Recombinant Production Cell Line Glycoengineering Services

Cell Line Glycoengineering for Recombinant Production

Recombinant proteins, vital in medicine and industry, often require modifications for enhanced properties and fewer side effects. Glycosylation as a common modification significantly affects these properties. Glycan changes in therapeutic proteins, like antibodies and fusion proteins, are crucial for safety and effectiveness. The choice of expression system plays a significant role in glycosylation, as different hosts have distinct glycosylation repertoire, leading to diverse glycosylation profiles. Current expression platforms for recombinant proteins often result in heterogeneous and non-human glycans. To address this, glycoengineering is increasingly used across various expression systems like mammalian cells, insect cells, yeast, and plants, and is crucial for improving biopharmaceuticals.

Fig.1 Glycosylation profiles of recombinant glycoproteins derived from different expression systems.¹

Technology Platforms for Cell Line Glycoengineering at Creative Biolabs

Creative Biolabs has developed advanced Genetic Glycoengineering platforms to engineer various host systems for the production of recombinant proteins with desired glycosylation patterns. We employ methods like gene knockdown, knockout, knockin, overexpression, and mutation to manipulate glycan heterogeneity, sialylation, fucosylation, and branching structures. Genome editing tools, such as TALENs and CRISPR/Cas9 system, enable precise and efficient glycoengineering. We provide these techniques and services with the aim of offering solutions for cell line glycoengineering.

Recombinant Production Cell Line Glycoengineering Services at Creative Biolabs

Various production systems, encompassing bacteria, yeast cells, plant cells, insect cells, and mammalian cells, have become accessible for the production of recombinant glycoproteins suited for a wide array of applications. Creative Biolabs harnesses cutting-edge technology and experienced experts to deliver an array of glycoprotein production systems and provide tailored glycoengineering services in these systems to meet the specific needs of our clients.

• Bacteria Glycoengineering
  Bacteria have been increasingly used for glycoengineering applications since the discovery of the pgl N-glycosylation pathway in bacteria. Genes responsible for glycan synthesis, transporters, glycosidases, and glycosyltransferases have been introduced into bacteria to establish new glycosylation pathways. For instance, by transferring C. jejuni PglB into bacteria like E. coli, N-linked glycoproteins engineered with specific bacterial sequons (D/E-Z-N-X-S/T) can be produced. Enzymes like α2,6-SiaT can introduced to produce humanized glycoproteins with terminal α2,6 sialylated N-glycans. Additionally, human-like O-glycosylation pathways can be engineered in bacteria by incorporating foreign O-OSTs, such as PglL/O, allowing the production of various mucin-type O-glycans like Tn, T, sialyl-Tn (STn), and sialyl-T (ST) glycans.
• Yeast Glycoengineering
  Yeast natively produces high-mannose and hypermannosyl N-glycans and performs O-mannosylation. To humanize the yeast N-glycosylation, yeast-specific glycosylation can be removed by elimination of yeast glycosyltransferases like Och1p and MNN1 and interfering with the lipid-linked oligosaccharide (LLO) biosynthesis pathway. For yeast-specific O-glycosylation, the key is to prevent its initiation by eliminating protein-O-mannosyltransferases (PMT) activities through PMT knockout or PMT inhibitors. Then, different glycosyltransferases and glycosidases are introduced to produce human-type hybrid- and complex-type N-glycans and O-glycans.
• Plant Cell Glycoengineering
  In plant cells, N-glycoengineering involves removing non-human glycans, such as eliminating β1,2-xylose and α1,3-fucose attachments by inhibiting XylT and FUT11/12. As plants lack mammalian-type O-glycosylation, the prevalent O-linked glycans in plants are found on Hyp residues. O-glycoengineering focuses on preventing the formation and extending Hyp residues. Expanding glycosylation capabilities in plant cells includes overexpressing heterologous glycosylation enzymes like GnT-III/IV/V for multi-antennary complex N-glycans and GalNAcT2 to initiate mucin-type O-glycan formation, along with genes involved in the sialylation pathway, enabling the production of well-defined human-like glycans.
• Insect Cell Glycoengineering
  Glycoengineering in insect expression systems, including the insect-based baculovirus expression systems, primarily targeted the modification of N-glycosylation processes. N-linked glycoengineering focuses on suppressing the insect-specific N-glycans with α1,3 core fucose by inhibiting the fused lobes (FDL) enzyme. Introducing mammalian glycosylation capabilities into insect systems mainly involves expressing enzymes for N-glycan elongation, like human GlcNAc-TI/II and GalT-I, along with the sialylation pathway, to generate complex-type N-glycans with terminal sialylation. These modifications can be achieved through insect cell lines or baculovirus vectors. However, O-linked glycosylation in insect cells remains an area requiring further exploration and development.
• Mammalian Cell Glycoengineering
  Mammalian cells, especially Chinese hamster ovary (CHO) cells, are prevalent in the production of recombinant protein therapeutics. In glycoengineering non-human mammalian cells, strategies include eliminating non-human glycans like α1,3 galactose (α1,3 Gal) and N-glycolylneuraminic acid (Neu5Gc) found in mouse cells. Efforts are also directed at reducing glycosylation heterogeneity, maintaining consistent glycosylation profiles, enhancing sialylation, and eliminating core fucosylation. These goals are achieved through various techniques, including enzyme knockdown or knockout (e.g., FUT8, GnTs) and overexpression (e.g., β1,4-GalT, α2,6-ST) to produce customized glycoprotein products and other glycoconjugates.

Advantages of Our Services

• Comprehensive platforms and various strategies in glycoengineering
• Cutting-edge techniques to precisely regulate gene expression
• Efficient glycoengineering across various expression systems
• Custom-designed glycoengineering for specific therapeutic needs
• Professional team with extensive experience in cell glycoengineering

Creative Biolabs leads the way in glycoengineering platforms across various cell species for recombinant production. We have an array of glycoengineering strategies to tailor glycan profiles in glycoproteins, offering customized solutions to address specific needs. If you have any glycoengineering requirements, don't hesitate to contact us for more information.

Reference

1. Khan, Amjad Hayat, et al. "Humanizing glycosylation pathways in eukaryotic expression systems." World Journal of Microbiology and Biotechnology 33 (2017): 1-12.

Related Services:

1. Strategies of Genetic Glycoengineering