Released N-glycan analysis of a therapeutic antibody

N-glycan analysis is critical to the characterization of therapeutic monoclonal antibodies. In this article, from Issue 9 of the Analytix Reporter, produced by Merck, a BIOshell™ Glycan HPLC column is used to analyze Cetuximab (Erbitux^®) N-glycans delivering a fast, high-resolution, and reproducible glycan identification using HILIC.

Introduction
Therapeutic monoclonal antibodies (mAbs) have seen an explosive growth since the first mAb was approved by the US FDA over thirty years ago. In fact, over the past five years, therapeutic antibodies have become the best-selling drugs, and they continue to grow in terms of new approvals and targets.

In this article, a BIOshell™ Glycan HPLC column is used to analyze Cetuximab (Erbitux^®) N-glycans labeled with procainamide. BIOshell™ Glycan HPLC columns are specifically engineered to deliver a fast, high-resolution, and reproducible glycan identification using HILIC.

Experimental
Glycan Release and Labeling - PNGase Fast Kit was used for glycan release with FASP (filter aided sample prep). The released glycans were labeled.

Results
In this study, cetuximab was used as a model therapeutic mAb to analyze released N-glycans. This mAb is a chimeric mouse-human IgG1 monoclonal antibody, against the epidermal growth factor receptor (EGFR). Cetuximab is used to treat head, neck, as well as colorectal cancers. The antibody is N-glycosylated both in the fragment crystallizable (Fc) and fragment antigen binding (Fab) regions. There are numerous studies and reports showing the attachment of N-glycans to mAbs and the subsequent effect of the attachment on various biological and physicochemical processes, leading to safety and quality issues. Some of the processes affected by the glycosylation include enhancement of the structural integrity of the mAb, serum half-life, antibody-dependent cellular toxicity (ADCC), anti-inflammatory activities, immunity, and antigen recognition. This clearly indicates towards the important need for understanding glycosylation patterns.

Conclusion
Characterizing and monitoring the glycosylation pattern of a therapeutic mAb is required by regulatory authorities to ensure efficacy and safety of the drug. While analysis and identification of glycans can be challenging because of their structural complexity, this article has shown that a BIOshell™ Glycan HPLC column was able to elucidate the complex glycosylation of cetuximab after an appropriate glycan release and labeling protocol. Another key consideration in glycan analysis is the deglycosylation protocol. While there is a fast method that significantly saves time, it is recommended to compare the results with the traditional overnight digestion and choose the one that gives more efficient deglycosylation.

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