Delineating the Transcriptional and Phenotypic Impact from Biotherapeutic Glycoengineering.

Glycosylation is critical to biopharmaceutical activity, stability, and pharmacokinetics. While production cells can be engineered to produce better protein glycoforms, glycans decorate thousands of host cell proteins, and it remains unclear how glycoengineering impacts the host cell. To decipher the cell response to glycoengineering, we studied a library of 166 glycoengineered CHO-K1 cell clones representing 54 different glycosyltransferase modifications. Through integrated analysis of glycomics, RNA-Seq, and phenotypic data, we discovered that glycoengineered mutants clustered into three distinct groups (wild-type-like, Moderate, and Substantial) based on their glycosylation patterns. Different glycosyltransferase families exhibited distinct phenotypic signatures: St3gal modifications increased growth rate and cell density, B4galt knockouts affected cell size, and Mgat knockouts enhanced cell viability. Notably, we found specific cellular reprogramming associated with each glycosyltransferase family, including alterations in energy metabolism, stress responses, and DNA repair mechanisms. These findings were validated in an independent set of 30 glycoengineered CHO-S cell lines, expressing a panel of 10 recombinant proteins. Our extensive analysis reveals phenotypic changes resulting from glycoengineering, identifies their molecular bases, and provides crucial insights for controlling glycosylation during therapeutic protein production.