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FDA Promotes Rapid Sterility Testing for Human Gene Therapy Products in its Draft Guidance for Investigational New Drug Applications (INDs)

This week, FDA’s Center for Biologics Evaluation and Research (CBER) published six draft guidances relating to gene therapy, three of which cover products for specific disease categories (hemophilia, rare diseases, and retinal disorders), and three of which address manufacturing and clinical study design issues related to gene therapy: chemistry, manufacturing and control (CMC) information in INDs, long term follow-up study design, and testing of retroviral vector-based products.

In a press release, FDA Commissioner Scott Gottlieb, M.D., highlighted “rapid advancements” and “great promise” in the gene therapy space, saying the guidances “are aimed at fostering developments in this innovative field.”  Dr. Gottlieb acknowledged that for some gene therapies, FDA “may need to accept some level of uncertainty” at the time of approval regarding questions related to durability of response, as well as product manufacturing and quality.  He acknowledged the need, however, to assure patient safety and to assure that potential risks are adequately characterized and benefits are adequately demonstrated.

The draft guidance is titled, "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). Draft Guidance for Industry. July 2018. Clicking on the link will bring up a complete PDF of the guidance document.

The purpose of the draft guidance is to inform sponsors how to provide sufficient CMC information required to assure product safety, identity, quality, purity, and strength (including potency) of the human gene therapy investigational product.

When finalized, the draft guidance will supersede the document entitled “Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),” dated April 2008.

Because the draft guidance recommends the use of rapid microbiological methods (RMM) for in-process and finished product sterility testing, it is important to understand FDA's position in this regard. As such, I will highlight specific sections in the draft guidance and where appropriate, provide additional interpretation and comment.


Human gene therapy products are defined as all products that mediate their effects by transcription or translation of transferred genetic material or by specifically altering host (human)genetic sequences. Some examples of gene therapy products include nucleic acids, genetically modified microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, and ex vivo genetically modified human cells. Gene therapy products meet the definition of “biological product” in section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the prevention, treatment, or cure of a disease or condition of human beings.

The FDA requires all sponsors of investigational new drug products (DPs), including investigational gene therapy products, to describe the CMC information for the drug substance (DS). FDA may place the IND on clinical hold if the IND does not contain sufficient CMC information to assess the risks to subjects in the proposed studies. The CMC information submitted in an IND is a commitment to perform manufacturing and testing of the investigational product, as stated. However, FDA acknowledges that manufacturing changes may be necessary as product development proceeds, and sponsors should submit information amendments to supplement the initial information submitted for the CMC processes. The CMC information submitted in the original IND for a Phase 1 study may be limited, and therefore, the effect of manufacturing changes, even minor changes, on product safety and quality may not be known. Thus, if a manufacturing change could affect product safety, identity, quality, purity, potency, or stability, sponsors should submit the manufacturing change prior to implementation.

What is Required in the IND Application

The IND should include specifications with established acceptance criteria for safety testing at Phase 1. Safety testing includes tests to ensure freedom from extraneous material, adventitious agents, microbial contamination, and replication competent virus. Information on some common safety test methods is provided in more detail in section 3.2.S.4.2, Analytical Procedures.

To maximize the sensitivity of safety testing, it is important that sponsors perform each test at the stage of production at which contamination is most likely to be detected. For example, tests for mycoplasma or adventitious viruses (in vivo or in vitro) should be performed on cell culture harvest material (cells and supernatant) prior to further processing, e.g., prior to clarification, filtration, purification, and inactivation.

Additional testing will depend on the type of gene therapy product and the phase of clinical development. These tests may include assays to assess product characteristics, such as identity, purity (including endotoxin and contaminants, such as residual host cell DNA, bovine serum albumin (BSA), DNase), and potency/strength.

Sponsors should provide a description of all the analytical procedures used during manufacturing to assess the manufacturing process and product quality. In the original IND submission, descriptions should have sufficient detail so that FDA can understand and evaluate the adequacy of the procedures. FDA recommends that sponsors develop detailed SOPs for how analytical procedures are conducted at early stages of product development as a part of the sponsor's quality system. FDA acknowledges that, during product development, analytical methods may be modified to improve control and suitability. However, assay control is necessary during all phases of clinical development to ensure product quality and safety and to allow for comparability studies, following manufacturing changes.

Safety testing on the DS should include microbiological testing, such as bioburden (or sterility, as appropriate), mycoplasma, and adventitious viral agent testing, to ensure product quality. Guidelines and/or procedures for many safety tests have been described in detail, elsewhere (e.g., bioburden [1] sterility [2] mycoplasma [3], adventitious agent testing, and tests for specific pathogens [4]).

Sponsors should list DP specifications in the original IND submission. The testing plan should be adequate to describe the physical, chemical, or biological characteristics of the DP necessary to ensure that the DP meets acceptable limits for identity, strength (potency), quality, and purity. Product lots that fail to meet specifications should not be used in clinical investigation without FDA approval. For early phase clinical studies, FDA recommends that assays be in place to assess safety (which includes tests to ensure freedom from extraneous material, adventitious agents, and microbial contamination) and dose (e.g., vector genomes, vector particles, or genetically modified cells) of the product.

Sponsors should describe the analytical procedures used for testing the DP. If the analytical procedures are the same as those for the DS, sponsors do not need to repeat this information unless there is a matrix effect from the DP on assay performance.

Product Release Testing

FDA recommends that product release assays be performed at the manufacturing step at which they are necessary and appropriate. For example, mycoplasma and adventitious agents release testing is recommended on cell culture harvest material. In addition, sterility, endotoxin, and identity testing are recommended on the final container product to ensure absence of microbial contamination or to detect product mix-ups that might have occurred during the final DP manufacturing steps (e.g., buffer exchange, dilution, or finish and fill steps).

If a DP is frozen before use, FDA recommends that sponsors perform sterility testing on the product prior to cryopreservation so that results will be available before the product is administered to a patient. However, if the product undergoes manipulation after thawing (e.g., washing, culturing), particularly if procedures are performed in an open system, sterility testing may need to be repeated.

FDA also recommends that the results of in-process sterility testing be incorporated into the acceptance criteria for final product specifications.

Alternative Microbiological Methods and Rapid Sterility Tests

Analytical procedures different than those outlined in the United States Pharmacopeia (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 tests), and rapid endotoxin tests. FDA recommends that sponsors plan to demonstrate equal or greater assurance of the test methodology, compared to a compendial method, prior to licensure, as required under 21 CFR 610.9.

For reference, Sec. 610.9, Equivalent methods and processes, states the following:
Modification of any particular test method or manufacturing process or the conditions under which it is conducted as required in this part or in the additional standards for specific biological products in parts 620 through 680 of this chapter shall be permitted only under the following conditions:
(a) The applicant presents evidence, in the form of a license application, or a supplement to the application submitted in accordance with 601.12(b) or (c), demonstrating that the modification will provide assurances of the safety, purity, potency, and effectiveness of the biological product equal to or greater than the assurances provided by the method or process specified in the general standards or additional standards for the biological product; and
(b) Approval of the modification is received in writing from the Director, Center for Biologics Evaluation and Research or the Director, Center for Drug Evaluation and Research.
FDA recognizes that the compendial sterility test may not be suitable for all products. For example, rapid sterility tests may be needed for ex vivo genetically modified cells administered fresh or with limited hold time between final formulation and patient administration.

For ex vivo genetically modified cells that are administered immediately after manufacturing, in-process sterility testing on sample taken 48 to 72 hours prior to final harvest is recommended for product release. For such products, aside from an in-process sterility test, we also recommend that sponsors perform a rapid microbial detection test, such as a Gram stain, on the final formulated product and a sterility test, compliant with 21 CFR 610.12, on the final formulated product.

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. Although the results of the sterility culture performed on the final product will not be available for product release, this testing will provide useful data. A negative result will provide assurance that an aseptic technique was maintained. A positive result will provide information for the medical management of the subject and trigger an investigation of the cause of the sterility failure. The sterility culture on the final formulated product should be continued for the full duration (usually 14 days) to obtain the final sterility test result, even after the product has been administered to the patient.

In all cases where product release is prior to obtaining results from a full 14-day sterility test, the investigational plan should address the actions to be taken in the event that the 14-day sterility test is determined to be positive after the product is administered to a subject. Sponsors should report the sterility failure to both the clinical investigator and FDA. FDA recommends that sponsors include results of the investigation of cause and any corrective actions in an information amendment submitted to the IND within 30 calendar days after initial receipt of the positive culture test result.

In addition, be aware that a product may sometimes interfere with the results of sterility testing. For example, a product component or manufacturing impurities (e.g., antibiotics) may have mycotoxic or anti-bacterial properties. Therefore, FDA recommends that sponsors assess the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71>, Sterility Tests.


The draft guidance is a welcome indication that the FDA accepts and encourages the use of alternative and rapid microbiological methods, specifically for sterility testing of short-lived products, such as advanced therapy medicinal products (ATMP; gene and cell therapy). It is appropriate that the guidance document aligns fairly well with 21 CFR 610.12; however, there are some areas that require further clarification.

For example, the use of the 19th Century Gram Stain should be viewed only as a measure of gross contamination because low levels of microbial contaminants would never be observed from a loopful of finished product.

Next, a finished product sterility test that is compliant with 21 CFR 610.12 does not require a 14 day incubation via the compendial USP Sterility Test.

In fact, 21 CFR 610.12 states the following:
Advances in technology in recent years have allowed the development of new sterility test methods that yield accurate and reliable test results in less time and with less operator intervention than the currently prescribed methods. Some examples of novel methods include the Adenosine Triphosphate (ATP) bioluminescence, chemiluminescence, and carbon dioxide head space measurement. Manufacturers may benefit from using such sterility test methods with rapid and advanced detection capabilities. Accordingly, we have amended § 610.12 to promote improvement and innovation in the development of sterility test methods, to address the challenges of novel products that may be introduced to the market in the future, and to potentially enhance sterility testing of currently approved products. This final rule provides manufacturers the flexibility to take advantage of methods as they become available, provided that these methods meet certain criteria.
Because some of these new technologies do not reply on microbial growth, or more raid growth-based methods may be validated to provide an equivalent sterility test result in less than 14 days, the full USP <71> incubation period may not be necessary.  

The teachings in 21 CFR 610.12 do allow for the sterility testing of material other than the finished drug product in its final container. The May 3, 2012 Federal Register Vol. 77, No. 86, Page 26162 – 26175 provides additional guidance on what alternative material may be comprised of, e.g., bulk material or active pharmaceutical ingredient (API), in- process material, stock concentrate material), as appropriate, and as approved in the biologics license application (BLA) or BLA supplement.

Additional guidance was provided in the same CFR reference (see page 26165 in the CFR):
As discussed in the preamble to the proposed rule (76 FR 36019 at 36021), certain allergenic and cell and gene therapy products may need to be tested for sterility at an in- process stage or some other stage of the manufacturing process (e.g., intermediate, API, bulk drug substance) instead of the final container material because the final container material may interfere with the sterility test. Likewise, as discussed in the preamble to the proposed rule, some cell therapy products and cell-based gene therapy products may need to be tested for sterility at an in-process stage or some other stage of manufacturing process because low production volumes may result in an insufficient final container material sample for sterility testing or a short product shelf-life may necessitate administration of the final product to a patient before sterility test results on the final container material are available.
Therefore, based on 21 CFR 610.12, it may be appropriate to test an alternative material and not necessarily the finished product, as long as this strategy is justified and acceptable to FDA. For these reasons, the draft guidance should be clearer on whether a sterility test on the final formulated product is a recommendation or an expectation.

Furthermore, the statement regarding demonstrating the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71>, should be extended to alternative or rapid sterility tests. These studies are, in fact, an expected part of the qualification of the alternative method and is usually conducted during the Method Suitability phase of the validation process.

Overall, the draft guidance is further assurance from the FDA that rapid and alternative methods are encouraged for use, especially on product that is short-lived or needs to be administered prior to obtaining results from the compendial sterility test. Although the document is specifically focused on gene therapy products in support of IND applications, the recommendations can be used for the routine release of the same or similar ATMPs.

Training on Rapid Sterility Testing of Gene and Cell Therapy Products

I will be speaking on this same topic during the PDA Europe Conference on Pharmaceutical Microbiology, which will be held in Berlin, Germany on October 15-16. More importantly, I will provide a comprehensive overview of rapid methods, including validation strategies, technology reviews, regulatory acceptance and applications during a two-day training course immediately following the conference on October 17-18. For more information, please visit


  1. USP<61> describes membrane filtration, plate count, and most probable number methods that can be done to quantitatively determine the bioburden of non-sterile DPs. Although 21 CFR 211.110(a)(6) does not specify a test method, it requires that bioburden in-process testing be conducted pursuant to written procedures during the manufacturing process of DPs.
  2. Sterility testing may be performed on the DS when it cannot be performed on the DP, as outlined in the final rule: Amendments to Sterility Test Requirements for Biological Products (May 3, 2012; 77 FR 26162 at 26165). Sterility tests are described in 21 CFR 610.12 and USP<71> Sterility Tests.
  3. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, July 1993.
  4. Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications, February 2010.


  1. I am considering using a rapid sterility method (growth-based) for an LVV DS. However, I have not found clear guidance outside of knowing to validate the method against the compendial <71> method.

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