Development and production of innovative biotherapeutics demand bioprocesses that consistently yield a high-quality product. However, current methods to determine product quality do not necessarily capture the actual mix of product and related impurities in cell culture supernatant, but rather what can be captured after purification. We developed a highly-sensitive method that can be applied to the detailed characterization of cell culture supernatants from bioreactors without a falsifying pre-purification step.

Membrane proteins and associated complexes currently comprise the majority of therapeutic targets and remain among the most challenging classes of proteins for analytical characterization. Through long-term strategic collaborations forged between industrial and academic research groups, there has been tremendous progress in advancing membrane protein mass spectrometry (MS) analytical methods and their concomitant application to Amgen therapeutic project progression. Herein, I will describe a detailed and personal account of how electrospray ionization (ESI) native mass spectrometry (nMS), ion mobility-MS (IM-MS), reversed-phase liquid chromatographic mass spectrometry (RPLC-MS), high-throughput solid phase extraction mass spectrometry, and matrix-assisted laser desorption ionization mass spectrometry methods were developed, optimized, and validated within Amgen Research.

Our study delves into characterization of product quality attributes associated with the Bispecific Antigen-Binding Biotherapeutic (BABB) molecule. We employed microfluidic-based capillary zone electrophoresis in conjunction with mass spectrometry (MS), to identify fragmentation/clipping sites within BABB therapeutics. We comprehensively investigated both predominant and low-abundance post-translation modifications via native MS. Lastly, we demonstrated the implementation of covalent labeling and MS, to characterize BABB’s subtle conformational changes under native vs. thermally stressed conditions.

A state-of-the-art, integrated, multi-instrument automated system was designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid- and microplate-handling robotics and utilities, integrated LC-MS, along with data analysis software to perform sample purification, preparation, data acquisition, and data analysis as a seamless integrated unit. The results are verified and formatted for expert curation directly in the cloud.

MS-based MAM for analytical testing of mAbs has the potential to improve efficiency and provide more detailed information on PQAs as compared to conventional methods. This presentation will overview USP reference standards and tools to support mAb characterization and analytical control strategies. Case studies, including multidimensional assessments of monitoring changes in PQAs upon forced degradation and the correlation between MAM and conventional methods, will also be discussed.

Forced degradation studies are an integral part of protein therapeutics research and development. Identification of the protein degradation pathways and the characterization of the higher-order structure (HOS) is critical in estimating the biological function, efficacy, and safety of biotherapeutics. A powerful tool for precise and high throughput characterization of the biotherapeutics is therefore of high value in the industry. This talk will delve into recent advances in characterization tools for assessing HOS with a focus on case studies involving force degraded antibodies.

rAAVs have grown from a proof-of-concept approach into a highly promising DNA delivery vector for the treatment of monogenic disorders during the past decades. Yet, the use of these vectors is not without hurdles when it comes to meet a biopharmaceutical`s high quality standard. The current demand for cell & gene therapies is anticipated to spur additional developments in the sector as it was the case for mAbs 20 years ago. This talk aims to shed some light on the current status of the analytical characterization panel and to show some pain points that need to be addressed in the future.

In vitro characterization of therapeutics binding to their targets is critical at each stage of drug development. With increasing complexity of therapeutics and challenging targets, more diversified technologies and methods are needed. In addition, there is an increasing need to understand the binding interactions that can better reflect what may happen in vivo. This presentation will focus on case studies that demonstrate the application of various molecular interaction characterization tools to enable drug development.

Surface plasmon resonance (SPR) is a well-established method to characterize biomolecular interactions and an important biotherapeutic characterization tool. Recently, a 10-fold difference in binding affinities was unexpectedly discovered when an antibody:antigen interaction was characterized using two different anti-human Fc (AHC) antibody surfaces. Multiple mAb:antigen interactions characterized using diverse human IgG capture surfaces revealed that use of inappropriate capture surface has a profound effect on the accuracy of ka, kd and KD values. This talk will provide insight on the source of this discrepancy and also present attributes of a good AHC antibody to perform capture-kinetic analyses on any label-free biosensor.

I will present the latest advances in end-to-end automation, digitalization and data management of the protein analytics workflows in the pharma research and early development (pRED) unit of Roche, focusing on mass spectrometry analysis of complex antibody-based drug candidates. It is routinely applied to a variety of sample types and throughput, from early binder screening to clone selection and bioprocess development.

Analytical Quality-by-Design offers a systematic and robust approach to the development of analytical procedures involving all stages of the product’s lifecycle. The presentation will overview FDA guidance and newer ICH guidelines on analytical control strategy including method development, validation, and lifecycle management. Special emphasis will be placed on the analytical procedures commonly used in in-process control, release and stability for biotherapeutics.

T cell dependent bispecifics (TDBs) have emerged as a promising cancer immunotherapeutic modality, which redirect T cells to eliminate tumor cells by co-engaging CD3 on T cells, and tumor antigen on tumor cells. The effector function characterization strategies for our TDBs will be presented herein, and case studies will be provided to evaluate the potential impact of residual effector function of Fc mutations on the efficacy and safety of TDBs.