Gene therapy developers face numerous analytical challenges, from discerning what product qualities are critical attributes to the need for fit-for-purpose analytical methods for viral vectors that offer a combination of speed, sensitivity, and resolution. Leveraging a contract research organization (CRO) partner with specialized expertise and awareness of evolving technological and regulatory development that functions as part of their customers’ internal analytical teams can lead to accelerated timelines and a higher probability of program success.
Short List of High-Level Challenges for Viral Vector–Based Therapies
Gene therapy based on viral vector delivery is still in its infancy, and like all other emerging technologies faces several high-level analytical challenges. One of the biggest is gaining the necessary depth of understanding of these novel materials. What is the best approach for analytically identifying viral vectors, and how does that approach scale so that it continues to provide valuable information? Making the right choices is central to the development of these products.
The in vivo outcomes of gene therapy drug products are becoming increasingly important in establishing the longevity and persistence of these treatments from the payer perspective. Gaining a platform understanding through detailed molecular characterization and interfacing that knowledge with clinical outcomes (safety and efficacy) is, therefore, essential, but can be difficult to achieve.
More specifically, what features of the molecules dictate both positive and negative aspects of the infectivity, productive transcription, and persistence of the therapeutic product in the body must be understood. Liquid chromatography (LC) and mass spectrometry (MS) are increasingly important because applying different methods to establish in vivo outcomes is critical.
Particular to viral vectors is the issue of full versus partial versus empty capsids. Production platforms that maximize full at the expense of partial and empty capsids are needed, as are methods for efficiently and effectively separating the desired full capsids from the undesirables.
Fortunately, because the application of analytics is really dictated by the product itself, there is a great deal of flexibility from a regulatory perspective. The key is to go back to the fundamentals of controlling quality for the purpose of known safety and efficacy, and safety is, ultimately, the priority for these products.
Analytics of Process Changes
Analytics are essential for understanding the impact of process changes on product quality attributes and safety and efficacy. Analytical tools (notably including potency assays) selected at the front end are important to enable full understanding of process changes and to correlate them with the outcomes of those assays for a limited number of batches.
With the necessity of making decisions on the basis of analysis of only a handful of samples, it is necessary to gather as much detail and establish as many correlations as possible to better control the process and outcomes. Platform methods are ideal because they enable the amalgamation of data from different batches and apply that amassed understanding in addition to the data from the actual runs under evaluation.
Evolution of Understanding
Gene therapy is not only an emerging field, but a rapidly evolving one, including from the point of view of analytics. There are some standard methods, but many methods are still evolving, and some are just being developed. In some cases, the current gold standard is not practical or appropriate, so alternatives are highly sought after, but analytical method development typically requires large sample sizes and is both time-consuming and costly. With viral vectors based on adeno-associated virus (AAV), for which more than a dozen serotypes are currently being leveraged in developmental programs, there is also the need to address serotype-specific issues.
The goal is to identify faster platform methods with sufficient resolution and sensitivity. LC- and capillary electrophoresis (CE)-based MS methods are emerging that require much smaller sample quantities and that offer much higher throughput. Work has also been conducted using ion exchange (IEX) chromatography for these purposes.
Improvements in capsid separation using these new techniques, however, are creating new questions that must be answered. Both IEX and CE-based methods reveal multiple peaks believed to represent partial capsids that have not previously been resolved. These results reflect the evolution of what is possible to monitor and what will become expected over time as understanding increases.
Wide Range of Expertise Required
While much of the initial analytical work on gene therapies was developed from a biology-focused perspective, analytical and protein chemists now have significant influence on current development and clinical thinking. In fact, the interface between this broad array of experts is critical, given the complexity of gene therapies and our current limited understanding.
For instance, while fluorescence and antibody detection are common tools for biologists, there are inherent biases in those methods for quantitation and consistency, because the xenogeneic reagent must be remade periodically. In contrast, with a chemical approach, such as MS, the right controls and approaches remain consistent for the lifetime of the product. Researchers are beginning to appreciate the advantages and disadvantages of these different approaches and how they can be used together to establish the optimum analytical package and understanding to drive gene therapy candidates forward.
As importantly, gains on the research side are translated into clinical understanding that then feeds back into the research end, enabling the design of more efficacious and practical platform solutions. Indeed, given the complexity of these products and the need to understand clinical parameters, it is essential to have more people at the table that can contribute different perspectives to the development of effective analytical solutions and strategies.
Material Limitations and Orthogonal Analyses
Adapting techniques used for monoclonal antibodies (mAbs) poses challenges for viral vectors, because access to large sample volumes is not an issue with most mAbs. With viral vectors, there is limited material available, which limits the ability to conduct orthogonal analyses for every characteristic or molecular feature.
The right approach for a given viral vector is often determined by the product yield. Analytical ultracentrifugation (AUC) could be appropriate for a high-yield vector, but not for a vector with a poor yield that is early in the development process. The key is to consider the current point in the project and what will be needed in the future to select the most appropriate method(s) at any given development stage.
In general, there is some consensus forming around the numbers of samples and key assay types that go with them. A full/empty analysis must be included, and one that is based on AUC and orthogonal LC methods will be highly advantageous. Where AUC is not possible, size-exclusion chromatography (SEC)–multiple-angle light scatterings (MALS) is often applied. Aggregation is often evaluated today using differential scanning fluorimetry, due to its high throughout and sensitivity.
On the molecular characterization side, peptide maps generated using MS have become typical for viral vector identification. For AAV vectors, there are three viral proteins, and there may also be variants of each (e.g., sliced, truncated) that must be identified, and MS is the preferred method. If product-related variants are present, they can initially be detected using a simple SDS-PAGE gel electrophoresis analysis and quantified using reverse-phase chromatography. Subsequently, intact MS is leveraged to orthogonally confirm those results.
Moving Regulatory Expectations
While regulatory requirements for viral vectors are probably less rigorous than those for mAbs, the level of rigor is increasing rapidly. Regulators’ expectations are moving as analytical capabilities improve and as they better understand the need for more effective analytical monitoring and characterization to ensure product consistency.
For instance, analysis of posttranslational modifications (PTMs) on viral proteins was not required even a few years ago, but regulatory agencies require this information in later application packages and are increasingly expecting it in the initial data package. They do not occur as frequently as for mAbs, but end-terminal modifications (e.g., phosphorylation, oxidation, deamidation, glycosylation) and their quantification are important.
In addition, there is an evolution away from a biology-centric mentality about gene therapy development to one with an emphasis on clinical outcomes, which requires much greater detail with respect to product characterization. Regulators want to understand what clinical outcomes are being driven by different aspects of the manufacturing process and are consequently steadily raising expectations. They continue to be driven by the goal of ensuring product quality and safety by mandating manufacturing consistency.
Rapid Evolution of Gene Editing
Viral vectors are not the only vehicles for delivering gene therapies. Much work is being done to develop non-viral approaches, including gene editing using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) proteins.
As with viral vector solutions, there has been significant evolution in CRISPR-Cas approaches and the associated analytics. Protein-based work has led to the development of many different proprietary Cas enzymes, and, similar to AAV, the proprietary nature of Cas is a dependency for most companies. Differences in the Cas protein at the molecular level influence product safety and efficacy and thus must be thoroughly characterized. Critical material and product analyses must be performed and correlated to outcomes for both ex vivo and in vivo product application.
Mass spectrometry is once again becoming a critical analytical tool. In some cases, a single base change could lead to a single amino acid change indicating a successful gene edit. MS has the sensitivity and resolution needed to analyze that level of change.
Meaningful Tech Advances
With the high level of complexity associated with gene therapy, the ability to integrate disparate pieces of information and understand the many aspects involved is crucial. Even seemingly small advances, such as an LC separation method that provides desired information on full versus empty capsids while also affording insights into the presence of other product-related impurities can be really powerful for understanding the potential clinical impact. Being able to apply these newer analytics on the clinical front to identify these correlations will drive the field forward.
In addition, these small advances and the growing diversity of analytical tools are making it possible to generate the volume of data really needed to help the field of gene therapy evolve and advance to the next stage. The bioinformatics approach will allow us to be much more systematic and predictive, as has occurred with mAbs. The sector is just at the beginning, but it is on a similar trajectory.
Support from an External Internal Analytical Development Group
Many of the companies advancing novel gene therapies are small companies operating with little information, internal infrastructure, or resources. They often have experts in biology, generally with mAb experience, but lack analytical knowledge and capacity specific to viral vectors. They consequently turn to external service providers for assistance.
Because AAV and other viral vector–based gene therapies are not standardized products, the typical CRO model isn’t sufficient; a different level of effort is required. Established polymerase chain reaction (PCR) analyses aren’t sufficient. Small biotechs need a trusted partner that can work closely with their internal teams to rapidly solve problems and accelerate product development.
BioAnalytix (now ProtaGene) takes a thoughtful, interactive, and iterative approach and acts as an internal analytical development group. We interact with our customers as if we were part of their team and treat each project as a collaborative exercise. We begin with a philosophical discussion, spending significant time upfront building trust in our capabilities before customers ever commit to a project. We then provide more specifics, homing in on what analytics are needed.
It is a methodic and precise practice followed by implementation. These interactions with the whole customer team continue as the gene therapy candidate progresses through development and include not only analytics issues, such as method development, but additional advice on the full scope of work and information needed to keep the project moving forward.
For many customers, their first project is confirmation that the trust we build is indeed deserved, which then leads to more projects. In fact, every client that BioAnalytix has ever worked with has continued to work with us, including individuals that move from one company to another, because that type of trust and effective, collaborative relationships are so important in this field.
The strategy at BioAnalytix is to offer an integrated protein and gene therapy collaborative testing service. With the recent merger of Protagen Protein Services with GeneWerk, we now offer the most advanced integrated set of analytical services capabilities in biologic development, especially in gene and cell therapy–related testing, from early development through drug approval and onto commercialization.
Integrating the analytic platforms and expertise of BioAnalytix, Protagen Protein Services, and GeneWerk makes it possible to design, generate, and deliver integrated, best-in-class analytical data packages and provide strategic support along the full development spectrum from developability, lead optimization, clone selection, and process development to vector safety, gene editing targets, sequence analysis, and gene expression.
Plans are also underway to significantly expand BioAnalytix’s laboratory capabilities to include GeneWerk’s technology platforms and analytical solutions, including the determination of fusion sequences adjacent to known DNA or RNA fragments in minimal tissue samples down to the single-cell level.