N & O-Glycosylation Assessment

Creative Biolabs has established a platform to predict a series of post-translational modifications of therapeutic proteins, such as antibodies, blood factors, hormones and interferons. Glycosylation is one of the common post-translational modifications. The carbohydrate attaches the amino acid residues at the nitrogen atom of Asparagine for N-Glycosylation and the oxygen atom of Serine and Threonine for O-linked glycosylation. When immunoglobulin V-genes contain Asparagine resides in the CDRs, an N-glycosylation motif may be formed during selection, of which approximately 20% antibodies are glycosylated in the variable regions.

N & O-Glycosylation

Correct glycosylation is important for antibody folding, stability, solubility, potency, pharmacokinetics and immunogenicity. Unexpected glycans in or near binding surface such as CDRs may interfere with the binding region or cause steric hindrance and hence decreasing binding affinity. Glycan structure differs in composition and branching, inducing further heterogeneity, which needs to be characterized and controlled during the production. In microbial systems, glycosylation doesn’t occur due to the lack of glycosylation pathways.

Protein glycosylation depends on the expression system, cell line, cell growth conditions and nutrients. Once the desired or an acceptable glycosylation profile has been established, the process criteria should be carefully maintained and followed. During the selection of alternative candidates, N-glycosylation motifs in the binding surface should be possibly excluded. When the glycosylation at a specific residue cannot be tolerated due to the bioactivity or safety issues, protein engineering will be performed to determine the effect of substitute the glycosylation site. 

N-glycosylation usually occurs in the motif N-X-S/T where X is any other residues except Proline. However, not all N-residues of such motifs are glycosylated and still over a thousand of other unique sites that are not consistent with this motif are known to be glycosylated. O-glycosylations of Serine and Threonine don’t share any clear common pattern. In Creative Biolabs, a boosting decision tree ensemble algorithm is applied on determined glycosylation sites of various antibodies to predict the O-glycosylation potential. We will clearly mark the sites with known glycosylation or lack thereof in highlight. All the antibodies are characterized in house within 2-3 weeks as customer desires.

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