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Immunoassays are an essential bioanalysis tool used throughout cell and gene therapy development and manufacturing to evaluate viral vector titer and immunogenicity responses to gene therapy treatment. The two most commonly used vehicles for introducing genetic therapies into mammalian cells are adeno-associated virus (AAV) and lentivirus vectors, consequently AAV titer, lentiviral titer, and gene therapy immunogenicity ELISA assays are commonly developed for this purpose. However, lengthy assay development and sample analysis time using traditional ELISA methods often causes delays in program schedules.
Utilization of robust and innovative immunoassay platforms for bioanalysis in gene therapy development remains a critical need to generate high quality data and support efficient method development including the use of tools such as design of experiments (DOE), data driven decision-making, and release tests for both CMC and final product manufacturing.
Gyrolab® automated microfluidic, nanoliter-scale immunoassay solutions dramatically shorten time to results to within one hour, use reduced sample volumes, and improve data quality for AAV- and lentivirus-based gene therapy immunoassays during development, and are ideally suited for compressed cell and gene therapy development timelines.
PRECLINICAL AND CLINICAL STUDIES
The rapid increase in gene therapies in development, from preclinical studies through bioprocess development and manufacturing through clinical studies, has accelerated the search for efficient gene therapy bioanalytical methods that deliver reliable results to instill confidence in data driven decision making and meet regulatory needs. Reductions in analysis time can ensure programs meet tight development timelines.
We will look at how immunoassays in gene therapy bioanalytical testing are optimized, including:
Oxford Biomedica has established itself on the gene therapy stage, being the first to administer lentiviral vectors in vivo and as the first lentiviral vector containing product to gain approval for advanced therapy in the US using a unique LentiVector® enabled technology (Novartis Kymriah®). Over 500 patients have been treated with gene-based therapeutics by Oxford Biomedica or its partners, and a number of new therapies are in development. The team at Oxford Biomedica saw that by using the Gyrolab system they would be able to run an assay 24X faster and with 10X less sample compared to using ELISA. See blog post, "Putting a New Spin on Immunoassays for Gene Therapy - Part 1"
Cell and gene therapy development demands for speed, precision, reproducibility, and GxP compliance in bioanalytical methods are met with Gyrolab microfluidic, CD-based assay technology.
Centrifugal force and a flow-through affinity microcolumn format eliminates lengthy incubations and minimizes matrix interference. The nanoliter-scale format saves reagents and samples, resulting in a cost effective and highly reproducible immunoassay that produces quality data over broad dynamic ranges. Gyrolab BioaffyTM CDs combined with Gyrolab automated systems dramatically reduce hands-on time and shorten processing times, leading to considerable gains in productivity.
Advancing new cell and gene therapies through preclinical and clinical studies requires specialized expertise in regulatory requirements, study design and execution, and scale-up vector manufacturing technologies. Many, if not all, pharma and biotech companies secure outsourcing CRO and CMO partners for development through commercialization of gene therapies. CRO/CMO partners face additional timeline and quality demands for immunoassay workflows, since they are conducting GLP- and GMP-compliant studies, often with fixed timelines specified by commercial sponsors.
Gyrolab immunoassays meet stringent criteria for regulatory-compliant CRO or CMO bioanalysis:
The measurement of anti-vector or transgene antibodies using a robust and reproducible immunoassay is an integral bioanalytical component in preclinical gene therapy studies to understand the potential for immunogenic reactions in human clinical use. Measurement of pre-existing anti-drug antibodies (ADA) may also be used to pre-screen subjects in clinical studies to avoid neutralizing antibodies potentially affecting efficacy. Cytokine biomarker immunoassays may also be used as an indicator of an inflammatory innate immune response to indicate potential for a “cytokine storm” response. Gyrolab ADA software guides the development of automated immunoassays for ADA analysis.
Gyrolab preclinical and clinical study immunoassays:
Optimizing conditions for viral vector production is a slow and laborious process and no real-time, in-line vector titer assay is available. Quantifying functional viral titer for lentiviral or AAV vector production is time-consuming and costly — the common approach (TCID50 assay) can take up to 6 weeks for an infectivity result, by which time the bioreactor run has been completed. A physical lentiviral titer or AAV titer is sufficient for most applications, and a functional titer can be calculated from a physical titer. As a result, immunoassays are routinely used to determine dose strength, for example by p24 quantitation as a measure of the physical titer of lentiviral-based therapies in process development and manufacturing release testing.
An important objective of the manufacturing and purification of gene therapies is to minimize co-purifying impurities of host cell proteins (HCPs) that are potentially toxic or immunogenic. Immunoassays based on polyclonal antibodies are routinely used to monitor the level of impurities in-process samples and for batch release tests. HCP measurement is a critical regulatory requirement and having an open platform that has the flexibility to measure HCPs from new model systems is important. Gyrolab immunoassays analyzing impurity proteins from HEK 293, a commonly used host cell line for gene therapy vector production, can be developed using commercially available reagents.
Viral vector titer and characterization
Host Cell Protein (HCP) impurity analysis
Cell culture and other process-related impurities
Gyrolab p24 Kits, Gyrolab AAVX Kits, and Gyrolab AAV9 Kits provide plug and play, miniaturized lentiviral p24 titer, and AAV titer immunoassays for use on all Gyrolab platforms. Kits include everything needed to generate 96 or 4800 data points, including capture reagent, detection reagent, standard curve samples, wash buffer, sample dilution buffer, and Gyrolab Bioaffy CD(s). Used together with Gyrolab systems, 96 data points are delivered in 80 minutes to accelerate lentiviral vector bioanalysis during production and bioprocessing.
Gyrolab systems meet all these criteria for advanced gene therapy bioanalytical assay performance and speed needed to succeed in cell and gene therapy development. Gyrolab systems are well established in pharma and both preclinical and clinical CROs and support a wide range of applications including immunogenicity, IgG titer, HCP analysis, and viral vector titer.
This white paper provides an overview of the viral vector landscape and its use in cell and gene therapy. The requirements for immunoassays in viral vector bioprocess analytics are discussed in detail and Gyrolab case studies from viral vector analytical labs are presented.
In this webinar, Andrea Monterola, Ph.D. from Viralgen Vector Core, and James Norton from Meira GTx, present how Gyrolab was used in viral vector downstream process development to determine AAV capsid titers and process-related impurities (HEK 293 HCP and endonuclease).
In this webinar, Tomasz Witkos, Ph.D., Scientist at AstraZeneca talked about how miniaturized immunoassays can be used for measuring purified AAV as well as in-process samples and how the immunoassay experimental setup can be modified for testing many different AAV serotypes.
Contact us to discuss your gene therapy immunoassay needs: