred to as a microbial data deviation (MDD) is often associated with the aforementioned guidance documents, as well as with United States Pharmacopeia (USP) <1117>, but these references either exclude microbiological tests and biological assays or intimate the difficulties associated with resolving a microbiological OOS. A better guideline to use when conducting an MDD investigation is the FDA aseptic processing guidance, Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice. Although this guideline addresses positive sterility results, it can be judiciously applied to investigate an MDD for a non- sterile raw material, product, and assay.
CONDUCTING AN OOS INVESTIGATION
The FDA guidance for conducting an OOS investigation applies to chemistry-based laboratory testing of drugs regulated by the Center for Drug Evaluation and Research (CDER). It is directed toward traditional drug testing and release methods. These laboratory tests are performed on active pharmaceutical ingredients (APIs), excipients, and other components, in-process materials, and finished drug products to the extent that current good manufacturing practices (cGMP) regulations (21 CFR 210 and 211) and the Federal Food, Drug, and Cosmetic Act (FDCA) [Section 501(a)(2) (B)] apply. The principles in this guidance also apply to in-house testing of drug product components that are purchased by a firm. This guidance can be used by contract firms performing production and/or laboratory testing activities on behalf of the marketing authorization holder. This guidance does not apply to biological assays (for example, in vivo, immunoassays) or microbiological testing.
Specifically, the FDA OOS guidance discusses how to investigate OOS test results, including the responsibilities of laboratory personnel, the laboratory phase of the investigation, additional testing that may be necessary, when to expand the investigation outside the laboratory, and the final evaluation of test results. It is important to remember, however, that FDA guidance documents, including the OOS guidance, do not establish legally enforceable responsibilities. Instead, guidance documents describe the Agency’s current thinking on a topic, and should be viewed only as recommendations unless specific regulatory or statutory requirements are cited. The use of the word “should” in Agency guidance documents means that something is only suggested or recommended, but establishes the current thinking of the agency.
Analytical OOS Investigations
FDA regulations require that an investigation be conducted whenever an OOS test result is obtained (21 CFR 211.192). The purpose of the investigation is to determine the cause of the OOS result. The source of the OOS result should be identified either as an aberration of the measurement process or an aberration of the manufacturing process. Even if a batch is rejected based on an OOS result, an investigation is necessary to determine whether the result is associated with other batches of the same drug product or other products. Batch rejection does not negate the need to perform the investigation. The regulations require that a written record of the investigation be made, including the conclusions and follow-up.
Responsibility of the Analyst. In accordance with the cGMP regulations in 21 CFR 211.160(b)(4), the analyst should ensure that only those instruments meeting established performance specifications are used, and that all instruments are properly calibrated. Systems not meeting suitability requirements should not be used. If reference standard responses indicate that the system is not functioning properly, all data collected during the suspect time period should be properly identified and should not be used. The cause of the malfunction should be identified and, if possible, corrected before a decision is made whether to use any data before the suspect period.
Analysts should check the data for compliance with test specifications before discarding test preparations or standard preparations. When unexpected results are obtained and no obvious explanation exists, test preparations should be retained, if stable, and the analyst should inform the supervisor. An assessment of the accuracy of the results should be started immediately.
If errors are obvious, such as the spilling of a sample solution or the incomplete transfer of a sample composite, the analyst should immediately document what happened. Analysts should not knowingly continue an analysis they expect to invalidate at a later time for an assignable cause (that is, analyses should not be completed for the sole purpose of seeing what results can be obtained when obvious errors are known).
Responsibility of the Laboratory Supervisor. The supervisor’s assessment should entail:
• Discussing the test method with the analyst; confirming analyst knowledge of, and performance of, the correct procedure.
• Examining the raw data obtained in the analysis, including chromatograms and spectra, and identifying anomalous or suspect information.
• Verifying that the calculations used to convert raw data values into a final test result are scientifically sound, appropriate, and correct; determining whether unauthorized or nonvalidated changes have been made to automated calculation methods.
• Confirming the performance of the instruments.
• Determining that appropriate reference standards, solvents, reagents, and other solutions were used and that they met quality control specifications.
• Evaluating the performance of the test method to ensure that it is performing according to the standard expected, based on method validation data and historical data.
• Fully documenting and preserving records of this laboratory assessment. The assignment of a cause for OOS results will be greatly facilitated if the retained sample preparations are examined promptly. Hypotheses regarding what might have happened (for example, dilution error, instrument malfunction) should be tested. Examination of the retained solutions should be performed as part of the laboratory investigation.
Laboratory management should ascertain not only the reliability of the individual values obtained, but also the significance these OOS results represent to the laboratory quality assurance program. Laboratory management should be especially alert to developing trends, and upper management should appropriately monitor these trends and ensure that any problem areas are addressed.
Laboratory error should be relatively rare. Frequent errors suggest a problem that might be due to inadequate training of analysts, poorly maintained or improperly calibrated equipment, or careless work. Whenever laboratory error is identified, the firm should determine the source of that error and take corrective action to prevent recurrence. To ensure full compliance with the cGMP regulations, the manufacturer also should maintain adequate documentation of the corrective action. When clear evidence of laboratory error exists, laboratory testing results should be invalidated. When evidence of laboratory error remains unclear, a full-scale OOS investigation should be conducted by the manufacturing firm to determine what caused the unexpected results. It should not be assumed that OOS test results are attributable to analytical error without performing and documenting an investigation. Both the initial laboratory assessment and the following OOS investigation should be documented fully.
Full-Scale OOS Investigation
When the initial assessment does not determine that laboratory error caused the OOS result, and testing results appear to be accurate, a full-scale OOS investigation using a predefined procedure should be conducted. This investigation may consist of a production process review and/or additional laboratory work. The objective of such an investigation should be to identify the root cause of the OOS result and take appropriate corrective and preventive action. A full-scale investigation should include a review of production and sampling procedures, and will often include additional laboratory testing. Such investigations should be given the highest priority. Among the elements of this phase is evaluation of the impact of OOS result(s) on already distributed batches.
Production Review
The investigation should be conducted by the quality control unit (QCU) and should involve Manufacturing, Process Development, Maintenance, and Engineering. In cases where manufacturing occurs off-site (for example, performed by a contract manufacturer), all sites potentially involved should be included in the investigation. Other potential problems should be identified and investigated.
The records and documentation of the manufacturing process should be fully reviewed to determine the possible cause of the OOS result(s). A full-scale OOS investigation should consist of a timely, thorough, and well-documented review. A written record of the review should include, at a minimum:
• A clear statement of the reason for the investigation
• A summary of the aspects of the manufacturing process that may have caused the problem
• Results of a documentation review, with the assignment of actual or probable cause
• Results of a review made to determine whether the problem has occurred previously
• Description of corrective actions taken
If this part of the OOS investigation confirms the OOS result and is successful in identifying its root cause, the OOS investigation may be terminated and the product rejected. A failure investigation that extends to other batches or products that may have been associated with the specific failure must also be completed (21 CFR 211.192). If any material was reprocessed after additional testing, the investigation should include comments and the signatures of appropriate production and QC personnel.
OOS results may indicate a flaw in product or process design. In such cases, it is essential that redesign of the product or process be undertaken to ensure reproducible product quality.
Retesting. The sample used for the retesting should be taken from the same homogeneous material that was originally collected, tested, and yielded the OOS results. For a liquid, it may be from the original unit of liquid product or composite of the liquid product; for a solid, it may be an additional weighing from the same sample composite prepared for the original test.
Retesting is indicated when instrument malfunctions or sample handling problems (for example, a dilution error) are suspected. Decisions to retest should be based on the objectives of the testing and sound scientific judgment. Predefined retesting plans should include retests performed by an analyst other than the one who performed the original test. A second analyst performing a retest should be at least as experienced and qualified in the method as the original analyst.
The practice of testing into compliance is unscientific and objectionable under cGMP. The maximum number of retests to be performed on a sample should be specified in advance in a written standard operating procedure (SOP). The number should be based on scientifically sound principles and should not be adjusted depending on the results obtained. The firm’s predetermined retesting procedures should contain a point at which the additional testing ends and the batch is evaluated. If the results are unsatisfactory at this point, the batch should be considered suspect and rejected or held pending further investigation [21 CFR 211.165(f)]. Any deviation from this SOP should be rare and done in accordance with 21 CFR 211.160(a), which states that any deviations from written specifications, sampling plans, test procedures, or other laboratory control mechanisms shall be recorded and justified. In such cases, before starting additional retesting, a protocol should be prepared (subject to approval by the QCU) that describes the additional testing to be performed and specifies the scientific and/or technical handling of the data.
In the case of a clearly identified laboratory error, the retest results would substitute for the original test result. All original data should be retained, however, and an explanation recorded. This record should be initialed and dated by the involved persons and include a discussion of the error, and supervisory comments. If no laboratory or calculation errors are identified in the first test, there is no scientific basis for invalidating initial OOS results in favor of passing retest results. All test results, both passing and suspect, should be reported and considered in batch release decisions.
Resampling. Resampling involves analyzing a specimen from any additional units collected as part of the original sampling procedure or from a new sample collected from the batch, should that be necessary. The original sample from a batch should be sufficiently large to accommodate additional testing in the event an OOS result is obtained. It may be appropriate to collect a new sample from the batch. Control mechanisms for examination of additional specimens should be in accordance with predetermined procedures and sampling strategies [21 CFR 211.165(c)].
When all data have been evaluated, an investigation might conclude that the original sample was prepared improperly and was therefore not representative of the batch quality [21 CFR 211.160(b)(3)]. Improper sample preparation might be indicated, for example, by widely varied results obtained from several aliquots of an original composite (after determining there was no error in the performance of the analysis).
Resampling should be performed using the same qualified, validated methods that were used for the initial sample. If, however, the investigation determines that the initial sampling method was inherently inadequate, a new accurate sampling method must be developed, documented, and reviewed and approved by the QCU [21 CFR 211.160 and 211.165(c)].
Reporting Testing Results
Averaging—Appropriate Uses. If the sample can be assumed to be homogeneous (that is, an individual sample preparation designed to be homogeneous), using averages can provide a more accurate result. In the case of microbiological assays, the USP prefers the use of averages because of the innate variability of the biological test system.
If a test consists of a specific number of replicates to arrive at a result (for example, a high-performance liquid chromatography [HPLC] assay result is determined by averaging the peak responses from a number of consecutive, replicate injections from the same preparation), then the assay result would be calculated using the peak response average. This determination is considered one test and one result, distinctly different from the analysis of different portions from a lot—intended to determine variability within the lot—and from multiple full analyses of the same homogenous sample.
The use of replicates to arrive at a single reportable result, and the specific number of replicates used, should be specified in the written, approved test method. Acceptance limits for variability between the replicates should also be specified in the method. Unexpected variation in replicate determinations should trigger remedial action as required by 21 CFR 211.160(b)(4). If acceptance limits for replicate variability are not met, the test results should not be used.
If a series of complete tests (full run-throughs of the test procedure), such as assays, are part of the test method, it may be appropriate to specify in the test method that the average of these multiple assays is considered one test and represents one reportable result. In this case, limits on acceptable variability between the individual assay results should be based on the known variability of the method and should be specified in the test methodology. A set of assay results not meeting these limits should not be used. These appropriate uses of averaging test data should be used during an OOS investigation only if they were used during the original testing that produced the OOS result.
Averaging—Inappropriate Uses. All individual test results should normally be reported as separate values. Where averaging of separate tests is appropriately specified by the test method, a single averaged result can be reported as the final test result. In some cases, a statistical treatment of the variability of results is reported (for example, in a test for dosage form content uniformity), and the standard deviation (or relative standard deviation) is reported with the individual unit dose test results.
Averaging can conceal variations in different portions of a batch, or within a sample, for example, when performing powder blend/mixture uniformity or dosage form content uniformity determinations. In these cases, testing is intended to measure variability within the product, and individual results provide the information for such an evaluation.
For additional testing performed during an OOS investigation, averaging the result(s) of the original test that prompted the investigation and additional retest or resample results obtained during the OOS investigation may hide variability among the individual results. These data can be misleading when some of the results are OOS and others are within specifications. The laboratory must provide all individual results for evaluation and consideration by the QCU, which is responsible for approving or rejecting, for example, drug products or in-process materials (21 CFR 211.22).
Outlier Tests. The cGMP regulations require that statistically valid QC criteria include appropriate acceptance and/or rejection levels [21 CFR 211.165(d)]. A value that is markedly different from the others in a series obtained using a validated method may qualify as a statistical outlier. An outlier may result from a deviation from prescribed test methods, or it may be the result of variability in the sample. It should never be assumed that the reason for an outlier is error in the testing procedure rather than inherent variability in the sample being tested.
Outlier testing is a statistical procedure for identifying from an array those data that are extreme, and the possible use of outlier tests should be determined in advance. It should be written into an SOP for data interpretation and be well documented. The SOP should include the specific outlier test to be applied, with relevant parameters specified in advance, and should specify the minimum number of results required to obtain a statistically significant assessment from the specified outlier test.
For biologic assays having a high variability, an outlier test may be an appropriate statistical analysis to identify those results that are statistically extreme observations. The USP describes outlier tests in the General Chapter on “Design and Analysis of Biological Assays.” In these cases, the outlier observation is omitted from calculations. The USP states that “arbitrary rejection or retention of an apparently aberrant response can be a serious source of bias . . . the rejection of observations solely on the basis of their relative magnitudes is a procedure to be used sparingly.”
An outlier test is only a statistical analysis of the data obtained from testing and retesting validated chemical tests with relatively small variance, and when the sample being tested can be considered homogeneous. It will not identify the cause of an extreme observation and should not be used to invalidate the suspect result. Outlier tests have no applicability in cases where the variability in the product is what is being assessed, such as for content uniformity, dissolution, or release rate determinations. In these applications, a value perceived to be an outlier may in fact be an accurate result of a nonuniform product.
When using these practices during the additional testing performed in an OOS investigation, the laboratory will obtain multiple results. It is critical for the laboratory to provide all test results for evaluation and consideration by the QCU in its final disposition decision. In addition, when investigation by a contract laboratory does not determine an assignable cause, all test results should be reported to the customer on the certificate of analysis (COA).
Concluding the Investigation
To conclude the investigation, the results should be evaluated, the batch quality should be determined, and a release decision should be made by the QCU. The SOP should be followed in arriving at this point. Once a batch has been rejected, there is no limit to further testing to determine the cause of the failure so that a corrective action can be taken.
Interpretation of Investigation Results
The QCU is responsible for interpreting the results of the investigation. An initial OOS result does not necessarily mean the subject batch fails and must be rejected. The OOS result should be investigated, and the findings of the investigation, including retest results, should be interpreted to evaluate the batch and reach a decision regarding release or rejection (21 CFR 211.165).
When an investigation has revealed a cause, and the suspect result is invalidated, the result should not be used to evaluate the quality of the batch or lot. Invalidation of a discrete test result may be done only upon the observation and documentation of a test event that can reasonably be determined to have caused the OOS result.
When an investigation indicates that an OOS result is caused by a factor affecting the batch quality (that is, an OOS result is confirmed), the result should be used in evaluating the quality of the batch or lot. A confirmed OOS result indicates that the batch does not meet established standards or specifications, and should result in the batch’s rejection, in accordance with 21 CFR 211.165(f), and proper disposition.
For inconclusive investigations that do not reveal a cause for the OOS test result, the investigation changes from an OOS investigation into a batch failure investigation, which must be extended to other batches or products that may have been associated with the specific failure (21 CFR 211.192). For inconclusive investigations that do not confirm the OOS result, the QCU might still ultimately decide to release the batch. Any decision to release a batch despite an initial OOS result that has not been invalidated should come only after a full investigation has shown that the OOS result does not reflect the quality of the batch. In making such a decision, the QCU should always err on the side of caution.
An assay result that is low, but within specifications, should raise a concern. One cause of the result could be that the batch was not formulated properly. Batches must be formulated with the intent to provide not less than 100% of the labeled or established amount of active ingredient [21 CFR 211.101(a)]. This situation includes where the analytical result meets specifications, but caution should be used in the release or reject decision.
All records pertaining to the OOS test result should be retained. Records must be kept of complete data derived from all tests performed to ensure compliance with established specifications and standards (21 CFR 211.194).
MICROBIOLOGY OOS INVESTIGATIONS
No definitive guideline exists on how to conduct a microbiological OOS investigation. The FDA OOS guidance essentially excludes microbiological and biologic assays; the ICH Q7A OOS guidance states that OOS investigations are not normally needed for in- process tests that are performed for the purpose of monitoring and/or adjusting the process; and USP <1117> intimates the difficulties associated with resolving a microbiological OOS. In fact, the term “microbiological OOS” is better served by the term “MDD.”
One guideline, the FDA aseptic processing guidance, addresses an MDD, albeit it is focused on sterile products. As sterility is the most critical attribute of a product, however, application of these stringent investigational principles can be applied to other products or raw materials to ensure that the decision to retest or fail a product has been made with the greatest scientific scrutiny and the lowest risk. When applied judiciously, the guidance is an effective tool that can be used to perform an MDD investigation.
The aseptic processing guidance identifies seven areas that should be scrutinized when conducting an MDD (Figure 20.1). Four additional areas of investigation not included in the guideline complete the cycle for investigating an MDD, and include API source, excipient source, satellite facilities (if used), and laboratory (in-house or contract).
he principles of conducting an analytical OOS investigation heretofore described should be invoked throughout an MDD investigation, including proper documentation, collection of data, and interpretation of data by the QCU and final disposition by the QCU and company management. In both instances, the investigation must be conducted using sound scientific principles, and the conclusion must be based on empirical data that are elucidated during the process. The underlining assumption of an MDD is that it evaluates the microbiological disposition of a product or material, and the product must be placed on hold until it is proven that the MDD is a result of laboratory or operator error that does not affect product integrity.
Standard Operating Procedures and Protocol
The firm should have an SOP that describes how an MDD investigation will be conducted, and should include a section for preparing a protocol subject to approval by the QCU that describes the additional testing to be performed and specifies the scientific and/or technical handling of the data. The protocol should be based on scientifically sound principles and should clearly state when retesting is appropriate. It should indicate that any unsatisfactory results incriminate the batch, which must be rejected or held pending further investigation [21 CFR 211.165(f)]. Any deviation from this SOP should be rare and done in accordance with 21 CFR 211.160(a), which states that any deviations from written specifications, sampling plans, test procedures, or other laboratory control mechanisms shall be recorded and justified. In such cases, before starting additional retesting, a protocol should be prepared (subject to approval by the QCU) that describes the additional testing to be performed and specifies the scientific and/or technical handling of the data.
Identification (Speciation) of the Organism
Microorganism isolates should be identified to the species level. Microbiological monitoring data should be reviewed to determine if the organism is found in laboratory and production environments, personnel, or product bioburden. Advanced identification methods (for example, nucleic acid based) are valuable for investigational purposes. When comparing results from environmental monitoring and product/production positives, both identifications should be performed using the same methodology. Identifying the same organism from environmental and product/production is helpful in determining the source of the contamination but by itself does not prove it. Additional data must support the findings.
Based on the species, an initial determination of the source can be made. For example, isolation of Staphylococcus hominis would indicate the source to be human skin while Bacillus sphaericus would implicate an environmental source. Interpretation of data by a qualified microbiologist is an essential element of the MDD investigational process.
Record of Laboratory Tests and Deviations
Review of laboratory deviations and investigations can help to eliminate or to implicate the laboratory as the source of contamination. If the organism is seldom found in the laboratory environment, product contamination is more likely than laboratory error. If the organism is found mainly in the laboratory and seldom in the production environments, product contamination may still be more likely than laboratory error. Laboratory test and deviation data review from at least one year before the MDD is suggested to develop a comprehensive understanding of the dynamics associated with the laboratory testing procedures and environment.
Proper handling of deviations is an essential aspect of laboratory control. When a deviation occurs, it should be documented, investigated, and remedied. If any deviation is considered to have compromised the integrity of the microbial testing, the test should be invalidated immediately without incubation.
An MDD result can be viewed as indicative of production or laboratory problems, and the entire manufacturing process should be comprehensively investigated as such problems often can extend beyond a single batch. To more accurately monitor potential contamination sources, maintaining separate trends by appropriate categories such as product, container type, filling line, sampling, and testing personnel is recommended. Where the degree of test sample manipulation is similar to that during production and an MDD is found, the production process may be implicated more than the laboratory.
Microbial monitoring of the testing area of the laboratory and personnel can reveal trends that are informative. Upward trends in the microbial load in the testing area of the laboratory should be promptly investigated as to cause, and corrected. In some instances, such trends can appear to be more indicative of laboratory error as a possible source of an MDD.
Where a laboratory has a good track record with respect to errors, this history can lower suspicion of the laboratory as a source of contamination as chances are greater that the contamination arose from production; however, the converse is not true. Specifically, where a laboratory has a poor track record, firms should not assume that the contamination is automatically more attributable to the laboratory and consequently overlook a genuine production problem. Accordingly, it is essential that all MDDs be thoroughly investigated.
Monitoring of Production Area Environment
Trend analysis of microorganisms in the critical and immediately adjacent areas is especially helpful in determining the source of contamination in an MDD investigation. Consideration of environmental microbial data should not be limited to results of monitoring the production environment for the lot, day, or shift associated with the suspect lot. Results showing little or no recovery of microorganisms can be misleading, high microbial counts. It is therefore important to look at both short- and long-term environmental trend analyses. A review of at least the past year of environmental monitoring data from the production area is useful in determining trends and overall performance of the area. It can help identify areas that are more susceptible to microbial contamination, facilitating corrective and preventive action (CAPA).
Monitoring Personnel (Laboratory and Production)
The review of data and associated trends from daily monitoring of personnel can provide important information indicating a route of contamination. The adequacy of personnel practices and training also merit significant review and consideration. Trending these data can help pinpoint specific technicians and operators who may be the source of the contamination, and appropriate remedial actions can be taken, including decertifying them to perform their jobs, if necessary. In certain instances, the technician or operator may be removed and asked to undergo comprehensive training and evaluation to ensure he or she meets standards established in corresponding SOPs.
Product Bioburden
A review of product bioburden is required to determine if adverse bioburden trends have occurred in the past. One-year data analysis will help ascertain if the bioburden levels contributed to the MDD. Excipients and APIs that show increased levels of microbial activity may be implicated as the source of the MDD, and remedial actions should be taken, including bioburden-reduction procedures or a change in the supplier. High-quality starting materials are necessary to produce high-quality finished products. Production Record Review
Complete batch and production control records should be reviewed to detect any signs of failures or anomalies that could have a bearing on product integrity. The investigation should include:
• Events that could have impacted a critical zone.
• Batch and trending data that indicate whether utility and/or support systems are functioning properly. For example, records of air quality monitoring for filling lines could show a time at which there was improper air balance or an unusually high particle count.
• Review of information on construction or maintenance activities that could have had an adverse impact.
• Review of production records from at least the past year to determine if trends were apparent.
Manufacturing History (Including Media Fills Where Applicable)
The manufacturing history of a product or similar products should be reviewed as part of the investigation. Past deviations, problems, or changes (for example, process, components, equipment) are among the factors that can provide an indication of the origin of the problem. Depending on the product(s) produced, sterility, bioburden, personnel, and environmental data from at least the past year should be reviewed to determine if trends were apparent.
API Source
As part of the total quality system, microbiological testing should be performed on every batch of API received into the firm’s plant(s) to confirm the microbiological acceptability of the material and to generate data for trending purposes. If a monograph exists for the API and includes microbial testing, those tests should be performed. For APIs with no microbial monographs, the firm should determine what microbiological tests are critical in assuring the microbial integrity of the material and perform the appropriate tests, including bioburden, microbial limits, endotoxin, and sterility testing. As part of the MDD investigation, a microbiological review of at least the past 15 batches should be conducted to ascertain both the species of organisms that are inherent in the material and any trends that may exist.
Confirmation of the indigenous microbes in the API with those found in the affected product implicate the API as a probable root cause of the organism, and appropriate preventive measures in the manufacture of the API should be conducted:
• Suspend delivery of new API lots to the firm
• Place all existing in-house lots of the material on quality assurance (QA) hold
• Place all in-process finished product lots containing the API on QA hold
• Require a written investigation from the manufacturer, including CAPA
• Reinspect the API facility
• Increase incoming testing and inspection of the material
• Consider elimination of the manufacturer as a provider of the API
• Conduct a formal investigation, including risk analysis, of the MDD to determine the disposition of the product and any affected batches of products from any of the firm’s production plants using the API
Excipients
It is highly recommended to perform incoming microbiological testing on excipients to ensure their integrity. Although it may not be feasible to test every lot or batch of excipient, they should all be tested on a regular basis as governed by the firm’s supplier certification SOP. The same process and criteria for investigating an API MDD should be applied to the excipient manufacturer, including a risk analysis. Microbial testing for certain products known not to harbor microorganisms (for example, concentrated acids and bases) may be waived. As part of the MDD investigation, a microbiological review of at least the past 15 batches should be conducted to ascertain both the species of organisms that are inherent in the material and any trends that may exist. Purified water or water for injection should be treated as an excipient, and a review of the microbial water quality from the past year should be included in the investigation.
Satellite Facilities
Some firms use multiple production facilities to formulate products, (for example, they may weigh the API and/or excipients in one facility and transport them to another facility, either off site or at the same site). Each facility must be treated in the same manner as the main production facility, and the areas to be investigated should be the same as described earlier:
• Speciation of the organism from the API/excipient/environment
• Production area environment
• Personnel (laboratory and production)
• Production record review
• Manufacturing history (including media fills)
Trend analysis is critical in ascertaining whether the organism has been associated with a satellite site, and the satellite should be under continuous microbiological surveillance to ensure it supports the microbial integrity of the process.
Contract Laboratories
Many firms use contract laboratories to test APIs, excipients, and bulk and finished products, and ask the laboratories to conduct an investigation of an MDD. Whenever possible, the firm should request that they be allowed to conduct the investigation of the laboratory; this request is not to indict the laboratory, but only serves the best interest of the firm. Generally, a contract laboratory has many clients, and serving one client over the others is not in their best interest. This philosophy is applicable not only to microbiological testing but also to analytical testing and to contract manufacturers. Allowing a firm access to the inner workings of the laboratory may be the best method to uncover the source of an MDD, or OOS in the case of analytical deviations.
If the laboratory does not allow outside personnel to conduct an investigation, the firm should require the laboratory to provide written documentation of these items:
• Receiving area. The physical condition of the receiving area may be indicative of the manner in which the laboratory operates. Good laboratories have well-defined areas that are organized, and materials are quickly logged in and sent to the appropriate departments. Sample log-in, whether manually recorded or electronically recorded, is a critical component of the area, and an SOP should cover sample receipt and tracking.
• Sample staging area. Similarly to the receiving area, the sample staging area should be well organized and clean, and this should be documented.
• Autoclave cycles. Autoclave cycles should be reviewed for the past year to determine if any excursions occurred.
• Cleaning agents and processes. Cleaning agents and frequencies should be reviewed for the past year, and trends should be documented.
• Environmental isolates. One-year environmental monitoring data should be reviewed and the organisms found compared to those found in the product. Additionally, the isolated organisms should be reviewed in relation to the cleaning agents being used. If Bacillus are frequently found and no sporocidal agent is used, there is a possibility that Bacillus spores may be a potential problem.
• Personnel. Personnel may contribute to an MDD, and the records from the past year of technicians conducting the testing should be thoroughly reviewed. Organisms associated with the technicians should be compared with those found in the MDD. If retraining is required, the training records should be reviewed.
• Incubators. A review of the cleaning and environmental monitoring of and organisms found in the incubators should be conducted. Incubators are a major source of environmental excursions and may contribute to an MDD.
• Filters (including high-efficiency particulate air [HEPA] filters, if used). Cleaning, maintenance, and environmental monitoring of filters from the past year should be reviewed and the organisms found compared with those found in the MDD.
• Purchased media. The manufacturer of the purchased media should be asked to provide a one-year review of the batches used to test the product in question. Media producers occasionally issue a recall of contaminated media, which can be a source of the MDD.
• Equipment. A one-year review of the microbiological history of biohazard hoods, work top spaces, pipettes, hockey sticks, and other areas and tools should be conducted to determine if they contributed to the MDD.
• Sterility testing. A one-year review of the anteroom, sterility suite, HEPA filters, and failure rate should be conducted, and any organisms should be compared with those in the MDD.
• General supplies. Many supplies are shipped in cardboard boxes that are a major source of contamination. Sampling of the cardboard may reveal the source of the MDD.
• Media preparation and growth promotion. A one-year review of media preparation and growth promotion should be conducted and correlated with the autoclave cycles used and test organisms used for the media preparation and growth promotion, respectively. Although not a common source of contamination, these areas should be well controlled as an overall investigational assessment.
• Training. Properly trained technicians are the most important component of well- functioning laboratories. Their records should be examined to ensure they received appropriate training for the tests they are performing. Aseptic training may be the most critical area to examine.
• Observation of testing. If possible, the firm should be allowed to observe the technician(s) who originally performed the testing perform a mock testing from start to finish, including receipt, preparation, handling, dispensing, diluting, and incubating samples. Minor inconsistencies may be revealed, which could be the source of the MDD.
CASE STUDY
A contract testing laboratory encountered an MDD in a sterility test. After conducting an MDD investigation, the firm was allowed to observe the laboratory’s technicians perform a mock test. As part of the test, 20 ampules were pooled into sterile containers before filtration. The sterile containers were urine sample containers that were sterilized by gamma radiation; they were shipped to the laboratory in a large cardboard box containing a polyethylene bag filled with 1000 containers. The containers were not individually wrapped as it was cost-efficient to purchase them in bulk. The laboratory had recently made the change from individually wrapped containers to the bulk package containers. The laboratory was asked to sample the inside, outside, and flaps of the cardboard box, and the polyethylene bag. Two samples were found to harbor the offending microorganism, which allowed the original test to be invalidated and retesting to be performed. Had the firm not been within the core observing the testing, the source of the contamination might not have been found, and the product would have been rejected. The laboratory discontinued purchasing in bulk and returned to purchasing individually wrapped sample containers. Witnessing the operation enabled appropriate CAPA to be conducted: the corrective action was to sample the cardboard and invalidate the original testing and retest according to USP <71>; the preventive action was to purchase individually wrapped sample containers.
OBJECTIONABLE MICROORGANISMS
Many companies believe that if their nonsterile product meets the USP requirements, it will be safe from FDA dispute. This belief is correct, and the manufacturer is responsible for all contents of its drug product. Should question arise over the appropriateness of a particular organism, the manufacturer is expected to have a justification for the presence of that organism, preferably as part of the batch release document. 21 CFR 211.113 requires that medicines should be “free of objectionable microorganisms.”
USP <1111> provides little specific guidance other than “The significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential hazard to the user.” The USP recommends that certain categories be routinely tested for total counts and specified indicator microbial contaminants, including natural plant, animal, and some mineral products for Salmonella, oral liquids for E. coli, topicals for P. aeruginosa and
S. aureus, and articles intended for rectal, urethral, or vaginal administration for yeasts and molds.
Other than these listed microorganisms, no others are mentioned as objectionable in the USP. FDA has a publication of objectionable microorganisms that is used for the food industry but can also be applied to pharmaceutical sciences: the Bad Bug Book. This publication details numerous microorganisms that may present a health hazard, and whose presence may invoke a rejection or recall of a product or excipient; however, the difficulty facing the industry is determining how objectionable a microorganism actually is. For products that are not labeled as “sterile,” circumstances exist where an objectionable microorganism could be acceptable for product release based on a risk assessment analysis.
Similarly to an MDD, little guidance exists to conduct a risk assessment for objectionable microorganisms. FDA suggests that once all organisms grown in the total count studies (total aerobic as well as total yeast and mold) are identified, a qualified microbiologist would conduct a risk analysis on the presence of those organisms in the medication. This risk analysis should incorporate a minimum of five separate analyses (Figure 20.2).
Absolute Number of Organisms Seen
Most products have microbial specifications for total bacterial counts. When the counts exceed these limits, even if the organisms identified are nonpathogenic or objectionable, an MDD should be conducted, most likely resulting in the rejection of the batch and possibly other batches. Although high numbers of nonpathogenic organisms may not pose a health hazard, they may affect product integrity. An unusually high number of organisms seen in the product may indicate a problem during the manufacturing process, or an issue with an excipient or API. The high bacterial counts may indicate that the microorganisms are thriving in the product. For a preserved product, this indication could suggest that the product’s preservative system is not functioning, is missing, or incorrectly formulated.
The Characteristics of the Microorganism
The characteristics of the microorganism can be determined by textbook, library, or Internet searches, and should include synonyms of the organism. The Bad Bug Book is an excellent reference for determining whether the organism is a known pathogen or an objectionable. The potential for the organism to cause spoilage of the product is another important factor. If the substances used by the microorganism for growth are found in the product, the risk is great that it will degrade the product, supporting the rejection of the batch and associated batches.
A microorganism is objectionable if it has the potential to degrade the product’s stability. Evaluate the microorganism’s tolerance to unusual conditions, such as low or high pH, high salt concentration, high sugar concentration (osmotic conditions), low water activity, and growth temperatures. Organisms with a proclivity for harboring plasmid-mediated antibiotic resistance may be considered objectionable, particularly if the product is to be used in vulnerable patients with compromised immune systems.
Product Characteristics
The characteristics of the product affect the ability of the microorganism to grow within it. Some of these include anhydrous versus water based (that is, enough free water to support microbial growth) and container design and closure (that is, the container should be designed to minimize contamination and subsequent spoilage and should also retard access to the environment and prevent contamination from the environment). Special consideration should be given to an anhydrous medication’s exposure to water, providing the potential for microbial proliferation. And a review of the production records, environmental monitoring trends, and product complaints is also warranted, and comprises part of an MDD investigation.
Route of Administration
Some microorganisms that could be tolerated in a topical medicine could cause severe distress to a patient if taken orally or inhaled. Similarly, a medication orally administered can tolerate some microorganisms that would be devastating in a medication meant to be applied topically to abraded skin or to rashes. Inhalants, although not required to be sterile, sensitive area, and great care
should be taken in classifying any contaminate as “nonobjectionable.”
Patient Population
The targeted patient plays a critical role in assessing the risk of a potential objectionable microorganism. Microorganisms isolated from products intended for pediatric or immune-compromised patients present a greater risk to these patients than microorganisms in those products intended for use in relatively healthy patients. Burkholderia cepacia in cough medicine poses a greater risk than if it was found in a topical cream. Although the manufacturer can not control patient abuse or off-label use of the product, the reasonable use of it should be considered as part of the risk analysis. Certain patient populations may be exposed to increased risk if they use the medication harboring the particular microorganism.
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