How CSL Behring Worked With EMA/FDA To Develop Hemgenix Potency Assays
By Yan Zhi, Ph.D., CSL Behring

Gene therapy has become an exciting modality by offering the possibility of curing an underlying disease within a specific patient population. Adeno-associated viruses (AAV) have been widely used as delivery vehicles for in vivo gene therapy (GT) applications. A total of eight AAV-based in vivo GT products have been approved by FDA and/or EMA. They include:
- Glybera, initial approval in 2012,
- Luxturna, initial approval in 2017,
- Zolgensma, initial approval in 2019,
- Upstaza, initial approval in 2022,
- Roctavian, initial approval in 2022,
- Hemgenix, initial approval in 2022,
- Elevidys, initial approval in 2023, and
- Beqvez, initial approval in 2024.
Potency is a critical quality attribute (CQA) for AAV-based in vivo GT products. The FDA guidance (2011) “Potency Tests for Cellular and Gene Therapy Products” provides the agency’s recommendations. Sponsors can develop either an individual method or a matrix of methods to measure product potency. In general, a fit-for-purpose or qualified potency assay based on protein or even mRNA expression after vector transduction could be sufficient during clinical development stage for investigational new drug applications, while functional assay that directly evaluates the mechanism of action (MoA), fully validated per ICH guidance, is expected for a BLA. Potency measurement should correlate to the clinical efficacy of the CGT product and is required for product release, comparability, and stability studies. The same FDA guidance also outlines the scientific challenges associated with potency assays for GT products, including limited sample sizes, the lack of reference standards, limited stability, and potentially complex MoA.
In March 2023, the Alliance for Regenerative Medicine (ARM) and the American Society of Gene & Cell Therapy (ASGCT) released a report addressing potency assay-related development delays for cell and gene therapies in response to an October 2022 meeting with FDA. It is noted that, since the 2011 FDA guidance for potency tests for CGT products, new technologies have been developed. Ideally, sponsors want a bespoke and multifaceted approach to demonstrate product potency. In some cases, regulatory requirements for potency assays may create redundancies that exceed what is needed to demonstrate potency.
As pointed out in this report, GT products are designed to undergo a series of processing events, which at minimum would include cell transduction in the targeted tissue and, subsequently, therapeutic protein expression to achieve the desired therapeutical effect in the intended patients. As a result, it can be challenging to decide where in this cascade to measure potency. Measuring toward the beginning of this cascade (for example, simply looking at transduction efficacy) will not ensure a functional product at the end, while measuring toward the end of this cascade would introduce significant biological variability.
Hemgenix: An Assay Development Case Study
Hemgenix is a recombinant AAV serotype 5 (AAV5) based in vivo gene therapy drug indicated for the treatment of adult patients with hemophilia B (congenital factor IX deficiency) to significantly reduce the frequency of bleeding episodes and factor IX (FIX) replacement therapy. CSL Behring produces Hemgenix using a baculovirus expression system in an insect cell line purified into drug product through sequential downstream processing steps, including affinity capture, additional purification/polish, and viral/sterile filtration.
As a one-time intravenous infusion based on the nominal genome copy (GC) concentration per kg of patient body weight, Hemgenix was approved by FDA under priority review in November of 2022 and was granted a conditional marketing authorization in 27 EU countries by the European Commission (EC) in February 2023. In the BLA and MAA for Hemgenix, both the cell-based infectivity assay to measure AAV vector replication with the acceptance criteria and the cell-based relative potency assay to quantify FIX activity with the acceptance criteria are included for lot release and stability testing.
An established cell line from a human hepatocellular carcinoma is used to mediate production of functional hFIX-Padua protein after transduction. The activity of hFIX-Padua protein in transduced cell culture supernatants is assessed using a commercially available chromogenic factor IX activity assay kit and reported as a relative potency value compared to a product-specific reference standard with the established potency value.
Regulatory Feedback About Potency Assays During Hemgenix BLA/MAA Submission
During BLA/MAA procedure, regulatory feedback related to potency assay was received and addressed.
Some feedback was: “The applicant was requested to justify the use of genome copies per cell for potency assessment. It was recommended to use infectious particles for this assessment.”
It was clarified that vector GC per kg of patient body weight is directly correlated with GC concentration per a liver-derived cell, and that it is the established strategy to establish consistent dosing in clinical studies. In addition, the vector genome titer assay per quantitative PCR (qPCR) was validated per ICH guidance to be an appropriate measurement of product strength with the required accuracy and precision. The assessment of infectivity is monitored through the ratio of GC to infectious particle (ip). The infectious vector titer is determined by a cell-based in vitro assay, which has inherent higher assay variability than genome copy measurement by qPCR method. In addition, HeLaRC32 cells used in infectivity assays are different from the liver-derived cells used in potency assays. As a result, EMA accepted the justification of using genome copies per cell for potency assessment.
Other feedback was: “The applicant was requested to evaluate the feasibility of establishing analytical methods for release retesting in the EU.”
It was emphasized that Hemgenix is intended to treat a disease with an orphan indication and a limited patient population in the EU. The current manufacturing process has a limited batch size, and noticeable portion of each drug product (DP) batch would be depleted by QC release testing and reference samples. Conducting release retesting after importation into the EU would deplete an additional portion of DP batch. An increase in DP material demand to implement additional release specification per post-marketing commitment (PMC) — plus the possibility that new analytical methods could be introduced amid a rapidly evolving advanced therapies landscape — might additionally reduce the available material for patient treatment. It was also pointed out that the facility to manufacture Hemgenix was successfully GMP-inspected by the corresponding national competent authority and that the qualified person (QP) will certify that each production batch is in accordance with the requirements of MAA. As a result, EMA agreed to an approach with a reasonable lead time to establish the analytical methods required for DP release testing in the EU.
Additional feedback was: “The agency considered the potency assay which measures FIX activity as insufficient and requested the introduction of an assay to measure transgene expression.”
It was pointed out the extended characterization data has demonstrated the robust correlation between the current release potency assay based on FIX activities and FIX protein expression in the transduced cell supernatant after being normalized against product-specific reference standards for a wide set of development batches with potency value, which is much wider than the acceptance criteria of potency value for batch release. Therefore, it was demonstrated that the current activity-based potency assay has already ensured the proper control of the product for transgene expression across manufacturing batches, even if indirectly.
FDA’s request was also acknowledged. To ensure linkage between the FIX expression and potency release assays, the sample to be analyzed for FIX expression will be taken from the cell supernatant of the potency release assay. Due to the cell-based nature of this bioassay, the absolute concentration of FIX protein for the samples, control and reference standard are expected to be assay dependent. As a result, the reportable result in FIX expression assay will be the level of FIX protein expression normalized against the FIX protein expression of the product reference standard. A FIX expression assay will be validated per ICH guidance to support commercial product release testing. It was agreed with FDA to use a PMC to validate an assay for the measurement of FIX expression level and to add such validated assay into the release testing panel with a specification.
Potency Assay Transfer After EMA Approval Of Hemgenix
As a PMC with EMA, the cell-based relative potency assay is to be transferred to a lab in EU for DP release testing after product importation.
Primarily, it is important to have not only a clear understanding of the comprehensive list of all critical materials (for example an analytical cell bank and product-specific reference standard/control sample), but also a solid supply plan for such critical materials to meet the demands for both commercial manufacturing and assay transfer. Considering the complexity of cell-based potency, it is not uncommon for the receiving unit to require additional critical materials for technical staff training and the potential for a higher assay failure rate during and immediately after assay transfer compared to the sending unit.
Furthermore, the appropriate testing sample(s) are needed to show method comparability between the sending and receiving units after assay transfer. In principle, a testing sample must be representative of the commercial product. However, it might be difficult to directly use commercial batch for assay transfer due to the concern of “double testing.” Alternatively, a representative testing sample could be created by mixing more than one commercial batch.
Next, it is critical for the sending unit to provide a clear method standard operating procedure (SOP), which should contain detailed technical information and minimize subjectivity as much as possible to increase assay robustness between the sending and receiving units.
Finally, it is effective to have technical staff from the receiving unit to directly observe the assay performance at the sending unit for learning and to have technical staff from the sending unit available for assay troubleshooting at the receiving unit.
Conclusion
Collaborations between global regulators and industry sponsors are essential for a tailored paradigm to measure biological activities, including potency, of gene therapy products, which are designed to provide a blueprint for patients to make functional proteins using their own cellular machinery. The gene therapy industry is still in its infancy and must maintain its momentum so that more patients can benefit from this new therapeutic modality.
About The Author:
Yan Zhi, Ph.D., is a director of cell and gene therapy product owner in process engineering at CSL Behring responsible for the technical performance of Hemgenix to support worldwide markets. Past posts include scientific and analytical leadership roles at WuXi AppTec, Fujifilm, Charles River Laboratories, and Spirovant. She received her undergraduate degree at the University of Science and Technology of China and her Ph.D. in microbiology and molecular genetics at the University of California, Irvine.