Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography.
Glycosylation of the conserved asparagine residue in CH2 domains of IgG molecules is an important post-translational modification. The presence of oligosaccharides is critical for structure, stability and biological function of IgG antibodies. Effect of the glycosylation states of recombinant monoclonal antibodies on protein A and protein G chromatography was evaluated. Antibodies lacking oligosaccharides eluted later from protein A and earlier from protein G columns than antibodies with oligosaccharides using a gradient of decreasing pH. Interestingly, different types of oligosaccharides also affected the elution of the antibodies. Antibodies with high mannose type oligosaccharides were enriched in later eluting fractions from protein A and earlier eluting fractions from protein G. While antibodies with more mature oligosaccharides, such as core fucosylated biantennary complex oligosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal galactoses, were enriched in earlier eluting fractions from protein A and in the later eluting fractions from protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody binding affinity to protein A and protein G was not affected by the absence or presence of oligosaccharides. It was thus concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosaccharides were due to differential structural changes around the CH2-CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.
Gaza-Bulseco
Georgeen
G
Process Sciences Department, Abbott Bioresearch Center, Worcester, MA 01605, USA.
Hickman
Keith
K
Sinicropi-Yao
Sara
S
Hurkmans
Karen
K
Chumsae
Chris
C
Liu
Hongcheng
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C029152:G-substrate; D009419:Nerve Tissue Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A