Deep Dive: FDA Guidance on Gene Therapy Products: RCR Testing and Monitoring

June 4, 2021

In January 2020, FDA published a guidance document entitled “Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up.” In a recent blog post, we introduced the guidance document, providing a general overview of its contents. Now, we will begin diving into the specifics of the document, getting into the details of the FDA’s recommendations and what that means for Sponsors developing cell and gene therapies. First up: product testing.

Recommendations for Product Testing

In this section of the guidance document, FDA provides guidance on specific materials to be tested for RCR. The Agency recommends using a stably transfected Vector Producer Cell (VPC) bank system, when possible, to create a consistent source of retroviral vector derived from a single cell clone. The VPC bank system should include a Master Cell Bank (MCB) and a Working Cell Bank (WCB); and all transient transfection-produced cell banks should be qualified as detailed in the CMC guidance. FDA recommends RCR-specific testing be conducted on the VPC MCB, vector supernatant, end of production cells, and ex-vivo transduced cells (if applicable).

RCR testing of the MCB should include ecotropic (narrow host cell line) as well as amphotropic (wide host cell line) RCR when the VPC are derived, at any step, by transduction with an ecotropic retroviral vector. Furthermore, it is also advised that the testing be performed using a cell line known to be permissive to infection by an ecotropic RCR. A properly qualified WCB, on the other hand, does not require RCR testing unless the MCB RCR testing is found to be deficient.

RCR testing of the supernatant should be accomplished by amplification on a permissive cell line in an amount that provides a 95% probability of detection of RCR if present at a concentration of 1 RCR/dose equivalent. The amount tested should be enough to allow detection of a single RCR in an individual assay and be performed in triplicate. Additional information for determining appropriate test volume, assay sensitivity, replicate size, and number of replicates is provided in the appendix of the guidance document.

Cell testing should be performed on the lesser of 1% or 108 pooled vector-producing cells or ex-vivo transduced cells with a permissive cell line. For ex-vivo retroviral transduced cells, the guidance recommends testing the cells for RCR regardless of the length of time the cells are cultured following transduction unless documented manufacturing and clinical data support that the transduced cell product is consistently free of RCR. If data does support discontinuation of RCR testing, the guidance recommends that a sufficiently sized sample of the cell product is archived for at least six months after the product expiration date unless the ex-vivo transduced cells are tested for RCR at product release.

Assays for testing vector supernatant and cells should include co-culture on a permissive cell line. Alternative methods such as Polymerase Chain Reaction (PCR) may also be utilized for release of testing of ex-vivo transduced cells provided they are developed in consultation with FDA’s Center for Biologics Evaluation and Research (CBER). Additionally, development of RCR assays that support virus entry, amplification, and particle production is encouraged. The Agency also supports the development of more sensitive alternative methods that reduce the interference effect inherent in high titer vector preparations.

Development of a standard virus stock is recommended for:

  • Validation of assays used to detect the presence of RCR prior to licensure
  • Determining the size of retroviral supernatant replicates to be tested that will ensure detection of a single retrovirus
  • A positive control (infectious titer) to empirically determine the relative sensitivity of assay methods used for detection of RCR in retroviral vectors

Any standard virus stock developed should be characterized for growth kinetics in the cells used for the RCR assay and tested for stability.

Interested in learning more? This is only part two in our four-part blog series, so stay tuned because there is a lot more to come. Next up we will get into the details of the Agency’s recommendations for patient monitoring.

Trusted Experts in Cell and Gene Therapy Development and Regulation

Are you in the process of developing a cell and gene therapy product subject to the recommendations outlined in this guidance document? Our Cell and Gene Therapy Center of Excellence has experience with all aspects of cell and gene therapy product development and is ready to help you achieve successful interactions with FDA. Contact us today to learn more about our cell and gene therapy-specific services and qualifications.

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