Elucidation of the Reversible Self-Association Interface of a Diabody-Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling.

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

The Strengths of Total Holographic Video Microscopy in Detecting Sub-Visible Protein Particles in Biopharmaceuticals: A Comparison to Flow Imaging and Resonant Mass Measurement.

Determination of subvisible particle (SVP) content in biopharmaceuticals is a prerequisite to ensure the quality of liquid biopharmaceutical products. Here, we present a comparison of the recently introduced holographic video microscopy (total holographic characterization, THC) with two orthogonal and well-established analytical technologies: micro flow imaging (MFI) and resonant mass measurement (RMM). The capabilities of the THC were investigated under conditions commonly applied in drug product development. Three different antibody products were used at different concentrations and formulations to cover a wide range of realistic use-cases. The comparison was particularly focused on protein aggregates to investigate the applicability of THC to this critical class of particles in drug product development. Protein concentrations up to 100 mg/ml were investigated covering a broad range of viscosity and refractive indices, both important parameters in particle detection. The comparison reveals that THC is highly sensitive to detect protein aggregates in a size range from 0.5 µm to 10 µm. THC shows a significant superiority to FI and RMM in detecting heterogenous protein aggregates which often appear as transparent and porous particles. Additionally, THC needs very small sample amount of about 30 µl and short measurement times, making it applicable for early development stages and high-throughput approaches. These results show that THC is a valuable supplement to the existing particle characterization method portfolio in drug product development.

Trafficking of JC virus-like particles across the blood-brain barrier.

Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood-brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.