Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

Abstract

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N-and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N-and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc Nglycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi-and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N-and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest. K E Y W O R D S CHO-GS cells, culture media, EPO-Fc protein, intact glycopeptide analysis, site-specific glycosylation SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.

The Possible Role of Anti-Neu5Gc as an Obstacle in Xenotransplantation

“…The Cetuximab humanized murine antibody has been implicated in anti-Gal IgE dependent anaphylaxis in some treated patients (77) and exhibit Neu5Gc epitopes that induce anti-Neu5Gc antibodies in the Cmah −/− mouse (78). Neu5Gc glycosylations are also detected in Erythropoietin produced in CHO (79,80). In addition, Neu5Gc derived from animal components and present in the medium can also be incorporated into glycans present on recombinant products.…”

Nonconsensus motif directed chemical synthesis of glutamine‐based glycopeptides

“…As upcoming improved mass spectrometry (MS) methods have tremendously simplified the determination of naturally occurring glycopeptides, 12–14 a difficult preparation including either chemical or enzymatical degradation or enrichment of intact glycopeptides is still necessary. An enrichment by exploiting lectin affinity or anion exchange chromatography can though increase the number of detectable N ‐glycans; still, only small amounts of testing material can be provided 4,5,15 . Besides the common motifs in N‐ glycopeptides, where N ‐acetyl glucosamine (GlcNAc) is linked glycosidically to the side chain of asparagine (N‐X‐S/T, where X can be any amino acid except proline), 16 several nonconsensus glycosylation motifs have been found in mammal glycoproteins like for instance N‐X‐C or Q‐G‐T.…”

“…Thus, this could be a great advance for biotechnology, if the considered glycopeptides show biological activity 21,24 . Hitherto, glycopeptides are also having yet a great influence in today's medicinal application as they are applied in the therapy of aggressive diseases like HIV, cancer, or bacterial infections 15,25–27 . Nevertheless, a synthetic approach, especially to glycopeptides with rarely found sequons, is highly important for the required drug development.…”

“…An enrichment by exploiting lectin affinity or anion exchange chromatography can though increase the number of detectable N-glycans; still, only small amounts of testing material can be provided. 4,5,15 Besides the common motifs in N-glycopeptides, where Nacetyl glucosamine (GlcNAc) is linked glycosidically to the side chain of asparagine (N-X-S/T, where X can be any amino acid except proline), 16 several nonconsensus glycosylation motifs have been found in mammal glycoproteins like for instance N-X-C or Q-G-T.…”

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography–Mass Spectrometry Peptide Mapping

“…In addition to these parameters, the glycosylation distribution at different sites is of relevance for EPO and related products. ,− The site-specific glycosylation information is essential for determining the actual molecule heterogeneity, e.g ., by proteoform profiling. , Several MAM methods measuring the glycosylation profiles at different EPO glycosylation sites have been reported. − …”