RESEARCH ARTICLE – Pharmaceutical BiotechnologyPhysical Stability Comparisons of IgG1-Fc Variants: Effects of N-Glycosylation Site Occupancy and Asp/Gln Residues at Site Asn 297
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
REFERENCES (46)
- et al.
pH dependent effect of glycosylation on protein stability
Eur J Pharm Sci
(2008) - et al.
Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc
J Biol Chem
(2012) - et al.
Pharmacokinetics of IgG1 monoclonal antibodies produced in humanized Pichia pastoris with specific glycoforms: A comparative study with CHO produced materials
Biologicals
(2011) - et al.
Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain
Biochem Biophys Res Commun
(2006) - et al.
Glycoproteomic analysis of antibodies
Mol Cell Proteomics
(2013) - et al.
Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs
J Am Soc Mass Spectrom
(2008) - et al.
Pharmacokinetic, pharmacodynamic and immunogenicity comparability assessment strategies for monoclonal antibodies
Trends Biotechnol
(2010) - et al.
Evaluation of effects of Fc domain high-mannose glycan on antibody stability
J Pharm Sci
(2012) - et al.
High-throughput biophysical analysis and data visualization of conformational stability of an IgG1 monoclonal antibody after deglycosylation
J Pharm Sci
(2013) - et al.
Comparative signature diagrams to evaluate biophysical data for differences in protein structure across various formulations
J Pharm Sci
(2013)