This presentation will discuss analytical method development strategies and methods to address critical attributes of rAAV, including quantification of empty and full particles and vector biological activity. Using HPLC, we have achieved separation of full and empty capsids, which allows accurate quantification. Potency matrix assays utilizing engineered cell line for gene therapy product characterization and release will also be discussed.

The development of mass spectrometric (MS) methods for characterization of adeno-associated virus (AAV) capsid proteins allows for the complete structural elucidation of constituent viral capsids in gene therapy development. Conventional peptide map and intact protein analysis, as well as a recently developed ZipChip capillary electrophoresis (CE)-MS method will be discussed in the presentation for various AAV capsid analyses, including serotype identification, confirmation of mutation and characterization of post-translational modifications.

Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. We use AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly AEX assay for the determination of empty and full capsid percentages.

Due to their intrinsic structural complexity and complicated manufacture processes, viral capsids or particles (e.g., adeno associated virus, AAV) are markedly heterogenous than well-established modalities (e.g., monoclonal antibodies, mAbs).  As such, novel analytical and characterization approaches are needed.  In this talk, both new methods and findings (e.g., post-translational modifications, PTM’s) will be presented.  Furthermore, I will also discuss analytical artifacts, which are common yet under-appreciated, thereby often leading to erroneous interpretation and counterproductive approaches. 

Deep IL-15 is Torque’s nanogel formulation of IL-15. When tethered to T cell drug products, Deep IL-15 creates sustained and controlled activation of T cells in the tumor microenvironment and minimizes systemic exposure. As a bioactive nanogel, Deep IL-15 presents interesting analytical challenges. A panel of assays have been developed for Deep IL-15 process control, release and characterization. The analytical development and extended characterization strategies will be discussed.

This presentation will outline phase appropriate CMC analytical control strategies for protein-based vaccines intended for successful regulatory submissions. Analytical approaches for the assay development, assay qualification, and characterizing the critical quality attributes (CQAs) relevant to phase 1/2 programs will be discussed. A case study will be presented to demonstrate the application of the regulatory accepted phase appropriate analytical strategies to support HIV vaccine development.

Methods for characterization and stability assessment of therapeutic monoclonal antibodies (mAbs) are well established. Same applies to protein subunit vaccine candidates. However, when working with conjugated products, this does not hold true. Here we present the challenges, findings, and at the end bordering of our expectation, in evaluation and identification of assays suitable for characterization, formulation and stability assessment of a conjugated vaccine candidate. We will present our experience with the conjugated HIV vaccine candidate.

A Liquid Chromatography-Mass Spectrometry method (LC-MS) was developed, validated, and implemented in Quality Control (QC) for the identity testing of acellular pertussis combination vaccines. A single LC-MS method is able to replace several antibody-based identity tests to enhance quality, increase efficiency, and reduce cost and cycle time.

The clinical development of adeno-associated virus (AAV) for gene therapy is an emerging field. AAV vectors can deliver genes to cells to address genetic defects or to enable cells in the body to produce therapeutic proteins that are intended to impact disease. This talk will describe formulation and drug product presentation considerations and challenges for AAV gene therapy candidates.

Strategies for development of formulations for therapeutic proteins and monoclonal antibodies have become reasonably well established over the last 20+ years. However, the emergence of new treatment modalities begs the question of how much of those strategies are still relevant. This presentation takes a look at the complexities brought on by conjugation of antibodies to small molecules, as well as specific concerns related to bi-specifics and gene therapy candidates from the formulation perspective. New formulation development plans must take into account not only established principles, but also prior knowledge surrounding the biophysical and biochemical differences for novel molecules.

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.

Stream-lining ADC process development activities is key for enabling fast-to-clinic timelines.  Increasingly, we find the need to implement mass spectrometry to characterize our ADCs during pre-clinical development activities.  The challenge is how to adapt the MS peptide map used for characterization to a high-throughput environment where it can be used to drive process development decisions.  We focused on alleviating two methodological pain-points: inefficient, time-consuming data analysis and sample preparation workflows. We leveraged a vendor-neutral mass spectrometry data analysis software suite to perform unsupervised data analysis in conjunction with automated peptide map sample preparation on a liquid handling robotic system.  In this talk, we will focus on how we’ve implemented these workflows and discuss the strategies we have utilized to ensure that we can increase capacity.

Size heterogeneity is a Critical Quality Attribute of monoclonal antibodies and is monitored through the product lifecycle, from assessments of developability, through pre-Clinical, and ultimately commercialization. USP is developing a series of monoclonal antibody standards which can be utilized in analytical assays to support method development, validation, transfer, training, and method performance monitoring. This talk focuses on characterization of size heterogeneity of these standards using various analytical techniques including Size Exclusion HPLC and SDS Capillary Electrophoresis.