Introduction

Many biologics products are used to diagnose, prevent, treat, and cure diseases and medical conditions. They are generally large, complex molecules which can be produced using biotechnology in a microorganism, plant or animal cell. Examples include blood and blood components, human tissue and cells, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, and advanced therapy medicinal products (ATMP), also known as gene and cell therapy products.

Gene therapy is the transfer of genetic material (DNA or RNA) into the cells of a patient’s body to treat the cause or symptoms of a specific disease. These can be used to reduce levels of a disease-causing version of a protein, increase production of disease-fighting proteins, or produce new/modified proteins. The genes can be introduced directly into a patient, packaged into artificially-created liposomes (sacs of fluid surrounded by a fatty membrane), or introduced in a viral vector.

Somatic cell therapy is the transfer of intact, live cells into a patient to help lessen or cure a disease. The cells may originate from the patient (autologous), a human donor (allogeneic) or from another species, such as an animal (xenogeneic). The cells are manipulated or modified ex vivo for subsequent administration to patients. The most promising drugs being developed today are based on Chimeric Antigen Receptor (CAR) T cell therapy. The CAR-T principle involves modifying the patient’s own immune cells (T cells) to express a receptor on their surface that recognizes tumor antigens on the surface of cancer cells. Once the receptor binds to a tumor antigen, the T-cell is stimulated to attack the cancer cells.

There are a number of challenges with these types of novel drug products. They usually possess a relatively short product shelf-life that must be administered to the patient before the end of a conventional 14-day sterility test. Batch sizes are also very small, and the compendia sterility test requirements for sample size cannot be achieved. Specifically, the more sample used for testing, the less drug is available for a successful clinical outcome. Fortunately, recent changes to regulatory policies and compendial chapters have taken these challenges into consideration.

Each of the references discussed below may be downloaded from our References Page.

FDA Final Guidance on Gene Therapy

In January 2020, FDA published its final guidance for industry, "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)." The guidance states, “Analytical procedures different than those outlined in the USP, FDA guidance, or Code of Federal Regulations (CFR) may be acceptable under IND if sponsors provide adequate information on test specificity, sensitivity, and robustness.”

Examples of alternative methods, which may be needed for live cells, include rapid sterility tests, rapid mycoplasma tests (including PCR-based) and rapid endotoxin tests. For these non-compendial tests, FDA recommends to qualify/validate them to ensure they are fit for their intended use.

The guidance also states for ex vivo genetically modified cells administered immediately after manufacturing, in-process sterility testing on sample taken 48 to 72 hours prior to final harvest is recommended for product release. This may include a Gram stain and a sterility test compliant with 21 CFR 610.12. Under this approach, the release criteria for sterility would be based on a negative result of the Gram stain and a no-growth result from the 48 to 72 hour in-process sterility test. When the USP <71> sterility test is used, the final product sterility test should incubate for the full 14-day duration, even after the product has been administered to the patient. However, it should be noted that 21 CFR 610.12 allows for the validation of a rapid sterility test, in which case the actual sterility test time to result may be significantly less than 14 days.

EU Guidelines for ATMPs

In May 2018, the EU published its new guidelines for ATMPs, "Rules Governing Medicinal Products in the European Union. Volume 4. GMP. Guidelines on Good Manufacturing Practice Specific to Advanced Therapy Medicinal Products.” The guideline applies to the manufacturing of ATMPs that have been granted a marketing authorisation and of ATMPs used in a clinical trial setting.

The guideline states, "[a]pplication of the sterility test to the finished product (Ph. Eur. 2.6.1) may not always be possible due to the scarcity of materials available, or it may not be possible to wait for the final result before the product is released due to short shelf-life or medical need. In these cases, the strategy regarding sterility assurance has to be adapted.” The guideline recommends to use alternative methods for preliminary results, combined with sterility testing of media or intermediates at relevant time points.

The use of validated alternative rapid microbiological methods according to Ph. Eur. 2.6.27 may also be considered, as long as method suitability for the product has been demonstrated. The guideline also states If the results of the sterility test of the product are not available at release, mitigation measures should be implemented, including informing the treating physician.

USP Informational Chapter <1071>

USP <1071>, Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk-Based Approach, became effective in December 2019. The chapter provides guidance when the compendial sterility test is unsuitable for product release due to short shelf life or immediate need, such as compounded sterile preparations (CSPs), positron emission tomographic (PET) products, and gene and cell therapies.

Rapid sterility tests (RSTs) should be risk-based and stakeholders should select their preferred technology considering time to result, specificity, limit of detection (LOD), sample size, and product attributes. The chapter refers to the 1% sampling plan in Ph. Eur. 2.6.27 and provides some examples. The chapter also recommends that rapid sterility tests or other rapid microbiological methods may be used as in-process controls prior to the final product release sterility test to provide early detection of gross contamination or probability that a product may fail sterility.

Finally, the chapter states that setting a limit of detection (LOD) of a single viable cell with all technologies is an unrealistic barrier of entry for any sterility test, especially when the signal is not the colony-forming unit that is amplified by cultural enrichment. We can agree with this but only as long as the method does not have a purported detection sensitivity level of a single cell. For example, some non-growth based rapid sterility tests have proven single cell detection capability (i.e., solid phase cytometry).

Considering a Rapid Sterility Test for ATMPs?

As can be seen by the newest policies and compendial recommendations, developing a comprehensive regulatory and validation strategy for ATMPs can be challenging. Our Consulting Team is available to assist with your ATMP rapid sterility test needs.