Protein higher order structure (HOS) is an important product quality attribute that governs the structure-function characteristics, safety, and efficacy of therapeutic proteins. Biophysics analyses help establish process and product knowledge, understand the impact of upstream and downstream process conditions, monitor product stability, and assess product comparability when process improvements are implemented. In this presentation, we are going to highlight contemporary biophysical methods for more efficient, higher resolution analysis of biopharmaceutical HOS with automated CD, DSC, microfluidic modulation IR, and NMR for protein characterization and comparability/biosimilarity studies for biopharmaceutical process and product development.

Here, we present an intact mass spectrometry (MS)-based assay that combines affinity capture with the SampleStream (SS) platform and FAIMS. The captured samples are directly loaded into the SS platform where each sample is injected within 30 seconds. FAIMS further offers improvements in signal-to-noise by separating ions prior to MS analysis. The established methods enable the high-throughput measurement of drug concentration and biotransformation for bispecific antibodies during preclinical studies.

Structural biology’s utility in drug discovery lies in its ability to rationalize targeting approaches for both large and small molecules projects, facilitate project execution, and to make these projects both more time- and cost-effective. Cryo-EM has revolutionized structural biology providing atomic resolution data to elucidate MOA, to map epitope/paratope, to modulate affinity, etc., in just a day’s time. A couple of examples on multi-specific drugs and ADCs will be discussed.

Antibody epitope characterization is an important component of therapeutic drug discovery as the binding site directly affects the biological activity. Chemical footprinting with mass spectrometry using carbenes generated from irradiation of diazirine-containing reagents was used to characterize antibody binding sites at the residue level. The structural resolution obtained allows panels of epitope-binned antibodies to be mapped and enables mechanistic understanding of function to support antibody therapeutic lead selection.

Gene therapy drugs using adeno-associated viruses (AAV) have been shown to be safe and effective in multiple clinical trials and two commercial products approved in the US. Understanding the physicochemical properties of these gene therapies is critical to patient safety. Here we used a capillary-electrophoresis Western system (CE-Western) to develop assays for characterizing process-related impurities and product attributes.

Fully-automated high-throughput analytical reversed-phase liquid chromatographic-mass spectrometry is essential to assess large panels of monoclonal and bispecific antibodies at research-stage. Here we present a modality- and target-agnostic MS method that affords the analysis of a 96-well plate in 41.4 mins., as compared to the traditional rpLC-MS method that would typically take 14.4 hrs. Moreover, we show examples utilizing HT LC-MS  to assess Fab glycosylation in discovery panels.

Identification of potential CQAs (pCQAs) that impact mAb target binding is important during the development of therapeutic mAbs. Here, we developed a novel competitive binding-MS strategy that enables high-throughput and multiplexed assessment of pCQAs directly from unfractionated and unstressed mAb drug samples. Specifically, by leveraging the differences in target binding capability under competitive binding conditions, the criticality of multiple mAb attributes can be simultaneously evaluated by quantitative mass spectrometry analysis.

The A_2A receptor (A_2AR), one of four adenosine G protein-coupled receptor subfamily members, shows exceptional expression and membrane trafficking. We created chimeric A₁ and A₃ receptors with A_2AR C-termini that show improved localization to the plasma membrane and bind to their selective ligands as well as native G proteins. In this talk, we will discuss the binding studies of purified receptors and cognate G_sa that elucidate key protein-protein interactions.

• Automated CD, DSC, microfluidic modulation IR, and NMR for protein characterization
• Best practices for analyzing and interpreting routine biophysical assays (DLS, DSC, SEC-MALS, and IR)
• What NMR methods are currently employed for product characterization and comparability/similarity assessments?
• What analysis software is used for analysis?
  
• How extensively are NMR data used in regulatory filings for biopharmaceuticals?
  

• Fast or thorough, can’t it be both?
• High throughput mass spectrometry-based analytics in biopharma
• Current challenges in instrumentation and analysis software for fully automated mass spectrometry applications in biopharma
• Role of native mass spectrometry in characterization of higher order structures and membrane proteins
• Applying mass spectrometry for high throughput epitope mapping, are we there yet?
  

During development of mAbs, it is essential to establish comparability of key properties, including higher-order structure, following manufacturing process changes. NMR spectroscopy is a non-destructive and data-rich analytical method that can report on these properties. Recent advances in NMR allow reproducible fingerprint spectra of mAbs to be collected. We show phase-appropriate applications of NMR in characterizing mAb comparability and process consistency, using unbiased statistical approaches to work without residue-specific assignments.

Intrinsically disordered proteins aggregation into fibrils is a common pathological feature of neurodegenerative diseases including Alzheimer’s Disease and Parkinson’s Disease. Recombinant preformed fibrils (PFFs) are a widely used tool to mimic disease initiation and progression. However, how monomeric Tau or a-syn is correlated with the corresponding PFF polymorphism and in vitro seeding potency, and how to precisely guide the formation of specific fibril conformers in vitro is currently unknown. Here, we are aiming to establish robust methods to screen PFFs’ seeding potency and understand critical properties that govern PFFs’ seeding potency in biological model systems.

Analytical ultracentrifugation (AUC) is an important technique that is routinely used for aggregate characterization and quantitation. This presentation will review key considerations for design and execution of a successful AUC experiment as well as discuss potential challenges and pitfalls. Emphasis is placed on the instrumentation and analysis methods used in the pharmaceutical industry for assessing aggregates in monoclonal antibody formulations.

Biopharma and food proteins are often exposed to high-pressure (HP) and low-temperature conditions during processing and storage. Sub-freezing temperatures, found during lyophilization or freezing, create aggressive environments that pose technical challenges for in situ measurements, both in the presence and absence of ice. Pressure can be used to force degradation in accelerated conditions, as well as to deconvolute cold denaturation from freeze denaturation effects on protein structure. HP-assisted small-angle neutron scattering (SANS) studies of induced unfolding and aggregation of IgG1 monoclonal antibodies will be presented. The advantages of SANS for freeze-thaw studies will be highlighted.