Fourier‐transform Infrared Spectroscopic Analysis of the Secondary Structure of Recombinant Humanized Immunoglobulin G

Abstract

We present our study on the secondary structure determination of aqueous and dried recombinant humanized immunoglobulin G (rhuIgG). For this purpose, we have examined two regions of the vibrational spectrum, the amide I and amide III, and curve-fitted the Fourier self-deconvoluted and original spectra, respectively. Structures calculated from the two different regions are in good agreement with each other and that of the general IgG molecule reported by others. From the spectra of the protein in aqueous solution, we discerned a predominantly β-sheet structure, approximately 60%, with the remainder in unordered motifs such as turns. Spectra for the dehydrated (i.e. spray-dried) protein yielded very similar results, indicating that removal of water did not significantly alter the secondary structure. The relatively high amount of β-sheet structure may be related to the resistance towards dehydration-induced structural alteration, based on the hypothesis that this motif is energetically favourable in the dried state. We also studied the effect of lactose, an excipient which was found to stabilize rhuIgG towards solid-state aggregation at elevated temperature and humidity. Formulation with lactose (20–60% wt%, dry basis) resulted in only minor changes in the protein's spectra and very similar calculated secondary structures, although the stability towards solid-state aggregation varied markedly.