Glycoproteins comprise an important class of biological compounds. The hallmark feature of a glycoprotein is the presence
of N-linked glycans, which exhibit a tremendous amount of structural complexity and diversity. It is this structural complexity
that makes N-linked glycans difficult to characterize. Current characterization workflows are tedious, time-consuming and
as such, are unsuitable for real-time bioprocessing applications. A microfluidic-based workflow that integrates and automates
glycan cleavage, purification, and chromatographic separation onto a microfluidic liquid chromatography–mass spectrometry
(LC–MS) chip for subsequent MS detection is a valuable approach to resolve these shortcomings in current methods.
Glycans' importance to bioprocessing
Protein glycosylation is an important class of post-translational modification often necessary for correct protein folding
and full biological function. More than one-third of recombinant protein drugs are glycoproteins, and antibodies are the largest
group of recombinantly produced glycoproteins. Glycans are covalently linked carbohydrate moieties of glycoconjugates such
as glycoproteins. N-linked glycans (N-glycans), the focus of this article, constitute one of the major classes of glycans
found on mammalian glycoproteins. N-glycans are most commonly attached to the nitrogen of asparagine present in the consensus
amino-acid sequence AsnXxxSer/Thr (where Xxx can be any amino acid except proline), as a result of the multistep enzymatic
process of glycosylation.
Recombinant monoclonal antibodies constitute a major class of anticancer therapeutics, and their N-glycans have earned much
attention in recent years. Antibodies include evolutionarily conserved sites of N-glycosylation, which contain heterogeneous
glycan structures of biantennary class. Glycan heterogeneity is a natural outcome of complex enzymatic synthesis pathways
where carbohydrate residues may be attached to each other in many different ways, thereby resulting in various linkages and
positional isomers. Table 1 provides a list of some of the N-glycans commonly found on natural and recombinant monoclonal
antibodies along with their structure, name, and monoisotopic mass.
Glycans are a source of heterogeneity that can affect the biological attributes of recombinant antibody therapeutics. In antibodies
possessing effector functions, N-glycans have been shown to be involved in the removal of cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC.) In vivo, after the Fab region of the antibody binds to the antigen, natural killer cells bind to the Fc region. This triggers the
release of cytotoxic agents that destroy the antigen. The lack of a core fucose in the Fc region of the antibody leads to
increased Fc-receptor binding and is correlated with up to 50 times higher ADCC activity (1).
Glycan heterogeneity varies with species, expression system, and cell-culture conditions. Chinese hamster ovary (CHO) cells,
a major expression host for many industrially manufactured human therapeutic antibodies, produce "human-like" N-linked glycans.
On the other hand, the "same" antibody produced in a mouse NS0 cell line can have very different, sometimes undesirable glycan
structures. NS0 cells contain α1,3-galactosyltransferase (α1,3GT), an enzyme that transfers α-Gal residues to N-acetyllactosamine
(LacNAc) termini of glycans. Antibody therapeutics with the Gal-α-Gal linkages (α-Gal epitopes) have been shown to cause immune
reactions (2). In Table 1, the glycan structure labeled G2 isomer shows the immunogenic Gal-α-Gal structure, while the structure
labeled G2 is the nonimmunogenic form.
Because glycoprotein heterogeneity can arise from incomplete synthesis, glycans can provide an important measure of production
process consistency. Enzymes involved in glycan synthesis can be sensitive to subtle changes in conditions, and manufacturing
processes rarely achieve 100% efficiency. Because variants may not be as safe and effective as the desired product, they must
be characterized and quantified. N-linked glycan characterization is becoming more routinely performed as an in-process test
during clone selection and screening of cell-culture conditions for recombinant glycoproteins derived from mammalian cell
lines. And, it is expected that regulatory agencies will more frequently request glycan characterization prior to product
Because glycosylation of recombinant monoclonal antibodies can influence their biological activity, efficacy and immunogenicity,
there is a growing interest in antibody design based on glycan engineering, and a growing need in drug discovery, drug development,
and bioprocessing for a fast and reliable separation and detection technique to characterize glycan microheterogeneity.