The past decade has seen a rapid expansion of the types of biotherapeutics being developed, applying a wider range of scientific approaches to address all aspects of human disease. This has led to the development of modalities ranging from mRNA vaccines to the subcellular (viral gene therapy, exosomal products) and the cellular and tissue (cell therapy, microbiome/live biotherapeutics). With this complexity comes an increase in the development challenges to manufacture and characterize these novel therapeutics.

This forum will focus on the application of various analytical technologies to solve modern problems in development, manufacturing, and control. Analytical tools enable the understanding of a therapeutics critical quality attributes and the control of these to ensure a consistent, safe and efficacious product. As the complexity of our biotherapeutics grows, so do the demands on our analytical tools. We will consider the application of novel technologies in development and the re-imagination of established approaches to solve emerging challenges. We will also discuss the challenges of introducing new technologies into development and quality labs and gaining regulatory acceptance for these approaches.

There have been significant interests in advancing analytical paradigms with unprecedented capabilities of real time measurement of critical process parameters (CPP) and critical product quality attributes (CQA) in biopharmaceutical development. The center piece of real time analytics is Process Analytical Technology (PAT). The PAT landscape at BMS Biologics Development features a broad spectrum of technologies, including vibrational spectroscopy, multivariate data analysis, multi-attribute chromatography, sensors, and automated-sampling technologies. The roadmap for strategic implementation of PAT tools entails identification of critical quality attributes, critical control points and critical process parameters with deliberate alignment of PAT technologies to the appropriate unit operations within bioprocess and with the right balance of technology capability and limitations. Our vision of PAT roadmap not only include the integration of analytical technologies into the bioprocess, but also extend to automated data-piping, analysis, aggregation, visualization and advanced process control. In-depth process understanding and advanced process control will enhance our quality by design (QbD) approaches in biopharmaceutical development and ultimately lead to great manufacturing sustainability.

The chimeric antigen receptor (CAR) T cell therapy technology is one of the most exciting and influencing therapeutic modalities that modern genetic engineering offers. At Takeda, we are actively developing the next-generation allogeneic cell therapy products with our induced pluripotent stem cell (iPSC) platform. As multiple cell therapy products are being derived from iPSCs, the need for holistic analysis of cell surface markers from various cell lines is imminent. However, the actual measurement of these marker proteins can be challenging because most marker proteins from T cells and NK cells are plasma membrane proteins with low abundance, poor solubility, and high sample complexity. To tackle these technical challenges and achieve analytical objectives, we developed and implemented a subcellular fractionation-assisted mass spectrometry(MS)-based proteomics workflow to identify and quantify surface marker proteins from various cell therapy products.

Host Cell Proteins (HCPs) are process-related impurities generated by the packaging cell line (HEK293T) during the lentiviral vector (LVV) manufacturing process. Since residual HCPs have the potential to affect product quality, safety, and efficacy; understanding the clearance of HCPs during the various stages of the LVV and drug product purification process is crucial.

Sandwich ELISA is the most commonly used method for monitoring HCPs; however, ELISA can only give information on the total amount of HCPs. Additionally, ELISA’s are also limited by the coverage of antibodies for the most abundant HCP species. Therefore, orthogonal methods such as 2-D fluorescence differential gel electrophoresis (2D DIGE), LC-MS and Capillary Electrophoresis (CE) can provide valuable complementary information to characterize, monitor and control HCP levels across the process.

2-D DIGE and LC-MS are time consuming and require specialization, making them not ideal for high throughput analyses. CE is less laborious, requires minimal training and can be scaled for high-throughput use. This presentation will focus on the use of capillary electrophoresis to track HCP clearance using two HEK293T-specific markers, E1A and SV40, to evaluate the potential impact of HCPs on DP quality through spiking experiments in small-scale transductions.

The supply of critical raw materials used for manufacturing and testing of biological and sterile products has been strained due to the COVID-19 pandemic and the associated development and manufacturing of vaccines and monoclonal antibody therapies. Reliance on single sourced materials has prompted Industry to rapidly develop strategies for implementation and registration of alternate materials and consumables, as well as think about long term strategies based on building increased knowledge and understanding of raw material critical attributes to better mitigate raw material supply constraints in the future. This forum will discuss the impact of raw material supply constraints and strategies aimed at reducing potential impact to supply concerns and/or gaining supply flexibility.

Supply of critical filters and single use components has been strained by the development and manufacturing of COVID-19 vaccines and monoclonal antibody treatments. To mitigate shortages of filters and other single use components, Amgen requested FDA for feedback on strategies to register second source filters to enable continue manufacturing. Overall submission strategy presented to FDA included filing post-change management protocols along with characterization data to support the change with a commitment to provide at-scale data at a later time. FDA feedback included additional considerations for filing these alternate filters.

Chromatography is used for the purification of monoclonal antibodies from harvested cell culture fluid (HCCF). Typically, one column is dedicated for each MAb, which results in resin being used to only 10-20% of its lifespan in pilot plant and clinical production. However, significant savings can be realized each year if resin is used for multiple products. In this study, a cleaning procedure “MabSelect SuRe Campaign Changeover Procedure (MSSCCP)” was developed at lab-scale that reduced protein carryover to below assay detectable limits allowing use of one column for multiple products. The re-use procedure was successfully implemented on pilot plant columns in Oct 2010 used for producing drug substance.