Key Takeaways
Release testing can constrain cell and gene therapies throughput even after production steps have been accelerated.
Sterility and mycoplasma testing create time-to-result challenges for short-shelf-life cell therapies.
Potency assays present a different bottleneck because they must be biologically meaningful, robust, and compatible with routine release.
Analytical methods mature throughout development and can require qualification, validation, transfer, and comparability work as programs approach commercialization.
Manufacturing expansion delivers its full value only when analytical testing, QC review, and QA disposition scale alongside production.
The Bottleneck Moves Downstream
Manufacturing speed does not by itself determine how quickly a cell and gene therapy product can reach a patient. The product must also be sampled, tested, reviewed, and formally released, and those activities operate on timelines of their own. For products with limited shelf lives, the interval available for release testing may be especially narrow. For more complex modalities, analytical methods must also demonstrate that the product has the required identity, purity, safety, and biological activity before a lot can move forward.
A multicenter cell study conducted by the NHLBI-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) illustrated how much of the total timeline can sit outside the core processing step: a cell-separation procedure that took approximately 90 minutes sat within an overall process of roughly seven hours once incoming testing, preparation, release testing, randomization, and delivery were included. The authors cautioned that the processing step alone could give a misleading picture of the time required to prepare and release the product.1
The same issue appears in current regulatory expectations for chimeric antigen receptor (CAR)-T cell products. Fresh CAR-T cells have a limited shelf life and consequently a limited window in which release testing can occur. That window has to accommodate sampling, testing, reporting of quality control (QC) results, quality assurance (QA) review, shipping, and receipt at the clinical site.2
As manufacturing becomes faster and more scalable, the constraint can simply move downstream unless analytical testing, QC review, and QA disposition expand with it.
Sterility Testing Creates a Clock Problem
Microbiological testing provides perhaps the clearest example of an assay colliding with the operating realities of cellular therapy. Traditional culture-based tests were developed in settings where products could wait for results. Some living cell products cannot.
A review of sterility testing for cellular therapies by NIH Clinical Center investigators James Gebo and Anna Lau described conventional sterility and mycoplasma testing timelines of 14 and 28 days, respectively, and noted that these timelines are incompatible with some short-shelf-life advanced therapy products, including products intended for fresh infusion.3 For these therapies, the issue is not simply whether a microbiological method can detect contamination. The method must provide a sufficiently reliable answer within the period available for release.
Product format can change that timing pressure. Fresh CAR-T products have a limited period between formulation and infusion, while cryopreservation can provide sufficient time for full release testing and greater scheduling flexibility.2 The release strategy therefore must be compatible with the way the product will be stored, shipped, and administered.
Rapid microbiological methods can shorten the testing window, but they do not remove the analytical burden. Alternative sterility methods still have to demonstrate appropriate performance, and noncompendial approaches require suitable qualification and validation. The challenge shifts from waiting for microbial growth to establishing that a faster method can reliably support release decisions.
For manufacturers seeking shorter cycle times, microbiological testing must fit within the product's usable life while still providing the confidence required for release.
Potency Is a Different Kind of Bottleneck
Potency presents a different analytical problem. Sterility testing is often constrained by the time required to obtain a result. Potency testing begins with a more fundamental question: what measurement adequately demonstrates the biological activity of this particular therapy?
Potency measurements for cellular and gene therapies are product-specific, and no single assay format applies across the category.4 Developers must therefore establish a strategy suited to the product's mechanism and biological activity rather than relying on a universal platform test.
CAR-T cells illustrate the difficulty. Their activity cannot always be represented by one analytical readout. Relevant approaches can include cell-killing assays, measurements of transduction efficiency, and cytokine-secretion assays, while products containing additional functional transgene elements may require separate measurements when those elements make meaningful contributions to biological activity.
Adeno-associated virus (AAV) gene therapies pose a different version of the same challenge. Potency must demonstrate meaningful biological activity within a vector system whose performance depends on both the vector and its therapeutic payload. Analytical reviews of recombinant AAV development identify potency as one of the areas where method design and standardization remain challenging, alongside characterization of vector identity, purity, and other critical attributes.5
Potency assurance also extends beyond the final lot-release result. It can involve manufacturing process design, process controls, material controls, in-process testing, and lot-release assays working together to establish confidence in product activity.6
For industrialization, the assay must be biologically meaningful, sufficiently robust, and compatible with routine release at the required manufacturing cadence.
When Assay Duration Collides with Product Shelf Life
For short-lived cell products, analytical strategy and manufacturing strategy cannot be developed independently. Some assays may take longer than the interval available between manufacturing and administration.
A 2024 review led by NIST researchers examining mechanism of action, potency, and efficacy in cell therapies discusses products for which potency results may not be available before the end of the product's shelf life and considers faster methods and staged release strategies as potential responses to that constraint.7
The difficulty is especially visible with fresh products because the release process competes directly with the product's remaining usable life. The maximum interval between formulation and infusion has to account not only for product stability but also for testing, review, shipment, and clinical-site handling.
That makes release strategy an early operating-model consideration. The approach must be compatible with whether the product will be administered fresh, supported through a staged release approach where appropriate, or given additional testing time through cryopreservation.
Cryopreservation can provide more time for release activities, but it changes other aspects of the manufacturing and product-control strategy. Additional considerations include thawing controls and assessment of risks associated with the cryoprotectant. Extending the analytical window therefore changes the operating model rather than eliminating the underlying release requirements.
Identity Adds Another Release Requirement
Identity testing contributes another distinct requirement to the release package. For patient-specific therapies, it sits alongside but does not replace the systems used to maintain chain of identity.
For CAR-T cell products, identity testing can address both the intended genetic modification and the cellular composition of the final product. Chain of identity serves a different purpose: maintaining the link among starting material, manufacturing intermediates, final product, and the intended recipient.
A manufacturer may therefore need to demonstrate what the product is biologically while also showing that it remains correctly linked to the appropriate manufacturing and patient pathway.
Gene therapy vectors likewise require identity confirmation as part of a broader analytical profile that can include vector identity, therapeutic gene identity, potency, purity, and safety attributes.
Release Is a Panel, Not a Single Assay
The analytical bottleneck rarely comes down to one test. Product disposition depends on a collection of measurements, and the combined workflow determines how quickly a batch can move forward.
CAR-T cell release can involve safety, identity, purity, biological activity, strength, sterility, mycoplasma, endotoxin, adventitious-agent testing, viability, vector-related attributes, and other product-specific characteristics. Vector manufacture introduces its own release package before the vector is used to produce the cellular product. Human gene therapy guidance similarly requires chemistry, manufacturing, and control information sufficient to address safety, identity, quality, purity, and strength, including potency.8
Even when no individual assay dominates the timeline, samples still must move through multiple methods, results must be reviewed, exceptions or investigations addressed when necessary, and QA must determine whether the lot can be released.
Gene therapy vectors add a similarly broad analytical package. Final-product characterization can include viral genome titer, functional or infectious measurements, vector and therapeutic gene identity, product-related impurities, contaminants, and other quality attributes.9 Each test adds another required result to the overall release sequence.
Improving one assay can therefore leave the overall bottleneck largely intact if the rest of the analytical and review workflow does not accelerate with it.
The Analytical Platform Has Its Own Development Curve
Release methods also mature as a program moves from early clinical manufacturing toward licensure. The analytical strategy that supports an early investigational study may not be sufficiently developed to support pivotal studies or commercial release.
For CAR-T programs, analytical assays may initially be fit for purpose and become progressively qualified and validated as development advances. Specifications and some methods can still evolve during early clinical stages as characterization data accumulate, while later-stage and licensure-supporting assays require a more mature analytical package.
Changes introduced later in development can create additional work. Replacing an assay, changing how an attribute is measured, or revising an acceptance criterion can require qualification, bridging, comparability assessments, or supporting data depending on the nature of the change.
A process may therefore reach operational maturity before the assays needed to characterize and release its product have done the same. Delaying analytical development shifts qualification, validation, transfer, and comparability work toward pivotal and commercial stages, where timelines are less forgiving and late method changes can complicate comparability, validation, and the regulatory evidence supporting product consistency.
AAV development faces similar pressures. Analytical reviews describe limited throughput, lengthy turnaround times, and a continuing need to improve or standardize methods used to characterize several vector attributes. As manufacturing expands, the analytical platform must mature with it.
Scaling Analytical Capacity Alongside Manufacturing
The practical capacity question for CGT manufacturers is not simply how many batches a facility can produce but how many batches the entire manufacturing and quality system can move to release.
Every additional lot generates more samples, more analytical work, more data to review, and more demand on QC and QA. Additional manufacturing suites, larger vector-production trains, or more parallelized cell-processing systems can increase physical production capacity, but released-product throughput still depends on analytical instruments, analyst availability, method scheduling, data reporting, review capacity, and lot disposition.
The release workflow for fresh CAR-T products makes this interdependence particularly visible. Testing and QA review sit in the same operational sequence as formulation, shipment, and clinical-site handling, so delays in one part of the system can consume time available elsewhere.
A similar mismatch can emerge during gene therapy process development. High-throughput manufacturing experiments can generate samples faster than established analytical methods can characterize them. Reviews of recombinant AAV process analytics identify limited throughput and lengthy turnaround times for some established methods, creating an analytical constraint even before a product reaches routine commercial release.10
Expanding manufacturing without expanding analytical capability can therefore move the queue downstream. A facility may gain bioreactor capacity or additional cell-processing slots while the true constraint shifts to sample testing, instrument access, analyst workload, QC review, or QA disposition.
Different assays will require different responses. Some may benefit from rapid alternative methods. Others may need more robust assay design, greater automation, earlier qualification and validation, or better standardization. Analytical capacity must be planned as part of manufacturing scale-up rather than treated as a support function expected to absorb additional volume after production expands.
Building Release Strategy into Cell and Gene Therapy Industrialization
Cell and gene therapy manufacturing continues to shorten and automate operations that once required longer and more manual workflows. The value of those improvements depends on whether the analytical and quality systems surrounding production can keep pace.
Release testing also supports more than the final disposition decision. Analytical methods feed process development, comparability assessments, stability programs, and the ability to expand manufacturing without creating a parallel backlog in QC.
As cell and gene therapy manufacturing becomes faster and more scalable, released-product throughput will increasingly depend on whether analytical development, testing capacity, QC review, and QA disposition can advance at the same speed. For advanced therapy manufacturers, meaningful capacity is the capacity of the full manufacturing and quality system to move product through release and into administration reliably.
References
1. Richman, Sara, et al. “Factors affecting the turnaround time for manufacturing, testing, and release of cellular therapy products prepared at multiple sites in support of multicenter cardiovascular regenerative medicine protocols: a Cardiovascular Cell Therapy Research Network (CCTRN) study.” Transfusion. 52: 2225–2233 (2012).
2. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products: Guidance for Industry. U.S. Food and Drug Administration. Center for Biologics Evaluation and Research. 31 Jan. 2024.
3. Gebo, James E T, and Anna F Lau. “Sterility Testing for Cellular Therapies: What Is the Role of the Clinical Microbiology Laboratory?” Journal of Clinical Microbiology. 58: e01492-19 (2020).
4. Potency Tests for Cellular and Gene Therapy Products: Guidance for Industry. U.S. Food and Drug Administration. Center for Biologics Evaluation and Research. Jan. 2011.
5. Green, Erica A, and Kelvin H Lee. “Analytical methods to characterize recombinant adeno-associated virus vectors and the benefit of standardization and reference materials.” Current Opinion in Biotechnology. 71: 65–76 (2021).
6. Potency Assurance for Cellular and Gene Therapy Products. Draft Guidance for Industry. U.S. Food and Drug Administration. Center for Biologics Evaluation and Research. 28 Dec. 2023.
7. Simon, Carl G Jr., et al. “Mechanism of action, potency and efficacy: considerations for cell therapies.” Journal of Translational Medicine. 22: 416 (2024).
8. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs): Guidance for Industry. U.S. Food and Drug Administration. Center for Biologics Evaluation and Research. Jan. 2020.
9. Clément, Nathalie, and Joshua C Grieger. “Manufacturing of recombinant adeno-associated viral vectors for clinical trials.” Molecular Therapy – Methods & Clinical Development. 3: 16002 (2016).
10. Gimpel, Andreas L, et al. “Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies.” Molecular Therapy – Methods & Clinical Development. 20: 740–754 (2021).
