of complete heavy chains and light chains linked by interchain disulfide bonds.This is a serious problem for IgG4because of the instabi-lity of the hinge region interchain disulfide bonds.65,67,70,71,78,79

Oligosaccharides

Glycosylation is an enzymatic reaction which occurs inside the cell.Oligosaccharide heteroge-neity,which can cause monoclonal antibody heterogeneity,is most likely introduced during oligosaccharide synthesis and processing.All IgGs have the conserved N-linked oligosaccharides on the two conserved asparagine residues in the CH2domain of the Fc region and approximately 15–20%also have N-linked oligosaccharides in the variable region.80–87The variable region oligosaccharide could be on either the light chain, the heavy chain,or both.18,45,86–104O-linked oligosaccharides have also been reported mostly located in the hinge region87–89,105–109or possibly on Fc region.97,110Low levels of nonglycosylated IgG were also observed,at least,for recombinant monoclonal antibodies.111,112

Wild type IgGs have a complicated oligosac-charide population on the conserved asparagine residues in the CH2domains.80–84Recombinant monoclonal antibodies expressed in various cell lines including CHO,NS0,hybridoma,and others normally have less complex oligosaccharide pro-files.The common structures of oligosaccharides of recombinant monoclonal antibodies are bianten-nary complex with a core fucose with zero,one,or two terminal galactose(Fig.1),and various truncated structures with the loss of the core fucose,GlcNAc,or both.48,63,113–127Oligosacchar-ides of high mannose have also been reported for several antibodies.33,62,97,103,111,128–131Sialic acid is normally present at a very low level for Fc oligosaccharides.47,101,102,111,132–136

While different subtypes of IgGs137,138and cell culture conditions33,93,130,134,139–143can affect oligo-saccharide structures,cell line45,46,137,138,141,144,145 is by far the most important factor that defines oligosaccharide structures.For example,recom-binant monoclonal antibodies from cell lines of murine origin commonly contain Gal a1-3Gal.46,101–103,131,132,136,138This affects the in vivo half-life of these antibodies significantly because of the presence of natural antibodies that recognize Gal a1-3Gal in the human body.146

Size heterogeneity of monoclonal antibodies caused by different oligosaccharide structures can be readily detected by mass spectrometry. Chromatography is also very useful in separation of monoclonal antibodies with different oligosac-charides.Monoclonal antibodies with additional Fab oligosaccharides are more hydrophilic and have been shown as an earlier elution shoulder on a HIC column.18The presence of sialic acids introduces additional negative charges and thus antibodies with sialic acids elute earlier on cation exchange chromatography.36,38,47There are multiple interactions between the

sugar mon N-linked oligosaccharide structures of recombinant monoclonal

antibodies.A:NGA2F(G0F);(B)NA1F(G1F);(C)NA2F(G2F).

JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008DOI10.1002/jps 2428LIU ET AL.