Guest Column | January 21, 2022

Proposed Solutions For Cell & Gene Therapy Validation Challenges

By BioPhorum


Cell and gene therapy (CGT) products are a class of advanced therapeutics that have tremendous potential to treat diseases. However, their manufacturing challenges are complex and successful commercialization of CGT products requires a multidisciplinary approach that integrates patient needs and product knowledge with the capability to commercially manufacture these complex products consistently and reliably.

The basic principles of current good manufacturing practices and general process validation concepts and practices apply to CGT manufacturing processes and analytical methods; however, following validation, concepts established for general biologics can result in challenges for CGT product validation due to their unique characteristics. Therefore, CGT validation requirements are still in development. Specific challenges in validating CGT product manufacturing processes include identifying quality attributes, establishing specifications, and validating analytical methods.

Many CGT products are novel personalized medicinal products manufactured in small batch sizes using relatively new technology. Given that CGT products are targeted for numerous modalities across a broad range of biomanufacturing processes, each has its own validation considerations.

Regulatory requirements for a controlled validation process are well established and have extensive guidelines. If these guidelines do not provide a clear route to address a specific CGT validation challenge, it is strongly recommended that direct communication with the relevant regulatory agency is initiated to discuss the challenge and potential resolution strategies. All proposed strategies should have a clear, detailed justification and fully documented evidence.

CGT-Specific Validation Challenges

While there are numerous challenges associated with CGT validation, as more experience and knowledge are gained on CGT manufacturing processes, some challenges will be resolved and others will be identified.

For example, when there is a limited supply of starting materials, validation of cell therapy manufacturing processes may use surrogate materials. When possible, you should consider complementing surrogate materials with samples from actual starting materials. Given the limited supply of starting materials and/or process intermediates, a concurrent validation may be acceptable where there is a strong benefit-risk ratio for the patient.

Due to the variety and complexity of CGT products, all solutions to these validation challenges should be considered against the specific product and aligned with the relevant regulatory agency before implementation.

Manufacturing Process Validation Challenges

Two of the main manufacturing process validation challenges relate to a limited batch history and designing process performance qualification (PPQ) for autologous therapies. The following is a sample of key challenges and proposed solutions for each.

Limited Number Of Batches At Commercial Scale Before And During PPQ

Companies may use different strategies to help them comply with industry guidelines for process validation in scenarios where limited manufacturing lots are produced to support the process control strategy.

Stage 1 – Process Design: The goal is to capture process knowledge while designing the commercial manufacturing process to deliver material that meets its quality attributes and use this information to establish the process control strategy for late-stage and commercial manufacturing. When only a few lots are expected, you should establish a strong manufacturing process as early as possible, complemented by analytical assays to assess critical quality attributes.

Stage 2 – Process Qualification: This stage confirms that the facility and process performance can produce material of consistent product quality at the commercial manufacturing scale. In gene therapies, fewer than three batches could provide sufficient material for launch and the market space. There may be the same scenario in cell therapy or different challenges depending on the cell therapy type. One scenario could be having one batch, per dose, per patient. Therefore, questions could include: Is it necessary to produce more lots at an undesirable cost burden without a defined need for the material? How are prospective acceptance criteria set with limited data for statistical analysis?

Stage 3 – Continued Process Verification (CPV): The recommended approach for CPV is like setting acceptance criteria for PPQs. It is suggested that you leverage data from an applicable platform or similar process and consider including earlier clinical batches or pilot-scale batches. A CPV monitoring program is typically implemented for Phase 3 or PPQ batches as the entry point to represent the commercial manufacturing history.

Designing PPQs For Autologous Cell Therapy

There are unique features among different modalities of products involved within the broader classification of CGT products – one of which is autologous cell therapy.

Limited Material Availability

Typical batch sizes are very small for autologous cell therapy products due to their personalized nature, and the amount of material (cells) is limited throughout the manufacturing process. The amount of material needed for extended characterization and stability testing during PPQs can reduce the available cells for dosing. Sometimes, the reduction can be such that the minimum required dose may not be achievable.

In cases where the resulting dose is above the acceptable level, it still leads to an ethical conundrum: return the cells to the patient or use the patient cells for characterization.

A common solution is to use surrogate cells from healthy donors as starting materials for PPQ batches. These are collected and processed using the same manufacturing process and tested using the same methods for patient cells. Surrogate cells do not need to be infused back with the final drug product (DP), so all the material is available for the extended testing needed during PPQs. If surrogate cells are used, you should demonstrate that the DP made using starting material from surrogate cells is representative of DP made from patient cells.

Wide Variability In Product Attributes

For autologous cell therapy, each product batch is made using cells collected from individual patients. This starting material can have a wide variability due to differences in patients, their disease state, and prior treatments. This can result in variability in process performance and product quality attributes. The combination of variability from starting material, the process, and analytics results in wide variability in the autologous cell therapy product that poses a unique challenge when setting appropriate acceptance criteria for PPQs.

To address this, it is important that contributions from various sources of variability during process development and process characterization are well understood. Data from a clinical study can be used to understand the total variability in the product, but controlled experiments might be necessary to tease out contributions from different sources.

Analytical Method Validation Challenges

Two of the main analytical method validation challenges relate to assay variability and potency assay validation. The following is a sample of key challenges and proposed solutions.

Assay Variability

Analytical test methods for CGTs tend to be complex and involve specialized techniques and novel instrumentation. For example, gene therapy product quality attributes are assessed for both the integrated capsid/DNA complex and for capsid and DNA as discrete entities. High assay variability is often encountered with these methods. CGTs inherently provide some unique challenges to variability.

For example, testing instances during validation and routine testing may be limited by batch size and batch frequency. Understanding assay variability requires sufficient material, time, and repetition to endure the many changes often experienced over a longer period. Such changes include variation among critical reagent lots and instrument/pipette calibration cycles. With limited opportunities for batch testing and/or short durations for validation execution, the assay may not be exposed to enough of these and other variables to realize true assay performance typically determined through empirical and statistical methods.

Potency Assay Validation

Potency is the ability of the product to effect a given result and generally reflects the therapeutic activity of the product. Potency assays are therefore based on the mode of action of the DP. For CGT products, the mode of action is complex, and more than one function is critical to their therapeutic activity. Therefore, targeting a single attribute for the potency assay is not sufficient. You should develop multiple assays or an assay matrix based on different product attributes that include at least one quantitative assay of the biological activity. The assay matrix should include assays that measure/demonstrate the ability of the vector to transfer the gene to the target cell, the expression of the transferred gene, and the biological effects of the expressed gene.


The manufacturing processes for CGT products are complex, as are the raw materials and analytical methods used during production and product release. Controlling these inputs is critical to ensuring the safety and efficacy of products. Process validation is one of the key elements required to successfully deliver safety and efficacy to patients. To do this successfully, development data, manufacturing history, and specifically designed studies are needed. It is also necessary to leverage the body of knowledge available to develop a sufficient and robust chemistry, manufacturing, and control package that creates a path toward commercialization.

Although the regulatory guidelines around validating CGT manufacturing processes are evolving, expectations for scientific robustness behind the validation strategies are unlikely to change. A strong understanding of the product and manufacturing process, together with reliable analytical methods, are paramount. There is also the need for a risk-benefit analysis when developing a robust process validation strategy through the life cycle of these novel therapies and to support successful commercialization.

This article is a summary of a recent BioPhorum publication on the topic. To read more, check out the full report, Cell and gene therapy validation challenges.