Biologics, an advancing therapeutic class for life-altering illnesses, require developable molecules with high expression and quality. Complex molecular formats like bi- and multispecific antibodies present challenges in clinical manufacturing due to modifications and impurities. We employ mass spectrometry and biophysical assays, including SEC-MS and micro-fluidic cIEF-MS, with automation for investigating poorly expressing multispecific antibodies. This integrated approach streamlines processes, providing valuable insights into structural properties and improving precision and throughput.

It has been demonstrated that integrating AI/ML methods with in-house experimental data can accelerate the antibody discovery process. Utilizing these methods, next-generation sequencing (NGS) data combined with biophysical assays can guide strategies for antibody lead selection. Furthermore, recent AI techniques, particularly those inspired by protein language models, can be effectively combined with experimentally annotated data for the antibody optimization process, such as affinity maturation.

I will highlight the methodological nuances and sensitivity involved in calculating structural and molecular descriptors for antibody developability assessment. I will discuss the impact of structure prediction methods, conformational sampling, underlying surface patch calculation methods, and more by presenting benchmarks against experimental developability data, including viscosity, polyspecificity, and aggregation. Additionally, I'll introduce an MD-based framework that leverages the outlined models to predict antibody developability risk flags in a high-throughput automated manner.

Antibodies possess effector functions through binding of the constant region (Fc) to Fcγ receptors (FcγR). We have profiled a panel of commonly used FcγR binding interface mutations and identified a preferred variant and isotype that silences effector functions and retains ProA binding. In addition, we identify an optimal combination of these and FcRn-enhancing mutations to extend half-life.

The determination and monitoring of critical quality attributes play a crucial role in the development of AAV-based gene therapies. For instance, accurately quantifying capsid titers is key to safely and efficaciously dose patients. Traditional capsid titer methods rely on ELISA which commonly suffers from relatively larger assay variability. Thus, requiring development of alternative high-throughput capsid titer methods to support AAV formulation screening and development studies.

Mass Spectrometry (MS) instrumentation has improved in recent years. Both high-resolution and routine MS have been increasingly used to characterize canonical IgGs and complex antibody-based products. Cyclic Ion Mobility (cIM-MS) for conformational characterization of native therapeutic monoclonal, bispecific, and ADCs will be discussed, as well as user-friendly benchtop MS for multiple-attribute monitoring studies and advanced MS workflows for accurate quantification of trace-level host cell proteins in antibody-based products.

Multi-attribute method by mass spectrometry (MAM) is an emerging analytical technology that allows for simultaneous monitoring of multiple quality attributes of therapeutic proteins on the level of individual amino acid residues in a single analysis. We will present the implementation of an automated MAM platform across six Novartis development laboratories and its application to streamline biopharmaceutical analysis and improve product and process development of mAb- and non-mAb-derived therapeutic proteins.

Vaccine-induced thrombotic thrombocytopenia is a dangerous complication that may occur upon the administration of Ad-based vaccines. This condition is associated with the formation of pathogenic monoclonal autoantibodies, which circulate in the patient’s blood and trigger thrombogenesis. In this talk, we will demonstrate how a wide range of MS-based techniques has been utilized for developing and structural/functional characterization of these pathogenic antibodies.

• AAV formulation development common challenges and strategies to support long-term stability. Lessons learned from different AAV serotypes
• Is it possible to develop a platform formulation that can be applied across all different AAV serotypes? Discuss novel and critical excipients, lyo vs liquid formulation
• Stability-indicating and extended characterization methods to characterize AAV drug product critical quality attributes (aggregation, full-empty ratio, genome titer etc)
• High-throughput and low-volume methods to support formulation screening and guide optimal formulation development​

Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) is utilized throughout mAb therapeutic development for aggregate characterization as an orthogonal technique to other size variant methods such as SEC. SV-AUC enjoys advantages such as the ability to analyze samples in their native matrix and broader dynamic range. However, establishing appropriate detection limits for low-abundance species remains a challenge. This session will describe preliminary work to establish detection limits for mAb aggregate characterization.

Site-specific deamidations of asparagine and glutamine residues in monoclonal antibodies can be observed using temperature perturbation and infrared two-dimensional correlation (2D-COS) methods. DOI: 10.1002/9780470027318.a9792 We will describe the rejuvenation of FTIR experiments using similar 2D-COS methods to characterize protein physical and chemical stability.

Numerous molecules are screened for optimal activity and developability in early drug development. Chemical and biophysical properties can affect activity—making them key for data interpretation—and provide a developability profile. Traditional chromatography-based methods often prove resource-intensive for this phase, where low volumes and numerous samples are analyzed. Here, we contrast data from traditional methods with those from more efficient, high-throughput approaches like chip-based capillary electrophoresis and spectroscopy.

Proteins are exposed to hydrostatic pressure (HP) in vivo and in processing conditions, e.g., freeze-thaw or sterilization. HP is a powerful tool to probe and drive protein-protein interactions (PPIs) of interest, and synergistic effects with salt have been previously reported. An empirical approach to scale combined HP and salt effects on PPIs is presented for SAXS data on ovalbumin solutions, applicable to other proteins and techniques in the literature.

Accurate estimation of affinity and kinetics is critical to developing potent therapeutic antibodies and for prediction of pharmacodynamics, yet this has become increasingly difficult given the extremely high potency and high binding-stability that can now be attained. We present optimized SPR-based methodologies that enable reliable characterization of such affinity complexes and report results for the Ranibizumab-VEGF complex.