Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Author:

Knight Michael J.1,Woolley Rachel E.2,Kwok Anthony2,Parsons Stuart2,Jones Hannah B. L.2,Gulácsy Christina E.2,Phaal Polly2,Kassaar Omar2,Dawkins Kieran1,Rodriguez Elizabeth1,Marques Andreia1,Bowsher Leo1,Wells Stephen A.3,Watts Andrew45,van den Elsen Jean M. H.25,Turner Alison1,O'Hara John1,Pudney Christopher R.25

Affiliation:

1. UCB, 216 Bath Road, Slough SL1 3WE, U.K.

2. Department of Biology and Biochemistry, University of Bath, Bath, U.K.

3. Department of Physics, University of Bath, Bath, U.K.

4. Department of Pharmacy and Pharmacology, University of Bath, Bath, U.K.

5. Centre for Therapeutic Innovation, University of Bath, Bath, U.K.

Abstract

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a ‘fingerprint’ of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg–mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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