October 2, 2018 PAP-Q3-18-CL-001
During the manufacture of viral vectors, impurities with varying compositions and physicochemical properties can be generated during both upstream and downstream processing steps or derived from raw and starting materials. Impurities originating from cell-culture media can include media components and other additives. Particular attention is required for safety testing of animal-derived raw materials and the cell and viral banks for potential adventitious viral and other microbiological contaminants.
Impurities from downstream processing can include surfactants and emulsifiers, chromatographic resin ligands and chemicals used for inactivating adventitious agents. Single-use biocontainers and other components are a potential source of extractables and leachables. Although manufacturing and purification processes typically utilize closed systems, the potential introduction of biological contaminants must be closely controlled and monitored.
Potential process-related impurities generated during manufacturing include proteins and nucleic acids derived from both the production cells and production viruses used to manufacture the therapeutic vectors. In addition, impurities related to the viral vector product itself, such as unpackaged viral vector genomes (RNA or DNA) and empty or partially filled capsids, can be generated. Process steps to reduce these impurities and methods to monitor their reduction have been developed and implemented. The acceptable limits of these impurities need to be established and included in the list that also includes the critical quality attributes of the viral vector drug product.
Given the broad range of impurities that can be present in viral vector drug substance/drug product, comprehensive analysis requires the use of diverse analytical techniques. Common methods include enzyme-linked immunosorbent assay (ELISA) using specific antibodies, quantitative polymerase chain reaction (qPCR) or digital-droplet PCR (ddPCR) assays with specific targeted amplicons, SDS-PAGE and capillary electrophoresis, HPLC, analytical ultracentrifugation (AUC), FACS and mass spectrometry.
While many techniques used for the characterization of viral vectors are similar to those used for conventional biopharmaceuticals, viral vectors present several unique challenges. One fundamental issue is the incomplete understanding of viruses and how they function, which can complicate certain analytical methods. Unlike conventional biologics, viral vectors comprise both protein and nucleic acid components. As a result, methods are employed to characterize the viral capsid or envelope and the encapsidated vector genome. qPCR and ddPCR can detect specific elements within the viral vector genome. Some targets are universal across different products with similar viral elements in common, while others are unique to specific genetic elements within the viral vector. An effective PCR strategy must distinguish between the therapeutic transgene contained within the viral vector and its cellular counterpart that resides in the production cells or cells used in QC for infectious titering. The concentration of the viral vector (viral titer) in the drug product is determined by qPCR or ddPCR. qPCR relies on a plasmid DNA standard curve to calculate the viral titer. The selection of the PCR target sequence, the design of the primer/probe and the conformation of the DNA template used in the standard curve all influence the robustness of the qPCR assay and can contribute to variability. For certain vector systems, the viral genome titer is used to calculate patient dosing; efforts are underway to reduce the variability associated with the qPCR assay by using ddPCR, which does not require a standard curve and instead allows absolute quantification of the vector genome titer. It is also important that full-length sequencing of the viral vector genome is performed to ensure that no deletions or rearrangements have occurred during manufacturing.
ELISA is used to quantify the number of viral particles and to verify the identity of the vector class and capsid. For example, conformational antibodies to specific AAV capsid serotypes are used to detect intact capsids (as opposed to free capsid proteins or subassemblies), and the p24 ELISA is used to quantify the lentiviral capsid. Both assays can be used to determine the total particle titer. Finally, the unavailability of serotype-specific antibodies makes it a challenge to quantify viral particles and provide serotype identity for chimeric serotypes of adeno-associated viral vectors.
ELISA or qPCR methods are used to measure the viral particle concentration in the product. Cell-based assays are also required to evaluate the infectivity of viral vectors. The infectious assays provide information on how many viral particles in the product are infectious. To establish the infectious assay, it is essential to identify the appropriate cell line that is permissive for transduction by the viral vector. Once the cell line is identified, a qualified QC reagent cell bank is established to support the infectious titer assay. In addition, the potency of the vector must be established; these cell-based assays are product specific: indicator cells are infected with the vector, and the expression of the transgene product is detected using methods that measure the expression/activity of the transgene product (e.g., RT-PCR, ELISA or FACS).
Replication-incompetent viral vectors are designed to be incapable of viral replication in the host. However, it is important to confirm that a replication-competent virus was not inadvertently generated via recombination during the production of the viral vector. Safety tests are designed to detect the presence of replication-competent vector and are performed on the final drug product using carefully selected cell lines. These are cumbersome assays but are critical to establish the safety of each viral vector product lot.
The type of viral vector (e.g., enveloped vs. non-enveloped) must also be taken into consideration. Enveloped viruses pose additional purification and storage issues. It is important to maintain the integrity of the vector during purification, sampling and handling, as it directly impacts its infectivity and the results in the QC laboratory. For example, with adenovirus, it is essential to establish that the fiber proteins are not damaged, because a loss of these proteins results in reduced vector infectivity and product potency. In addition, viral vectors can harbor DNA or RNA genomes. With RNA viruses, such as retroviral and lentiviral vectors, it is necessary to convert the RNA genome into DNA before quantification.
Challenges not unique to viral vectors but demanding deep understanding of both viral vector manufacturing and analytical processes include matrix effects, which can differ for samples taken at each stage of the production process. The different matrices can impact the performance of an assay and must be considered while developing and qualifying assays. For quantification of the residual host cell proteins, the field currently relies on a limited set of commercial kits that are available for residual host cell protein (HCP) analysis, and a need exists to develop more sensitive, customized methods to quantify and identify residual HCPs.
A significant consideration for viral vector analytics is the relatively small lot sizes, which limit the availability of sufficient material for method development, assay qualification/validation and stability testing. There is much less material made during viral vector manufacturing than the manufacturing of conventional biologics, such as monoclonal antibodies.
For projects on accelerated timelines (e.g., Fast Track and Priority Review designations), it is also necessary to factor in the time needed to produce sufficient material for the development of customized assays, to complete the analytical development and qualification work and to generate sufficient quantities of reference standard for each product. A clear understanding of the regulatory requirements and expertise to develop optimized, robust assays is therefore important.
The considerable time required for in-process, release and stability testing that rely on many assays and diverse technologies is a significant issue. Regulatory expectations for greater sensitivity, specificity and accuracy are pushing the limits of existing analytical technologies. There is a need to improve the efficiency and cost of method development, as well as the ongoing monitoring and testing of products and impurities using validated methods.
Additional methods are under development for the analysis of viral vectors. ddPCR for vector product and residual host-cell DNA analyses are two examples wherein reduced variability is observed compared with qPCR. AUC enables separation and analysis of empty and full AAV or adenovirus capsids in one step, with less variability compared with conventional approaches, which require two different methods (e.g., qPCR and ELISA). The adoption of new methods by industry and regulatory authorities, particularly those developed as alternatives to established cell-based assays, can be a slow process.
Automation of sample preparation can accelerate both the development of new methods and the analysis of in-process and product-release samples using current methods. It also has the added benefit of increasing reproducibility. Reducing assay times through the introduction of automation and high-throughput technologies allows for faster lot characterization and release time. Brammer Bio employs automation, including liquid handlers and robotic cell dispensing, to speed up sample preparation for a variety of assays. In addition, Brammer Bio uses novel approaches to rapidly quantify viral particles, such as Virocyt technology.
Brammer Bio works with many clients dealing with shortened development timelines and recognizes the need for greater efficiency in viral vector analytics. We develop innovative, reliable solutions that comply with global regulatory requirements while reducing the time needed to complete important assays in order to accelerate lot release time.
Additionally, Brammer Bio develops our own high-throughput analytical methods. For example, we established a custom high-throughput lentiviral vector infectious titer assay in which infected cell lysates can be directly used in the qPCR reaction, without having to purify the target cell DNA. This assay not only reduces the time required from 72 hours to less than 48 hours but also increases the number of samples that can be analyzed.
Assays for therapeutic viral vector product and impurity analyses must not only meet requirements for sensitivity, specificity and accuracy but also be highly robust. During method development, it is essential to consider the conditions under which the analysis will be performed once the vector enters into commercial production. Ease of implementation on analytical equipment that is modern, with high sensitivity, accuracy and throughput, is essential.
At Brammer Bio, we understand that our clients are moving quickly from early- to later-stage development phases and to commercialization. We develop robust assays that can support clinical development and commercial manufacturing. Rammer Bio’s development and QC teams work closely with the QA department during the development process to establish methods that will provide practical support for commercial GMP production. In addition, our assays are qualified for clinical manufacturing support and require a limited and defined amount of additional work for validation. This approach ensures a high probability of successful validation at later phases and reduces time to commercialization.
To further support the rapid development of effective and robust analytical methods for the characterization of viral vectors, we have established specifications for critical reagents used in the assays, conduct regular vendor audits and have comprehensive analytical procedures in place to evaluate incoming materials. If lot-to-lot variability is a possibility, bridging studies are conducted to confirm that the variability of the materials will not impact in-process, release or stability test performance. The Brammer Bio team takes great pride in our ability to support gene and cell therapy development from clinical trials to commercial supply, and in addition to our manufacturing capacity and expertise, the capability for viral vector analytical testing can ensure that these products are safe, pure and potent.
Dr. Snyder was the founder of Florida Biologix, which was spun out of the University of Florida in 2015 and merged to create Brammer Bio in 2016. Dr. Snyder has been investigating virus biology, vector development, cGMP manufacturing and analytical technologies, and viral vector–mediated gene transfer for over 32 years. Dr. Snyder received his doctoral degree in microbiology from the State University of New York at Stony Brook and obtained his BA in biology from Washington University in St. Louis.
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