RESEARCH ARTICLE – Pharmaceutical Biotechnology
Physical Stability Comparisons of IgG1-Fc Variants: Effects of N-Glycosylation Site Occupancy and Asp/Gln Residues at Site Asn 297

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ABSTRACT

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0–6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1613–1627, 2014

Section snippets

INTRODUCTION

Monoclonal antibodies (mAbs) are well established as the leading class of protein-based drugs because of their high target specificity and long half lives.1., 2. The majority of mAbs developed to date are IgG1 proteins, consisting of four polypeptide chains (two heavy and two light chains) that arrange into 12 Ig domains that form into a Y-shaped molecule with two antigen binding (Fab) regions and one crystallizable (Fc) region. The homodimeric, horseshoe shaped, Fc region contains two

Materials

Both the human IgG1-Fc sequence (comprising 446 amino acids with a theoretical molecular weight of 50,120.92 Da) and a point mutant of the IgG1-Fc protein (with 446 amino acids and a theoretical molecular weight of 50,148.96 Da) were prepared and expressed using a glycosylation deficient strain of P. pastoris as described by Xiao et al.26 The nonglycosylated variant of the IgG1-Fc was made by mutating the N-linked glycosylation site at Asn 297 (EU numbering) to Gln 297, thus eliminating the

Initial Characterization of the Different IgG1-Fc Glycoforms

A pictorial presentation of the five different types of IgG1-Fc proteins (diglycosylated, monoglycosylated and three different nonglycosylated forms) examined in this study is presented in Figure 1. HM IgG1-Fc glycoforms were expressed in yeast and purified using a combination of Protein G and CEX/HIC. Purified protein fractions of each variant from both CEX and HIC were combined to ensure the same material was being examined during biophysical studies (see Methods section). Expression of

DISCUSSION

Comparability exercises are routinely performed in the biopharmaceutical industry as a result of manufacturing process changes (e.g., cell culture or purification steps) or alterations in final product presentation (e.g., formulation composition or different packaging material) for protein based drugs under development or currently marketed.36., 37. In these studies, the critical quality attributes of the pre and postchange protein drug are evaluated in a head-to-head fashion to better

ACKNOWLEDGMENTS

The authors wish to acknowledge the NIH for the financial support of T.J.T. via NIH grant NIGMS RO1 GM090080, and S.Z.O via NIH biotechnology training grant 5-T32-GM008359. The authors also acknowledge King Saud University for the financial support of M.A. Alsenaidy. We also wish to acknowledge and thank Dr. Nadezhda Galeva of the KU Mass Spectrometry/Analytical Proteomics Laboratory for her efforts in acquiring the ESI–LC/MS spectra. The Waters Synapt G2 and NanoAcquity were purchased with

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