Currently more than 20 recombinant IgG antibody therapeutics are licensed, for treatment of a variety of diseases. Additionally, it is estimated that ~ 30% of new drugs likely to be licensed during the next decade will be based on antibody products. Protein engineering has been employed to "tailor" Mabs to activate selected pathways of action (effector mechanisms) considered to optimize clinical outcomes whilst minimizing their potential to be immunogenic. However, human IgG is a glycoprotein and the presence of oligosaccharides within the IgG-Fc has a profound influence on the effector mechanism(s) activated. Cell engineering has been applied to produce selected antibody glycoforms that promise to be more efficacious in vivo. Research has been translated to generate cell lines for commercial production and a new generation of antibody therapeutics are now entering the clinic. Advances in efficacy may widen the patient population benefiting from these drugs and/or reduce the cost of treatment.

IgG Antibodies - Basic Structure

Five classes of antibody are identified in human blood (IgM, IgG, IgA, IgD & IgE); however antibodies of the IgG class predominate and, to date, all licensed antibody therapeutics have been based on the IgG format. The IgG molecule is multifunctional and binds antigen (pathogen!) to form immune complexes that activate clearance (effector) mechanisms [1, 2]. There are four subclasses of IgG that each expresses a unique profile of effector activities. The basic structure of an IgG molecule is of two light and two heavy chains in covalent and non-covalent association to form three independent protein moieties connected through a flexible linker, the hinge region (fig. 1). Two of these moieties are identical in structure and each expresses a specific antigen-binding site (Fab); the third, (Fc), expresses interaction sites for ligands that activate effector mechanisms. These effector ligands include three structurally homologous cellular Fc receptor types (FcγRI, FcγRII, FcγRIII), the neonatal Fc receptor (FcRn) and the C1q component of complement.
The IgG-Fc region is a homodimer comprised of inter-chain disulphide bonded hinge regions, glycosylated C_H2 domains, bearing an N-linked oligosaccharide at asparagine 297 (Asn-297) and non-covalently paired C_H3 domains.

Effector mechanisms are severely compromised or ablated for aglycosylated or deglycosylated forms of IgG [1, 2]. Current evidence suggests that the oligosaccharide does not directly bind effector ligands but influences the conformation at the polypeptide interaction sites; multiple non-covalent interactions between the oligosaccharide and the C_H2 domain polypeptide influences the conformation of each, in a reciprocal manner [2,3].

Oligosaccharide Heterogeneity and the IgG Glycoforms

i) Normal polyclonal IgG
The oligosaccharide moiety is of the complex diantennary type comprised of a core heptasaccharide structure with variable addition of other sugar residues (fig. 2). A typical HPLC profile for released oligosaccharides is shown in figure 3; the minimal structure observed is a heptasaccharide having terminal N-acetylglucosamine residues (A), however, more commonly the octa-saccharide bearing fucose on the primary N-acetylglucosamine residue is observed (E & M). The variable addition of galactose generates further heterogeneity, e.g. B, α(1-6) arm; C, α(1-3) arm; D, digalactosyl; a minority (<10%) of oligosaccharides bear sialic acid residues. In spite of the low sialic acid content catabolism of IgG is not effected through the asialylglycoprotein receptor but through a unique FcRn mediated pathway.

ii) Recombinant monoclonal IgG
A majority of currently licensed antibody therapeutics is produced in Chinese hamster ovary (CHO) cells; mouse NSO and Sp2/0 cells are also used. The profile of oligosaccharides released from antibody produced by "wild type" CHO cells is predominantly comprised of fucosylated agalactosyl (G0F; peak E, fig. 3); fucosylated mono- and di-galactosylated oligosaccharides also being present (G1F & G2F; peaks F, G & H, fig. 3). Minor populations of other oligosaccharides, e.g. high mannose (Man_5-8), are invariably present. NSO and Sp2/0 cells also produce predominantly G0F oligosaccharides but may also add galactose α(1-3) galactose and N-glycolylneuraminic acid residues. The α(1-3) galactose residue is not expressed by humans and is immunogenic; it is ubiquitous in the environment and ~ 1% of circulating IgG is specific for this epitope. N-acetylneuraminic acid residues may be added but in α(2-3) linkage rather than α(2-6) as in normal human IgG. NS0 cells may also add the N-glycolyl form of sialic acid (N-glycylneuraminic acid) that may also be immunogenic. Neither CHO, NSO nor Sp2/0 cells have the capacity to add bisecting N-acetylglucosamine residues.