Non-Mammalian Expression System "Humanization" Service

N-glycosylation, as one of the most complex post-translational modification, influences the structural characteristics of the antibody Fc part, thereby potentially modulating effector function and pharmacokinetics. As the long-term pioneer as well as market leader in the field of therapeutic antibody development, Creative Biolabs has successfully established and commercialized a wide spectrum of functional assays to illustrate antibody bioactivity and its mechanism of action. Particularly, we offer this unique opportunity for our worldwide customers to adopt impeccable Fc engineering services.

Introduction of N-Glycosylation

Glycosylation, the attachment of a sugar moiety to a given protein backbone, is a largely conservative post-translational modification in multicellular organisms. In humans, it is estimated that more than 50% of proteins are glycosylated. In particular, serum glycoproteins form a heterogeneous glycosylation mixture of other homologous protein backbones. The Fc region of IgG1 contains a conserved N-glycosylation site at Asn297 of each CH₂ domain. Variations in the structure of these glycans lead to subtle changes in structure that have a significant impact on the interaction of IgG with the immune system. The remarkable success of therapeutic applications of IgG in the form of monoclonal antibodies and pooled IgG formulations has raised concerns about such glycoproteins. It is generally believed that oligosaccharides attached to the Fc region play a key role in the biological activity of IgG. Therefore, glycosylation has become a focus of great interest in many fields which has been designed to optimize antibody products.

Fig.1 N-glycans Types. (Yap, et al., 2017) Fig.1 Types of N-glycans.¹

“Humanization” of N-Glycosylation Pathway of Non-Mammalian Cells

Primarily mammalian cell lines have been used for the production of glycoengineered antibodies, but non-mammalian expression hosts such as yeast and plants genetically modified to allow for the production of antibodies with defined carbohydrate structures may be an option. In order to fully understand the different IgG activities, more than protein-protein interactions alone have to be considered. A single N-glycan residue significantly affects the biological activity of the mAb. Due to cost effectiveness, some non-mammalian cell lines have been used to produce therapeutic antibodies and they are highly tolerant of glycosylation engineering.

Creative Biolabs aims to humanize immunogenic glycoforms by eliminating enzymes responsible for the addition of non-mammalian glycans and subsequent introduction of mammalian glycan processing enzymes. Like mammalian cells, N-glycosylation of newly synthesized proteins in insects, yeasts, and plant cells is initiated by the oligosaccharyltransferase complex in the endoplasmic reticulum (ER), which transfers the oligosaccharide precursor Glc3Man9GlcNAc2 onto the growing polypeptide chain, more specifically, onto a suitable asparagine residue within the N-glycosylation consensus sequence (Asn-Xaa-Ser/Thr). Yeast- and plant-derived mAbs exhibit most of the uniform N-glycosylation profiles. Engineering of the respective pathways in non-mammalian expression hosts yielded significant results.

Phase 1: Analysis and Design (Weeks 1-3)

Client Consultation & Project Definition: We discuss your specific needs, target protein, host cell, and desired glycosylation profile. We define project goals, deliverables, and initial timelines.
Non-Mammalian Glycosylation Pathway Analysis: We thoroughly analyze the existing N-glycosylation pathway of your chosen non-mammalian host cell. This involves identifying key enzymes and their substrate specificities.
Human Glycosylation Pathway Design: Based on your target glycosylation profile, we design a strategy for introducing and/or modifying the host cell's machinery. This includes selecting human glycosyltransferases, glycosidases, and sugar nucleotide transporters.
Gene Design & Codon Optimization: We design the synthetic genes encoding the human enzymes, optimizing the codon usage for efficient expression in your non-mammalian host.

Phase 2: Genetic Engineering & Transformation (Weeks 4-7)

Gene Synthesis & Cloning: The designed human glycosylation pathway genes are synthesized and cloned into appropriate expression vectors compatible with your host cell.
Host Cell Transformation & Selection: The host cells are transformed with the engineered vectors. Selection markers are used to identify successfully transformed clones.

Phase 3: Clone Screening & Characterization (Weeks 8-12)

Primary Clone Screening: We screen a number of transformed clones to identify those expressing the introduced human glycosylation enzymes. This may involve techniques like PCR, Western blotting, or enzyme activity assays.
Glycosylation Profile Analysis: Promising clones are selected for detailed analysis of their protein glycosylation profiles. We use techniques like mass spectrometry to determine the glycan structures attached to a reporter protein or your target protein (if expressed at this stage).
Optimization & Iteration (if needed): Based on the initial glycosylation analysis, further modifications to the introduced genes, expression levels, or culture conditions might be necessary to achieve the desired human-like glycosylation. This may involve additional rounds of genetic engineering and screening.

Phase 4: Final Analysis & Delivery (Weeks 13-16)

Detailed Glycosylation Characterization: The clone(s) exhibiting the most promising human-like glycosylation profile are subjected to comprehensive analysis.
Data Analysis & Reporting: We compile all experimental data, including gene sequences, expression levels, and detailed glycosylation analysis, into a comprehensive report.
Delivery of Engineered Cell Line & Report: The engineered cell line capable of producing proteins with a more human-like N-glycosylation pattern, along with the detailed report, is delivered to you.

Highlights

Production of Recombinant Proteins with Human-Like Glycans
Reduced Immunogenicity
Enhanced Protein Functionality
Advanced Genetic Engineering Technologies
Customized Solutions for Various Host Systems
Expert Consultation and Guidance

Published Data

Summary: This research focuses on modifying the N-glycosylation pathway in Nicotiana tabacum to make it more suitable for producing therapeutic proteins. By using knockout technology, the researchers successfully removed two enzymes (XylT and FucT) that add non-human sugar structures to proteins. This modification improves the effectiveness and reduces potential immune reactions to plant-made drugs. The study's findings are a step forward in using N. tabacum for the large-scale production of essential medicines.

Analysis of N-glycosylation of purified VRC01 from transgenic NtFX-KO lines. (OA Literature) Fig.2 Purified antibody N-glycosylation analysis from transgenic NtFX-KO lines.²

Related Services

Different host organisms (e.g., bacteria, yeast, insect cells, plant cells, mammalian cells) have distinct glycosylation machinery, resulting in variations in the types and structures of glycans they attach to proteins. For therapeutic proteins, it is often desirable to produce proteins with human-like glycosylation patterns to optimize their efficacy and minimize immunogenicity. In addition to this service, we also provide a wide spectrum of host cell glycoengineering services, including:

For more detailed information, please feel free to contact us or directly send us an inquiry.

References

Yap, Sally SL, et al. "Dengue virus glycosylation: what do we know?." Frontiers in Microbiology 8 (2017): 283239. Distributed under Open Access License CC BY 4.0, without modification.
Göritzer, Kathrin, et al. "Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9." Frontiers in Plant Science 13 (2022): 1003065. Distributed under Open Access License CC BY 4.0, without modification.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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