Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics

Immunoglobulins (Igc) are soluble serum glycoproteins in which the oligosac charides play significant roles in the bioactivity and pharmacokinetics. Re combinant immunoglobulins (rIgG) produced in different host cells by recomb inant DNA technology are becoming major therapeutic agents to treat life th reatening diseases such as cancer. Since glycosylation is cell type specifi c, rIgCs produced in different host cells contain different patterns of oli gosaccharides which could affect the biological functions. In order to dete rmine the extent of this variation N-linked oligosaccharide structures pres ent in the IgGs of different animal species were characterized. IgCs of hum an, rhesus, dog, cow, guinea pig, sheep, goat, horse, rat, mouse, rabbit, c at, and chicken were treated with peptide-N-glycosidase-F PNGase Fl and the oligosaccharides analyzed by matrix-assisted laser desorption/ionization t ime-of-flight mass spectrometry (MALDI-TOF-MS) for neutral and acidic oligo saccharides, in positive and negative ion modes, respectively. The data sho w that for neutral oligosaccharides, the proportions of terminal Gal, core Fuc and/or bisecting GlcNAc containing oligosaccharides vary from species t o species; for sialylated oligosaccharides in the negative mode MALDI-TOF-M S;IS show that human and chicken IgG contain oligosaccharides with N-acetyl -neuraminic acid (NANA), whereas rhesus, cow, sheep, goat, horse, and mouse IgGs contain oligosaccharides with N-glycolylneuraminic acid (NGNA). In co ntrast, IgGs from dog, guinea pig, rat, and rabbit contain both NANA and NG NA. Further, the PNGase F released oligosaccharides mere derivatized with 9 -aminopyrene 1,4,6-trisulfonic acid (APTS) and analyzed by capillary electr ophoresis with laser induced fluorescence detection (CE-LIF). The CE-LIF re sults indicate that the proportion of the two isomers of monogalactosylated , biantennary, complex oligosaccharides vary significantly, suggesting that the branch specificity of beta 1,4-galactosyltransferase might be differen t in different species. These results show that the glycosylation of IgGs i s species-specific, and reveal the necessity for appropriate cell line sele ction to express rIgGs for human therapy. The results of this study are use ful for people working in the transgenic area.