Thermal Unfolding Curves of High Concentration Bovine IgG Measured by FTIR Spectroscopy

Protein J (2011) 30:395403 DOI 10.1007/s10930-011-9344-y

Thermal Unfolding Curves of High Concentration Bovine IgG Measured by FTIR Spectroscopy

V. Sathya Devi Denis R. Coleman

Jeremy Truntzer

Published online: 13 July 2011 Springer Science+Business Media, LLC 2011

Abstract The purpose of this research is to study the thermal unfolding of high concentration bovine Immunoglobulin G (IgG) under 26 different experimental conditions by Fourier Transform Infrared spectroscopy with improved purge conditions and software calculations. When bovine IgG (25200 mg/mL) was thermally denatured between pH 4.0 and 8.0, it was observed that at 25 mg/mL concentration, the protein exhibited maximum thermal stability at pH 6.0 and 7.0 as evident from the apparent Tm values. Increasing the concentration from 25 to 100 mg/mL at those pH values increased the thermal resistance of the protein by 23 C. But, at 200 mg/mL,

IgG showed a small decrease in its transition temperature. Presence of 100 mM Trehalose enhanced the Tm values at all conditions and possibly prevented the complete loss of IgG as insoluble aggregates at higher temperatures. Second derivative plots were constructed to explain the conformational changes of IgG during thermal unfolding.

Keywords Bovine Immunoglobulin G FTIR

spectroscopy Thermal unfolding High concentration

Water subtraction and bootstrapping

AbbreviationsFTIR spectroscopy Fourier transform infrared spectroscopyImmunoglobulin G IgGCD Circular dichroismDSC Differential scanning calorimetry NMR Nuclear magnetic resonance

V. Sathya Devi (&) D. R. Coleman (&) J. Truntzer

Coleman Softlabs, Inc., 296 Bay Road, Atherton, CA 94027, USAe-mail: [email protected]; [email protected]

D. R. Colemane-mail: [email protected]

Present Address:V. Sathya DeviIntegrated BioTherapeutics Inc., 21 Firsteld Road, Gaithersburg, MD 20878, USA

Present Address:J. TruntzerUC Irvine School of Medicine, Irvine, CA 92617, USA

1 Introduction

More than 20 therapeutic monoclonal antibodies have been approved by Food and Drug Administration (FDA) and hundreds of others are currently in various stages of development [23]. All of the approved therapeutic monoclonal antibodies belong to Immunoglobulin G (IgG) class which is the major immunoglobulin type in human sera. Stabilization of IgG especially at higher concentrations poses a major formulation challenge for commercial usage [26]. Because of their ease of administration and the costs, liquid antibody dosage forms are usually preferred over lyophilized forms. Among all the commercial antibody products, above half are stable enough to be formulated in liquid form [33].

The major challenge...