Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation.

Area of Science:

• Biotechnology
• Molecular Biology
• Biopharmaceutical Manufacturing

Background:

• N-linked glycosylation is a critical quality attribute (CQA) for monoclonal antibodies (mAbs), influencing immunomodulatory effects.
• Terminal galactose residues on mAbs impact antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC), serum half-life, and antiviral activity.
• MicroRNAs (miRNAs) have emerged as tools to modulate protein glycosylation in production cells.

Purpose of the Study:

• To characterize and validate miRNAs as engineering tools for modulating the degree of galactosylation in mAbs.
• To investigate the biological mode of action of miRNAs in regulating the galactosylation pathway.
• To assess the influence of miRNA-mediated glycosylation changes on bioprocess parameters.

Main Methods:

• Stable plasmid-based overexpression of specific miRNAs (e.g., mmu-miR-452-5p, mmu-miR-193b-3p for increased galactosylation; mmu-miR-7646-5p, mmu-miR-7243-3p, etc. for decreased galactosylation) in production cells.
• Characterization of miRNA biological mode of action on gene expression within the galactosylation pathway.
• Evaluation of miRNA effects on bioprocess-related parameters.

Main Results:

• Successfully identified and validated miRNAs for increasing or decreasing N-linked galactosylation.
• Elucidated the regulatory mechanisms of miRNA action on the galactosylation pathway.
• Demonstrated the influence of these miRNAs on critical bioprocess parameters.

Conclusions:

• Stable miRNA overexpression is a versatile tool for engineering N-linked glycosylation.
• This approach enables the modulation of galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production.
• miRNA-based engineering offers precise control over CQAs for enhanced therapeutic antibody function.