Analytical Characterisation of Biotherapeutics

Structure-function analysis of biotherapeutics is pivotal for evaluating key product quality attributes, but it requires extensive characterisation of enriched fractions by various methods. Online affinity chromatography combined with mass spectrometry (AC-MS) offers a quick and efficient way to simultaneously analyze binding and structural characteristics of mAb variants. We introduce novel AC-MS workflows with miniaturised target protein affinity columns connected to 1D or 2D-LC-MS setups, enabling early structure-function characterisation of mAbs and multispecifics.

This presentation introduces an advanced mass spectrometry workflow for host cell protein characterisation in biotherapeutics. The strategy delivers deep coverage, reliable quantification across a wide dynamic range, and clear regulatory alignment. By uniting scientific detail with compliance needs, it demonstrates how mass spectrometry can move beyond discovery into a practical platform for product development, comparability studies, and quality control.

Epitope determination is a critical step in structure-based drug development. For the past decade, X-ray crystallography, Hydrogen-Deuterium Exchange and Alanine scanning have proven invaluable for studying protein-ligand interactions. Recent advancements in Cryo-Electron Microscopy have revolutionized structural biology. We conducted a comprehensive comparison of three cutting-edge techniques: Ala-scan, HDX, and Cryo-EM single particle analysis. Our findings highlight the strengths and limitations of each approach.

The pharmacokinetics of therapeutic IgGs are significantly influenced by their interaction with the neonatal Fc receptor (FcRn), which facilitates extended serum half-life through pH-dependent binding. This interaction involves both affinity and avidity, with IgGs engaging FcRn at acidic pH and releasing at neutral pH. Advanced techniques like switchSENSE, Interaction Map and FcRn affinity chromatography provide insights into these interactions, aiding in the engineering of IgGs with optimised pharmacokinetic profiles and therapeutic efficacy.

Platform analytical approaches enable fast product development during early clinical stages while maintaining comprehensive analytical quality control testing. Adapting these methods for multispecific mAbs and antibody-related formats comes with several challenges, such as altered separation behavior, additional quality attributes and low concentration formulations for highly active products. Incorporating increased flexibility and new technologies in analytical methods will allow platform approaches to be used for advanced antibody formats.

ELN27 is a bi-specific soloMER molecule that targets two key pro-inflammatory cytokines implicated in the pathogenesis of inflammatory bowel disease (IBD). ELN27 demonstrates high-affinity, picomolar binding to both targets, and exhibits superior potency compared to currently approved and investigational mono-specific biologics across multiple experimental models of inflammation. These results support the continued development of ELN27 as a biomarker-guided, next-generation therapeutic candidate for the treatment of IBD.

Characterization of antibodies directed against proteins with multiple membrane-spanning domains or tumor-specific post-translational modifications is challenging due to the absence of suitable recombinant proteins. We used Virus-Like Particles (VLPs) and Membrane Vesicles (MVs) derived from cells expressing such challenging targets to determine antibody binding kinetics and epitope binning using biolayer interferometry (BLI).

In the development of AAV-based gene therapies, it is important to obtain a drug product with high purity and minimal levels of impurities. In this talk, we present two CE-Western assays for the analytical assessment of AAV, focusing on quantifying the relative stoichiometry of viral proteins (VP) and measuring residual baculovirus protein impurities, specifically GP64. The results were further supported by LC-MS analyses.

Flow induced dispersion analysis (FIDA), is an established method to determine diffusion coefficients of molecules with minuscule compound consumption. It is highly applicable to support various aspects of pharmaceutical development of peptides. As examples, the sensitivity allows us to study oligomer dissociation simulating dilution upon subcutaneous injection as an alternative to CG-MALS, and the versatility allows us to achieve binding coefficients to serum components such as albumin. Finally, we are also applying the technology to rapidly screen biophysical properties for SAR and formulation development.

This presentation will cover analytical strategy for a bispecific antibody structure and non-standard CQA monitoring. A method development for a reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry and application case study will be presented where mispairing species were characterized and quantified in both upstream and downstream bispecific mAb process development. Additionally, method robustness and qualification data will be discussed.