Abstract
Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian CHO cells rely on the heterogeneous N-glycosylation capacity of the cell. Recently, approaches for engineering the glycosylation capacities of mammalian cells for custom designed glycoforms have been developed. With these opportunities there is an increasing need for fast, sensitive, and global analysis of the glycoproteoform landscape produced to evaluate homogeneity and consistency. Here we use high-resolution native mass spectrometry to measure the glycoproteoform profile of 24 glycoengineered variants of EPO. Based on the unique mass and intensity profiles of each variant, we classify them according to similarities in glycosylation profiles. The classification distinguishes EPO variants with varying levels of glycan branchingand sialylation, which are crucial parameters in biotherapeutic efficacy. We propose that our methods could be of great benefit in the characterization of other glycosylated biopharmaceuticals, ranging from the initial clonal selection to batch-to-batch controls, and the assessment of similarity between biosimilar/biobetter products.
Highlights
Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian Chinese hamster ovary (CHO) cells rely on the heterogeneous N-glycosylation capacity of the cell
Attractive emerging strategies include the so-called GlycoDelete approach wherein complex N-glycans are reduced to trisaccharide stems[10,11], and a second approach wherein combinatorial knock out (KO) and knock in (KI) of various glycosyltransferase enzymes in CHO are used to create a cell with custom designed glycosylation capacity to produce a more homogenous glycosylation profile8,14(Fig. 1a)
To describe how individual KOs and KIs affect the overall EPO glycoproteome profile, we describe in detail two examples, namely C21 EPO, which represents a KO for the β1,6-N-acetyl-glucosaminyltransferase involved in the β6-branching of N-glycans, and C23 EPO, which represents a stacked KO of the β1,4-N-acetylglucosaminyltransferase isozymes A and B that control the β4-branching of N-glycans
Summary
Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian CHO cells rely on the heterogeneous N-glycosylation capacity of the cell. Attractive emerging strategies include the so-called GlycoDelete approach wherein complex N-glycans are reduced to trisaccharide stems[10,11], and a second approach wherein combinatorial knock out (KO) and knock in (KI) of various glycosyltransferase enzymes in CHO are used to create a cell with custom designed glycosylation capacity to produce a more homogenous glycosylation profile8,14(Fig. 1a) With biologics displaying such a complex glycosylation profile, an issue that needs to be addressed is the in-detail analytical assessment of the glycoproteoform heterogeneity. When compared to the relatively simple native MS spectra for therapeutic IgGs that typically display only a dozen of different glycoforms, the EPO native MS spectra exhibit hundreds of different glycoproteoforms[22] arising from the heterogeneity on its three N- and one O-glycosylation sites This makes compositional analysis of such spectra much more challenging
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
