Sensitive detection of immunoglobulin G stability using in real-time isothermal differential scanning fluorimetry: determinants of protein stability for antibody-based therapeutics

Moggridge, J., Biggar, K., Dawson, N. and Storey, K. B. (2017) Sensitive detection of immunoglobulin G stability using in real-time isothermal differential scanning fluorimetry: determinants of protein stability for antibody-based therapeutics. Technology in Cancer Research and Treatment, 16(6), pp. 997-1005. (doi: 10.1177/1533034617714149) (PMID:28602127) (PMCID:PMC5762059)

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Abstract

Protein instability is a major obstacle in the production and delivery of monoclonal antibody–based therapies for cancer. This study presents real-time isothermal differential scanning fluorimetry as an emerging method to evaluate the stability of human immunoglobulin G protein with high sensitivity. The stability of polyclonal human immunoglobulin G against urea-induced denaturation was assessed following: (1) oxidation by the free-radical generator 2,2-Azobis[2-amidinopropane]dihydrochloride and (2) in selected storage buffers. Significant differences in immunoglobulin G stability were detected by real-time isothermal differential scanning fluorimetry when the immunoglobulin G was stored in 1,4-Piperazinediethanesulfonic acid buffer compared to phosphate-buffered saline, with half-maximal rate of denaturation occurring at a higher urea concentration in 1,4-Piperazinediethanesulfonic acid than phosphate-buffered saline (Knd;PIPES = 3.56 ± 0.09 M, Knd;PBS = 2.94 ± 0.08 M; P < .01), but differential scanning fluorimetry did not detect differences in unfolding temperature (Tm;PIPES = 70.5 ± 0.3°C, Tm;PBS = 69.7 ± 0.2°C). The effects of 2,2-Azobis[2-amidinopropane]dihydrochloride-induced oxidation on immunoglobulin G stability were analyzed by real-time isothermal differential scanning fluorimetry; the oxidized protein showed greater sensitivity to urea (Knd;CNTRL = 3.96 ± 0.19 M, Knd;AAPH = 3.49 ± 0.07 M; P < .05). Similarly, differential scanning fluorimetry indicated greater thermal sensitivity of oxidized immunoglobulin G (Tm;CNTRL = 70.5 ± 0.3°C, Tm;AAPH = 62.9 ± 0.1°C; P < .001). However, a third method for assessing protein stability, pulse proteolysis, proved to be substantially less sensitive and did not detect significant effects of 2,2-Azobis[2-amidinopropane]dihydrochloride on the half-maximal concentration of urea needed to denature immunoglobulin G (Cm;CNTRL= 6.8 ± 0.1 M; Cm;AAPH = 6.4 ± 0.7 M). Overall these results demonstrate the merit of using real-time isothermal differential scanning fluorimetry as a rapid and sensitive technique for the evaluation of protein stability in solution using a quantitative real-time thermocycler.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Dawson, Dr Neal
Authors: Moggridge, J., Biggar, K., Dawson, N., and Storey, K. B.
College/School:College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
Journal Name:Technology in Cancer Research and Treatment
Publisher:SAGE Publications
ISSN:1533-0346
ISSN (Online):1533-0338
Published Online:12 June 2017
Copyright Holders:Copyright © 2017 The Authors
First Published:First published in Technology in Cancer Research and Treatment 16(6): 997-1005
Publisher Policy:Reproduced under a Creative Commons License

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