Integrated Microfluidic LC-MS

The authors describe a new microfluidic-based workflow that integrates and automates glycan cleaveage, purification, and chromatographic separation onto a microfluidic liquid chromatography–mass spectrometry chip for MS detection.
Mar 01, 2010

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

Table I
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 release (3).

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.