Analysis of the glycosylation profile of recombinant human R-Spondin 1 protein (rhRSPO1) produced by glycoengineered CHO-DG44 cells

• Grant number: 24/00036-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: August 05, 2024
• End date: February 04, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Mari Cleide Sogayar
• Grantee: Vitor Prado Colantoni
• Supervisor: Weston Struwe
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Oxford, England
• Associated to the scholarship: 22/01906-2 - Glycoengineering of CHO-DG44 cells for the production of recombinant human R-Spondin 1 (rhRSPO1) with high content of terminally sialylated N-linked glycan chains, BP.DD

Abstract

Cell lines derived from Chinese hamster ovary cells (CHO), particularly, the CHO-DG44 cell line, are the most widely used for production of biotherapeutic proteins worldwide due to their several advantageous characteristics, such as their high similarity with human cells in terms of post-translational modifications of proteins, despite having a low capacity for terminal sialylation of glycans attached to glycoproteins during the glycosylation process. This characteristic represents an important disadvantage of this expression system due to the positive effects of terminal sialylation on the plasma half-life, immunogenicity and, consequently, the biological activity of the proteins. Considering its wide use for the production of biopharmaceuticals under cGMP conditions and its several advantages for heterologous expression of recombinant proteins, the need to establish a CHO-DG44 expression platform that presents a high capacity for terminal sialylation of glycoproteins becomes evident. In order to address this issue, this project aims to establish a CHO-DG44 cell line capable of producing glycoproteins with a high content of terminally sialylated complex-type N-linked glycans through ectopic expression of two critical sialylation enzymes, namely: ²-galactoside ±-2,6-sialyltransferase 1 (ST6GAL1), which catalyzes the addition of ±-2,6 linked sialic acid molecules to galactose containing ends of N-linked oligosaccharide chains, and the UDP-N-acetylglucosamine 2-epimerase / N- acetylmannosamine kinase (GNE), which catalyzes the first two reactions of the sialic acid synthesis pathway. To evaluate the effects of recombinant ST6GAL1 and GNE overexpression in the glycosylation profile of CHO-DG44 cells, we decided to promote the ectopic expression of recombinant human R-Spondin 1 (rhRSPO1), a secreted glycoprotein known for its important role in regulating cell proliferation, differentiation and death through induction of the Wnt/²-catenin pathway. To analyze the glycosylation profile of the rhRSPO1 produced by the parental and glyco-engineered CHO-DG44 cells, the purified rhRSPO1 samples will be characterized using a glycoproteomics approach by native mass spectrometry.