Abstract
Successful development of protein therapeutics depends critically on achieving stability under a range of conditions. A deeper understanding of the drivers of instability across different stress conditions, will enable the engineering of more robust protein scaffolds. We compared the impacts of low pH and high temperature stresses on the structure of a humanized antibody fragment (Fab) A33, using atomistic molecular dynamics simulations, using a recent 2.5Å crystal structure. This revealed that low-pH induced the loss of native contacts in the domain CL. By contrast, thermal stress led to 5-7% loss of native contacts in all four domains, and simultaneous loss of>30% of native contacts in the VL-VH and CL-CH interfaces. This revealed divergent destabilising pathways under the two different stresses. The underlying cause of instability was probed using FoldX and Rosetta mutation analysis, and packing density calculations. These agreed that mutations in the CL domain, and CL-CH1 interface have the greatest potential for stabilisation of Fab A33. Several key salt bridge losses underpinned the conformational change in CL at low pH, whereas at high temperature, salt bridges became more dynamic, thus contributing to an overall destabilization. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR) to solvent, which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, and the VH and CL domains as interesting future targets for engineering stability to both pH- and thermal-stresses simultaneously.
Highlights
In the last 30 years, monoclonal antibody products have become the main drug class for new approvals in the pharmaceutical industry [1]
To determine which domains of Fab A33 are more susceptible to unfolding under low pH and high temperature, we first followed the RMSD of each individually aligned domain (VL, VH, calculated between residues 109–214 (CL) and CH1) along the simulations, as changes in RMSD are indicative of a conformational change
The trend in radius of gyration (Rg) matches that observed previously by small-angle xray scattering (SAXS) experiments, each Rg determined by SAXS was fractionally higher than those from Molecular dynamics (MD) because solution X-ray scattering is affected by the hydration shell around the protein [14]
Summary
In the last 30 years, monoclonal antibody products have become the main drug class for new approvals in the pharmaceutical industry [1]. Over 60 antibody-based drugs are on the market, representing half of the total sales, with over 550 further antibodies in clinical development [2]. They are used as therapeutic drugs to treat human diseases, mainly in oncology, autoimmune diseases and cardiovascular diseases. The use of antibody fragments, such as the antigen-binding antibody fragment (Fab) studied here, brings additional advantages, including deeper tissue penetration due to their smaller size, which has proven beneficial to treat tumors [3]. Fab fragments lack the Fc domain, and are not glycosylated which allows simpler and less costly. The lack of the Fc domain leads to their more rapid clearance in humans than for full antibodies
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