Dataset Information

Glycoprofile Comparison of SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines

ABSTRACT: This study assessed the N-glycosylation profiles of a full-length trimeric spike protein expressed in either HEK or CHO cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma Finder™ using HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of N-glycan generated by the two cell lines across the 22 known N-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic N-glycolylneuraminic acid at a functionally key N-glycosylation site was observed in the CHO derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific N-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants, to devise updated diagnostic tests and therapeutic vaccine designs

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Helen Wright

LAB HEAD: Jagroop Pandhal

PROVIDER: PXD054718 | Pride | 2025-09-15

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20200805_CHO_spike_gluC_glyco1.mzML	Mzml
	20200805_CHO_spike_gluC_glyco2.mzML	Mzml
	20200805_CHO_spike_gluC_glyco3.mzML	Mzml
	20200805_CHO_spike_tryp_glyco1.mzML	Mzml
	20200805_CHO_spike_tryp_glyco2.mzML	Mzml

Items per page:

1 - 5 of 19

Publications

Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines.

Wright Helen L HL Evans Caroline C Jackson Philip J PJ James David C DC Tee Kang Lan KL Wong Tuck Seng TS Dickman Mark J MJ Pandhal Jagroop J

Molecular biotechnology 20241001 9

Coronavirus SARS-CoV-2 spike protein remains a key focus of research due to a continued need for diagnostic and therapeutic tools to monitor and respond to new variants. Glycosylation of the spike protein is critical for the protein's functions in viral attachment and host cell entry. For scalable and cost-effective production of the spike protein, expression system-driven divergence in glycosylation patterns on recombinant spike proteins needs to be fully understood. This study assessed the N-g ...[more]

PMID: 39352566

Similar Datasets

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.