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Science

This report highlights the creation and study of a collection of specially engineered CHO cells that help us understand and improve how sugars attach to proteins—an important process for biology and biotech. For the first time, we measured how certain genes affect these sugar structures, uncovering new insights that go beyond what's in textbooks today. We also explored how changes in the sugar chains on antibodies influence their interactions with immune cells. Additionally, CHO cells in our study produced a key HIV protein (gp120) that reacted differently with various HIV-neutralizing antibodies, confirming that its sugars play a crucial role in these interactions. One of the engineered cell lines produced a therapeutic protein with uniform sugar structures, showing promise for improving its effectiveness in treatments. This work paves the way for more precise and efficient biotech applications.

Societal Impact

Our findings can benefit society by advancing the development of better biopharmaceuticals, such as more effective and safer therapeutic antibodies and vaccines. By uncovering how sugars on proteins influence their interactions with the immune system, our work can help improve treatments for diseases like cancer and infectious diseases, including HIV. The engineered CHO cells we created also provide valuable tools for the biotech industry, enabling the production of high-quality therapeutic proteins with consistent and optimized sugar structures, which can enhance drug effectiveness and reduce manufacturing costs. Ultimately, this research supports innovation in medicine, leading to improved healthcare outcomes for the public.

Technical Summary

We developed and characterized a comprehensive panel of CHO cell glycosylation mutants, enabling in-depth exploration of glycosylation's role in biotechnology. Using EPO-Fc and trastuzumab as model molecules, we quantitatively analyzed their N-glycans through LC-MS/MS to understand the effects of glycosylation gene disruptions. Notably, EPO-Fc displayed uniform Man9 glycans only when most α-mannosidases were inactivated, demonstrating a precise glycan modification. Trastuzumab produced in these mutants revealed how Fc N-glycans affect interactions with Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa) and antibody-dependent cellular cytotoxicity (ADCC), offering new insights into the glycan-dependent mechanism underlying Fc receptor binding and immune effector functions.

Additionally, CHO mutants expressing trimeric gp120 highlighted the role of gp120 glycans in interactions with broadly neutralizing anti-HIV antibodies, revealing distinct reactivity profiles. Furthermore, one mutant produced human β-glucocerebrosidase with uniform Man5 N-glycans, demonstrating its potential for therapeutic glycoengineering and improved drug efficacy. These findings establish the utility of our CHO glycosylation mutants for advancing therapeutic protein development and understanding glycan biology.

References

Ryan Haryadi, Kah Fai Chan, Pao Chun Lin, Yun Lei Tan, Corrine Wan, Wahyu Shahreel, Shi Jie Tay, Terry Nguyen-Khuong, Ian Walsh, Zhiwei Song (2024). Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research. Scientific Reports. 14:23068. https://www.nature.com/articles/s41598-024-73722-z