PEGylated proteins and many non-ionic polymeric surfactants and excipients, such as polysorbates and poloxamers that are used in formulation of biopharmaceuticals share the same building block which is polyoxyethylene. Characterization of PEGylated proteins and polymer excipients with a combination of liquid chromatography, charge reduced mass spectrometry and software-assisted composition analysis will be presented. This method offers advantages such as retaining intact polymeric structures, visualization, and fast profiling of polymeric species.

Most standard approaches used to prepare antibody conjugates suffer from non-quantitative, indiscriminate labeling. The value of introducing cargo at specific sites has become increasingly apparent. However, current site-specific labeling methods are not amenable to high-throughput screening and pose characterization challenges. I will discuss several bioconjugation techniques that we developed to enable the rapid, highly efficient, and site-specific labeling of full-length antibodies and how we characterize these conjugates.

As protein therapeutics become more complex, our analytical tools need to rise to the challenge. Unravelling these structural complexities by combining multiple chromatographic dimensions holds great promise. We will share some real-life examples of how this innovative approach dramatically improves our analytical understanding of novel conjugates and other biotherapeutics.

Alteration in spike protein glycosylation is a mechanism whereby viruses evolve to remain fit as they circulate in the human population. Glycosylation is inherently heterogeneous, making rigorous comparison of the complete glycosylated structures of viral proteins a statistical problem. In response, we have used high definition MSE (HDMSE) on a cyclic ion mobility MS instrument to produce optimal depth and confidence for calculating similarities of viral spike glycoproteins during evolution. 

Biologics development needs to summarize and explain very complex data - in this workshop we will show how LC-UV-MS experiment outcomes can be represented in shareable, web-based dashboards. Some of the most complex analyses are easily digestible, and become understandable by an entire organization, and reduce the burden on needing expert explanation. Usable for MAM, screening, system suitability, Protein Metrics’ Deep Query tools will be useful to any biotherapeutics organization.

Nanoparticle-based artificial antigen presenting cells (aAPCs) that present peptide-HLA complexes target specific T cell populations for expansion and enrichment. Secondary activating or tolerizing signals dictate the activity that T cells adopt. Adapting aAPCs for diverse patient populations requires identification and characterization of HLA-restricted peptides that are distinctly expressed on target cells. We present modular nanoparticle components, combined with a systematic approach for the identification and evaluation of antigen derived peptides, to target a variety of therapeutic indications.

The characterization of Live Biotherapeutic Products (LBPs) includes the examination of novel attributes outside of classical protein-based biotherapeutic properties. The FDA has set regulatory guidance for the evaluation of LBPs. Identification of novel attributes and the qualification of the release assays are critical to moving products into regulatory compliance. This presentation will discuss the development and qualification of assays used to dose LBPs.

A protein’s structure-function relationship refers to a trade-off between stability and bioactivity, molded by its evolution. Here, characteristics of residues in granulocyte-colony stimulating factor (G-CSF) are probed with 15N-1H HSQC NMR during a denaturing thermal melt. We confirm a previously observed aggregation-prone conformation change in loop AB and uncover a “switch mechanism” significant to bioactivity. Thus, we found residues H43, V48 and S63 to be pivotal to the stability-bioactivity trade-off.

The complexity of multi-specific biotherapeutics requires a comprehensive set of analytical techniques and an appropriate developability strategy to guide lead discovery and optimization. An overview of the integrated developability concept at Sanofi will be presented with a focus on chemical and physical stability of multi-specific drug candidates. The strategic overview will be augmented with case examples highlighting the challenges in characterization and developability of multi-specific molecules.

Multi-specific antibodies are rapidly becoming attractive modalities as they bind to multiple targets simultaneously, reducing side effects and toxicity build-up. Monitoring the impurity/stability/modification of large complexed molecules is vital during drug discovery and development to ensure the potency of the drug and can be done efficiently through capillary electrophoresis (CE) and intact mass analysis by CE-MS. Immunoaffinity purification-CE-MS can effectively monitor protein stability in vivo and identify the cleavage sites.

Triple-targeting formats hold great therapeutic promise but translation of concepts into active molecules is challenging both in obtaining differentiated functional activity, as well as meeting stringent developability criteria. We discuss the discovery and characterization of the components and final candidates of a tri-specific antibody format referred to as Triclonics that permits high-throughput in-format repertoire screening to result in active molecules that harness the developability characteristics of regular human monoclonal antibodies.

The AQS³pro system, built upon Microfluidic Modulation Spectroscopy, is an automated infrared platform optimized to determine biomolecule structure in a complex buffer and at formulation concentration. The AQS³pro combines a 24 or 96-well plate, a quantum cascade laser, built-in automation, and a microfluidic flow cell to produce highly precise, higher-order structure measurements for determining stability, aggregation, lot-to-lot similarity, and formulation optimization of biomolecules.

Bispecific antibodies efficiently trigger T-cells mediated cytotoxicity and many T-cell engaging biopharmaceuticals are in clinical development. Current analytical methods measuring T-cell activation are not optimal. Here we showcase an improved bioassay platform for reliable assessment of T-cell activation using CD3xCD19. Effector T-cells carry a reporter gen downstream the CD3 signalling cascade and a pair of engineered target cells is used as antigen-positive/-negative control

Extracellular vesicles (EV) have emerged as an important therapeutic modality for delivering pharmacological payloads via cell-derived lipid nanoparticles. Despite the considerable opportunities in disease intervention, the complex properties of EV impose unique challenges to analytical and process development, that is distinct from other classes of Advanced Therapy Medicinal Products. Here an overview of the current status and emerging trends in the analytical technologies for structural and functional characterization of EV are discussed.

In this study, we developed and evaluated a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ion-pairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis. Coupling to high-resolution mass spectrometric (HRMS) analysis, the established HILIC-MS method could provide in-depth analytical characterization for oligonucleotide and siRNA standards and their impurities.

Oncorus is a clinical-stage biotechnology company focused on developing next-generation viral immunotherapies for the treatment of cancer. While oncolytic viruses are potent tumor killing agents, their therapeutic benefit has largely been limited to intratumoral administration. Oncorus' pioneering synthetic virus approach involves encapsulating viral RNA genomes in lipid nanoparticles to enable intravenous delivery. This talk will provide an overview of analytical methods used to characterize synthetic viral RNA and LNP products.

Characterizing bispecific antibody binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches have limited usefulness for interpreting complex bispecific binding. To address these deficiencies, we developed and implemented a new dual-target binding assay using AlphaPlex® technology that accurately dissects the affinities of both target binding domains directly and simultaneously.