Xylt2 Knockout Eliminates O-Xylosylation and Enhances Bone Morphogenetic Protein 2 Production in Chinese Hamster Ovary Cells.

O-xylosylation of glycine-serine (GS) linkers in multispecific antibodies or fusion proteins introduces product heterogeneity, posing critical challenges for biomanufacturing quality control and elevating potential immunogenicity risks. This post-translational modification is primarily catalyzed by xylosyltransferase 2 (Xylt2) in chinese hamster ovary (CHO) cells. To address this, we generated Xylt2-deficient CHO cells via both clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and zinc finger nuclease (ZFN)-mediated gene knockout, which resulted in the complete elimination of O-xylosylation as verified by mass spectrometry in a GS linker-containing bispecific antibody. Furthermore, because heparan sulfate proteoglycans (HSPGs), key cell-surface receptors for ligand binding and internalization, rely on O-xylosylation for their proper biosynthesis and function, the Xylt2 knockout also enhanced the titer of human bone morphogenetic protein 2 (hBMP2), which undergoes HSPG-dependent cellular uptake, with up to a 2.5-fold increase from 9.62 to 34.09 µg/mL in engineered cells compared to wild-type CHO controls. Collectively, our results demonstrate that Xylt2-deficient cell lines provide a genetic approach to produce recombinant proteins without O-xylosylation, as well as for enhancing the titer of hBMP2.