The analysis of cell and gene therapies and oligonucleotide modalities requires more than traditional chromatographic or even ligand-binding methods can offer. These highly complex therapies require bioanalytical scientists with expertise in molecular biology and immunogenicity methodologies and the capability to perform cell-based assays and leverage flow cytometry in a good practice (GxP) environment.
More Than a Single Entity
Cell and gene therapies aim to prevent, treat, or cure diseases by correcting the underlying genetic defects: cell therapies aim to restore or alter subpopulations of cells or to use cells to transit a therapy through the body, while gene therapies replace, inactivate, or introduce genes into cells, either inside (in vivo) or outside (ex vivo) the body. The global cell and gene therapy market has constantly grown since the EMA approvals of Glybera® in 2012 and Strimvelis® in 2016 and is projected to grow at a compound annual growth rate above 24% between 2018 and 2024.1
Unlike chemical and biological drug substances, cell and gene therapies are not single, discrete entities. Determining the analytes in therapies comprising living cells or viral vectors delivering genes intended to express certain proteins can be quite challenging.
Cells can be measured and characterized, as can diagnostic biomarkers indicative of protein expression. A whole suite of immune responses must be evaluated, given that product formulations contain excipients in addition to the cells. Gene therapies and oligonucleotides present high levels of complexity and require sophisticated drug-delivery systems. While a standard antibody drug may require two or three assays, an oligonucleotide formulated with excipients and a novel delivery system will likely require 10–15.
With these next-generation therapies, it is important to consider how the product is dosed, the formulation components, and possible downstream effects. Comprehensive characterization requires specialists in flow cytometry, molecular biology, immunology, and other disciplines.
From Chromatography to Flow Cytometry
Just 15 years ago, an average bioanalytical lab largely relied on chromatographic methods. With the advent of mAb therapies, ligand-binding assays for immunogenicity (e.g., ELISA) became widely used. Today, cell-based assays, flow cytometry, and molecular biology–based methods, such as branch-chain DNA analysis, are important.
Flow cytometry has itself evolved to meet changing needs. Initially designed to detect cell types based on surface characteristics, flow cytometry is now combined with the detection of specific intracellular properties (intracellular flow cytometry) to characterize signaling networks at the single-cell level. Gating strategies required to identify the cell populations are developed by the bioanalytical scientist and must be implemented in a very manual process. Flow cytometry thus involves art as much as science and requires deep knowledge and understanding of the technique and the products under evaluation. One of the key workflows is centralizing the data review, and processing to a single team for a global trial can ensure consistency in the data. The European Bioanalysis Forum document on best practices for flow cytometry in a regulated environment2 provides invaluable guidance on traceability and comparability of data.
Skill and Capacity Shortages
The rapid advancement of bioanalytical needs for these new modalities has created both skill and capacity shortages. Bioanalytical scientists who were chemists and chromatographers are now learning molecular and cell biology. They must be flexible and adaptable, have a much broader skill set, and be more creative and dynamic as techniques evolve.
Bioanalytical scientists must do more than execute methods; they must keep the broader context, the end point the therapy should achieve, and its mechanism of action in focus as assays are developed to generate the appropriate data.
There is also a need for bioanalytical labs that can perform flow cytometry to the quality levels required to support regulatory filings. While hundreds of labs can perform chromatography and ligand-binding assays, far fewer have the required expertise in molecular biology or perform flow cytometry at the standards required for regulatory approval.
Innovation and Creativity are Required
Complicating the situation is the lack of specific regulatory guidance on cell-based assays and the use of flow cytometry for drug development applications. Guidance documents exist for chromatography and ligand-binding assays, but only a few white papers have been published on bioanalysis for cell and gene therapies. Regulators want to drive approvals of novel treatments, and in the absence of clear guidance, they will accept new methods provided that evidence shows that the treatments are robust and appropriate.
Both pharma companies and CROs must be innovative and develop techniques that will enable them to provide the required data and find solutions to new challenges — such as the cross-validation of a flow cytometry method in two laboratories — as they arise. Bioanalytical scientists working with clinicians can effectively solve problems. Because no one in the industry has long-term experience working with these methods, it is vital that the bioanalytical scientists that have made progress share their insights with others for the further advancement of techniques.
At Celerion, we are excited about the combination of flow cytometry with mass spectrometry and our ability to perform mass spectral analyses at the cellular level. When this technique becomes sufficiently sensitive for cell therapies, it will be a watershed moment. Cells will then be measured as standard analytes, and it will be possible to characterize them more clearly and completely as the immunologic responses they generate take effect in the body.
Reagent Quality Must be Addressed
One of the key challenges facing modern bioanalytical scientists is the variability in reagent quality. For both ELISA and cell-based assay reagents, a lot of time and effort is wasted on ensuring that lot-to-lot variability does not impact the results generated over time to support a program.
Patient Centricity Driving Future Advances
Two key trends in bioanalysis for the pharmaceutical industry — dry blood spots and microsampling — relate to the growing need for patient-centric solutions and the drive to obtain blood samples with limited pain, stress, and inconvenience. The ultimate goal is to enable secure pharmacokinetic analyses outside the clinic; patients could input their own drop of blood into a microfluidic testing device that would upload the results to the cloud.
The COVID-19 pandemic is accelerating the development of this type of solution, as it is the development of diagnostics, therapeutics, and vaccines for SARS-CoV-2. When we emerge from the pandemic, we will have the opportunity to apply what has been learned to eliminate inefficiencies in both bioanalysis and drug development.
At the Center of Drug Development
Although it is pharmaceutical companies that are creating novel treatments for patients, CROs play a crucial role in the development of those medicines and must consistently prioritize that mission. Everyone that works at a CRO should know what they are really working on; it isn’t a method, but a drug intended to improve or save lives.
Bioanalytical scientists are involved in screening drugs during the discovery phase to preclinical analysis, process development, clinical research, product release, and the development of methods for market surveillance programs following commercialization. Helping to bring novel modalities like cell and gene therapies to patients is a privilege.
References
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The global cell and gene therapy market is growing at a CAGR of over 24% during the forecast period 2018-2024. ReportLinker. 18 Nov. 2019. Web.
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van der Strate, Barry et al. “Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.” Bioanalysis, 9: 1253–1264 (2017).
