The 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, especially when preceded or followed by a finding of an adverse trend or atypically 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, are a particularly 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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