The Role of Microbiological Media in Maintaining Laboratory Data Integrity

• Bristol-Myers Squibb

In the pharmaceutical industry, the generation and collection of data is key to ensuring product quality and patient safety. As such, the integrity of this data is paramount. “Data integrity”, for the purposes of this discussion, is defined as the degree of accuracy, completeness and consistency of generated and collected data. ¹ The highest level of integrity must be maintained in all aspects of data generation, compilation and storage. Some of the most egregious data integrity issues encountered concern the intentional falsification of data. ² However, data integrity may be adversely impacted by unintentional actions as well.

Data integrity in a Quality Control (QC) microbiology laboratory offers a unique set of challenges as much of the testing and recording of data is performed manually. This increases the likelihood of common human errors. However, there are additional errors that can also occur that are less obvious and harder to detect. One of the most critical and commonly overlooked factors that can have significant impact on microbiological data integrity is the improper selection, handling and use of microbiological media. Microbiological media is used in testing for microbial limits, sterility, antimicrobial effectiveness, and bioburden and is critical in assessing the quality of raw materials, process waters and compressed gases, intermediate and finished product, as well as equipment and facility cleaning validation. ^3,4 Media is also used for aseptic process validation (i.e.; media fills) and environmental monitoring (EM) which are critical for assessing and maintaining appropriate microbiological control of the manufacturing environment. The improper selection, quality control, storage and handling, incubation, or use of compromised media can directly impact the ability of the media to provide reliable results. Tests in which an inappropriate or compromised medium is utilized may fail to recover the maximum number of organisms present and/or specifically targeted organisms. Consequently, a test result of “no growth” may appear to indicate a passing result, when in reality it may be a “false negative” result jeopardizing product quality and patient safety. Similarly, the occurrence of inaccurate results can have a serious negative impact resulting in unplanned facility shutdowns, loss of batches, product recalls, drug shortages, risk to the patient, and damage to the company’s reputation as well as possible monetary damages.

Studies have demonstrated that traditional culture based tests utilizing microbiological media can recover only 1% - 10% of the microorganisms actually present in any environmental sample. ⁵ Using improper or compromised media or failure to incubate the sample properly will further impede maximum recovery. Microorganisms present within pharmaceutical operations (e.g.; water and gas systems, environmental samples or product) often are highly stressed due to the inhospitable environment and residual chemical agents etc. ⁶ Therefore, it becomes very important that the quality of the media used for microbiological analyses be assured to maximize recovery. For certain tests in particular, such as anaerobic bioburden monitoring, factors such as media selection, handling and incubation parameters are critical. ⁷

Both United States Pharmacopoeia (USP) Chapter<1117>⁸ and EudraLex Volume 4 Chapter 6 ⁹ provide valuable information on the proper preparation, QC and storage of microbiological media. However, the discussion on this topic is limited. There is currently no official regulation or guidance document that addresses this particular topic in great detail. Because of the potential impact that improperly selected or handled microbiological media can have on the integrity of laboratory data, at minimum, a QC microbiology laboratory should consider the points below when utilizing any microbiological media for testing purposes. Some of these points are more commonly considered and are actively employed in many QC labs. However, there are others that are no less critical that are more obscure and may be overlooked. The following is a detailed listing of factors that can enhance or impede the level of integrity of any laboratory data generated using microbiological media.

Commonly considered points include but are not limited to:

Media Preparation:

1. Dehydrated media and ready-made media should be obtained from a qualified supplier.
2. When preparing media in-house, the manufacturer’s instructions should be followed exactly, a certified and calibrated balance should be used, and the volume and weights of components should be recorded.
3. The water used to prepare media should be of purified, deionized or distilled grade.
4. Certificates of analysis (CoA) or Certificates of Performance (CoP)containing information on expiry dating, recommended storage conditions and appropriate microorganisms to be used in QC testing should be obtained from the manufacturer and archived by the lab for both dehydrated and prepared (ready-to-use) media.
5. The shipping conditions used by the manufacturer/ supplier to ship media to sites for purchased media should be validated. In the absence of a site-conducted study, obtaining a documentation (e.g.; a certificate or study) from the manufacturer/supplier demonstrating the stability of the media under the shipping conditions of the manufacturer should be obtained. As all media to a certain extent are sensitive to various environmental factors such as temperature, humidity and light, improper shipping conditions can have detrimental effect on the quality or shelf-life of the media.
6. If heating is necessary to help dissolve the media, care must be taken not to overheat as all culture media are heat sensitive to some extent.
7. Sterilization of media should be performed as per the manufacturer’s instructions or user-validated parameters. The effects of the sterilization method and impact on the media is validated by sterility and growth-promotion testing of the media.

Media Storage:

1. Purchased, ready-made media should be stored according to the manufacturer’s instructions. Media prepared in- house must be stored under validated conditions as the ingredients are sensitive to various environmental factors. Agar media should not be stored at or below 0°C to prevent degradation of the media due to freezing. Stored media should be protected from exposure to light and excessive temperature. Before prolonged storage, agar plates should be placed in a sealed package or container to retard moisture loss.
2. All purchased and in-house prepared media should be labeled with the media type or name, lot and/or batch numbers, preparation date, storage conditions, expiry date, and package opening date.The person who prepared it should also be identified.
3. Expiry dating should be established for dehydrated media after opening of the bottle. In general, the manufacturer’s expiry date provided on the bottle is applicable only to the unopened bottle. The establishment of the expiry dating for opened containers ensures that the quality does not degrade during the open storage period.

QC Testing, Release and Use:

1. A sterility check should be performed on a portion of any media prepared in-house (recommended 2%). ³ For media that are not terminally sterilized (e.g.; Violet Red Bile Glucose Agar, Xylose Lysine Deoxycholate Agar; Enterobacteria Enrichment Broth Mossel), 100% of the lot should be pre-incubated followed by a visual inspection (in an appropriately controlled environment such as a biological safety cabinet) in order to perform the sterility check. Growth-promotion testing (GPT) for this media is performed after the pre-incubation stage. The use of a negative control during actual testing should not be considered a substitute for the pre-approval sterility check of the media.
2. The accurate performance of media prepared either in- house or by a manufacturer is demonstrated by performing GPT. Each lot of any prepared media must undergo GPT before use. If the lab receives two shipments of the same lot, both should be tested. In addition to GPT, selective and differential media should also be tested for inhibition and indicative properties as appropriate.
3. All media should be checked prior to use by visual inspection for the following: cracked containers or lids, dehydration (resulting in cracks or dimpled surfaces on solid media), excessive darkening or color changes, crystal formation (from possible freezing), an excessive number of bubbles, microbial contamination, status of redox indicators (if appropriate), lot number and expiry date checked and recorded and overall, cleanliness and integrity of the tubes and plates (lid should not stick to dish).This inspection will prevent adverse effects from damage or changes that may have occurred during shipping or storage.
4. For contact (RODAC®) plates, the presence of a raised agar level for surface should be verified before use. An improper agar level in contact plates will result inadequate sampling as all the surface of the agar may not fully contact the area intended to be sampled.

These points are equally critical, but less obvious:

Media Preparation:

1. Media should not be left in the autoclave after sterilization to allow for cooling of the liquid media. This practice can cause caramelization and breakdown of media components, adversely impacting the quality and performance of the media.
2. The pH of each batch of medium should be confirmed once the medium has returned to room temperature (20°–25°C). The reading should never be taken while the medium is hot or cold as it will affect the pH reading. It is also important to withdraw a sample for pH testing aseptically in order not to contaminate the medium. The user should ensure that the appropriate probes and calibration solutions are used for pH determination of both solid and liquid media.
3. Re-melting of an original container of solid media should be performed only once as many ingredients are heat sensitive. If a microwave or hot plate is used, the re-melting process needs to be validated for the type of media melted.
4. Molten agar medium should be held in a monitored water bath at a temperature of 45° to 50° for not more than 8 hours. A high purity water (e.g.; Purified Water) should be used in the water bath to prevent cross-contamination. The temperature of the molten agar should be recorded before use as excessive heat will damage or kill any organisms present in a sample to be tested (for example, when using the pour plate method).

Media Storage:

1. The in-use shelf life of microbiological media should be established, documented and scientifically justified. The manufacturer’s expiry date should also be verified by the lab to ensure it is valid under the lab’s storage conditions. Furthermore, periodic stability checks should be performed over the shelf life of media with a long expiry period.

QC Testing, Release and Use:

1. Appropriate reference cultures and environmental isolates reflective of the intended use of the media should be included in the GPT battery. Isolate selection should be based on risk to the product and/or process. Isolates should also be used in a manner that is reflective of where they are recovered in order to appropriately challenge the media. All selections and rationales for using them should be scientifically justified. Test organisms may be selected from the appropriate compendial test chapter, or be selected based on the manufacturer’s recommendation for a particular medium. Both reference cultures and environmental isolates should be checked for viability and identity before use in the GPT in order to ensure accurate qualification test results.
2. Media used for anaerobic bioburden testing should be pre-reduced and appropriately handled both before and during use. Exposure to aerobic environments should be minimized to the extent possible during test execution to ensure proper performance and accurate test results. Reference cultures and environmental isolates appropriate for these specific media and incubation conditions should be selected for use.
3. Incubators play a pivotal role in the successful outcome of a microbiological analyses. Incubation of GPT plates and tubes should be performed using appropriately qualified, calibrated, and maintained incubators. Periodic requalification of incubators should be performed with appropriate load configurations representing the worst case scenario.
4. When a batch of media does not meet the requirements of GPT, an investigation should be initiated to identify the root cause of the failure. Any batch of media that fails GPT for reasons other than substantiated laboratory error during testing (e.g.; an invalid GPT) is unsuitable for use.

In conclusion, apart from the preparation, QC and storage of microbiological media it is important for the QC microbiology laboratory to execute equal due diligence in the proper handling and storage of samples, calibration and maintenance of equipment, maintenance and storage of microbiological cultures, and training of personnel. The lab should also employ appropriate and robust verification of collected data as all of these factors including media contribute equally to ensure accurate, consistent, complete results and data integrity. By giving due attention and consideration to all of the points outlined in this article, particularly with respect to the application of the media used in various microbiological tests, a QC Microbiology Laboratory can increase and preserve the level of integrity of the testing data obtained with the ultimate goal of protecting product quality and patient safety.

References

1. M. Teeling. What is Data Integrity? Learn How to Ensure Database Data Integrity via Checks, Tests & Best Practices, 2012 (https://www.veracode.com/blog/2012/05/what-is-data- integrity)
2. M. Fritz; G. Toscano; and D. Jones. Data Integrity – Make sure this hot topic doesn’t burn you ... or your suppliers, contract manufacturers or contract laboratories. The Journal. Issue, 2014; 28: 38.
3. T. Sandle. Assessment of Culture Media in Pharmaceutical Microbiology. American Pharmaceutical Review. 2014; pp 1-6.
4. A. Cundell. Review of the Media Selection and Incubation Conditions for the Compendial Sterility and Microbial Limit Tests. Pharm Forum Nov/Dec 2002. 28(6):2034- 2041
5. C. R. Jackson; K.C. Randolph; S.L. Osborn; and H.L. Tyler. Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables. BMC microbiology. 2013; 13(1): 1.
6. A. Sage. Use of stressed population for application validation to better represent real world testing. American Pharmaceutical Review. 2013; pp 1-7.
7. J. Moldenhauer. Justification of incubation conditions used for environmental monitoring. American Pharmaceutical Review. 2014; pp 1-4. BioMed research international. 2013.
8. USP<1117>Microbiological Best Laboratory Practices. USP39-NF34 Page 1349.
9. EudraLex Volume 4 Chapter 6 Quality Control. pp 5-6.