Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Claire Dobson ,Neil A Ranson,Fraser Welsh,David Lowne,Peter Thornton,Joanne Arnold,Jonathan J Phillips,Anna Aagaard,Joanne Goodman,Arthur Lewis,Christopher Lloyd,Anna Ridderstad Wollberg,Lisa Vinall,Paul W A Devine,David C Lowe,Christopher F Van Der Walle,Daniel R Higazi,Andrew Buchanan,Alison E Ashcroft,Theodoros K Karamanos,Clare L Pashley,Tristan J Vaughan,Sheena E Radford,Alistair D Kippen,Lars‐Inge Olsson ,Bojana Popović ,M.g Iadanza

doi:10.1038/srep38644

Abstract

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

Highlights

Therapeutic antibodies must be able to withstand a range of stresses during manufacture
IgG expression levels (200 mg/L) were in the range typical for transiently expressed recombinant human IgGs25, unlike MEDI578, MEDI1912 showed colloidal instability, adsorption to filter membranes, and gelation, resulting in poor yields (
Sedimentation-velocity analytical ultracentrifugation (SV-AUC) revealed that MEDI1912 (1 mg/mL in formulation buffer (Methods)) self-associates to form dimers with small amounts of higher order oligomers (Fig. 1b), while dynamic light scattering (DLS) showed that MEDI1912 formed oligomers under all buffer conditions tested in a concentration-dependent manner (Supplementary Figure 1a)

Summary

Introduction

Therapeutic antibodies must be able to withstand a range of stresses during manufacture. Protein aggregation involving reversible self-association is an increasingly recognised problem affecting the bioprocessing of human therapeutic antibodies that influences both shelf life and efficacy[9,10,11]. This has become more common due to a growing trend towards formulations that allow sub-cutaneous administration routes. A variety of computational approaches[6,15,16] and simple experimental assays have been developed to identify antibody variants with increased aggregation propensity and decreased colloidal stability[17,18,19]. The HDX/ XL-MS and targeted mutagenesis methodology employed represents a powerful approach that could be used as a generic strategy to improve the robust and reproducible manufacture of antibody-based medicines and protein therapeutics

Methods

Results

Conclusion