Identifying Post-Translational Modifications in Monoclonal Antibodies

The analysis of post-translational modifications (PTMs)-such as glycosylation, oxidation, and deamidation-in monoclonal antibodies (mAbs) can be carried out at different levels.

Analysis of Intact Proteins and Large Fragments

The analysis of an entire mAb, or large fragments thereof, can provide information about the protein as a whole (such as mass, protein sequence, structural integrity, and PTMs), but cannot determine the precise location of a PTM. Therefore, it can be useful to digest a mAb into slightly smaller fragments, including the light and heavy chains (generated by disulfide bond reduction), Fab and Fc fragments (generated by papain cleavage), and the F(ab’)₂ fragment (generated by cleavage with pepsin or the immunoglobulin-degrading enzyme of Streptococcus pyogenes [IdeS]). These fragments are more amenable to chromatographic and mass spectrometric (MS) analysis, making the identification of particular modifications at specific domains easier.

Direct MS analysis of proteins can be used to interrogate modifications with relatively large mass shifts, such as C-terminal lysine (128 Da) or fucose (146 Da). Other modifications, however, give rise to very small or no mass differences; examples include aspartate isomerization (0 Da), deamidation (1 Da), or disulfide bridge reduction (2 Da). The broad charge envelope produced by large proteins precludes resolution of these small mass differences by direct MS analysis. However, coupling high performance liquid chromatography (HPLC) separation to MS analysis enables measurement of these subtle mass differences because they are chromatographically separated before MS detection. 

Peptide Mapping

The limitations of PTM analysis at the intact protein level can be overcome by digesting the protein (usually with trypsin), and then analyzing the peptides. This strategy is known as bottom-up analysis or peptide mapping. With peptide mapping, it is possible to identify and quantify PTMs. Many common PTMs cause a shift in reversed-phase LC retention relative to the native peptide (Table I).

One of the most important PTMs to characterize and quantify is glycosylation, which is important for therapeutic efficacy. Glycosylation renders a peptide more hydrophilic, meaning it can be easily resolved from the nonglycosylated peptide. Given the more polar nature of these peptides, however, reversed-phase LC typically cannot fully resolve mAb glycoforms. Hydrophilic-interaction chromatography (HILIC), however, has been used to baseline-resolve all glycoforms, including G1F isoforms. Under reversed-phase LC conditions glycopeptides will be eluted in the region of the peptides, whereas with HILIC they are separated in three regions: non-, O-linked, and N-linked glycopeptides.

To obtain detailed insight into glycans they must first be liberated from the protein or peptide backbone. Removal of N-glycans is carried out enzymatically using the deglycosidase PNGase F, which cleaves between the proximal N-acetylglucosamine (GlcNAc) residue and the protein, thereby converting the asparagine residue to aspartic acid.

Reference

• K. Sandra, I. Vandenheede, and P. Sandra, J. Chromatogr. A1335, 81–103 (2014).

Find this, and other webcasts, at http://www.chromacademy.com/Characterization-of-Protein-Biopharmaceutical-Post-Translational-Modifications.html (free until October 20).