N-Glycosylation and enzymatic activity of the rHuPH20 expressed in Chinese hamster ovary cells.

Abstract rHuPH20, a neutral pH-active hyaluronidase that degrades glycosaminoglycans under physiologic conditions, has six potential N-glycosylation sites. In this report, the rHuPH20 expressed in Chinese hamster ovary (CHO) cells was analyzed and characterized using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Removal of the N-linked glycans from rHuPH20 with PNGase F shifted the molecular weight from 66 kDa to approximately 52 kDa, its deduced molecular weight based on sequence analysis, suggesting that most, if not all, of the potential N-glycosylation sites are linked to oligosaccharides. Then the N-linked glycans released from the rHuPH20 by PNGase F were characterized by UPLC-FLR-MS, and the six N-glycosylation sites of the rHuPH20 were identified and characterized by UPLC-MS/MS at peptide levels. Subsequently, we found that the rHuPH20 increased the dispersion of locally subcutaneous injected drugs and the in vitro and in vivo bioactivity were decreased significantly after PNGase F treatment. In particular, rHuPH20 significantly augmented the absolute bioavailability of locally subcutaneous injected large protein therapeutics, while the bioavailability decreased after being digested by PNGase F. These results demonstrated that N-glycosylation is important for the bioactivity of the rHuPH20.