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Liu T, Li Y, Xu J, et al. N-Glycosylation and enzymatic activity of the rHuPH20 expressed in Chinese hamster ovary cells. Anal Biochem. 2021;632:114380doi: 10.1016/j.ab.2021.114380.
Liu, T., Li, Y., Xu, J., Guo, Q., Zhang, D., Song, L., Li, J., Qian, W., Guo, H., Zhou, X., & Hou, S. (2021). N-Glycosylation and enzymatic activity of the rHuPH20 expressed in Chinese hamster ovary cells. Analytical biochemistry, 632114380. https://doi.org/10.1016/j.ab.2021.114380
Liu, Tao, et al. "N-Glycosylation and enzymatic activity of the rHuPH20 expressed in Chinese hamster ovary cells." Analytical biochemistry vol. 632 (2021): 114380. doi: https://doi.org/10.1016/j.ab.2021.114380
Liu T, Li Y, Xu J, Guo Q, Zhang D, Song L, Li J, Qian W, Guo H, Zhou X, Hou S. N-Glycosylation and enzymatic activity of the rHuPH20 expressed in Chinese hamster ovary cells. Anal Biochem. 2021 Nov 01;632:114380. doi: 10.1016/j.ab.2021.114380. Epub 2021 Sep 11. PMID: 34520755.
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Anal Biochem. 2021 Nov 01;632:114380. doi: 10.1016/j.ab.2021.114380. Epub 2021 Sep 11.

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

Analytical biochemistry

Tao Liu, Yantao Li, Jin Xu, Qingcheng Guo, Dapeng Zhang, Lankun Song, Jun Li, Weizhu Qian, Huaizu Guo, Xinli Zhou, Sheng Hou

Affiliations

  1. Department of Oncology, Huashan Hospital, Fudan University, Shanghai, 200043, China; State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China.
  2. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China.
  3. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China; School of Pharmacy, Liaocheng University, Liaocheng, 252000, China; Shanghai Zhangjiang Biotechnology Co., Ltd, Shanghai, 201203, China.
  4. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China; School of Pharmacy, Liaocheng University, Liaocheng, 252000, China; Taizhou Mabtech Pharmaceuticals Co., Ltd, Taizhou 225316, China.
  5. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China; School of Pharmacy, Liaocheng University, Liaocheng, 252000, China.
  6. Waters Corporation, Shanghai, 201206, China.
  7. School of Pharmacy, Liaocheng University, Liaocheng, 252000, China.
  8. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China; School of Pharmacy, Liaocheng University, Liaocheng, 252000, China; Shanghai Zhangjiang Biotechnology Co., Ltd, Shanghai, 201203, China. Electronic address: [email protected].
  9. Department of Oncology, Huashan Hospital, Fudan University, Shanghai, 200043, China. Electronic address: [email protected].
  10. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, 201203, China; NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai, 201203, China; School of Pharmacy, Liaocheng University, Liaocheng, 252000, China. Electronic address: [email protected].

PMID: 34520755 DOI: 10.1016/j.ab.2021.114380

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.

Copyright © 2021 Elsevier Inc. All rights reserved.

Keywords: Bioactivity; Glycosylation; Subcutaneous injection bioavailability; UPLC-UV/MS/MS; rHuPH20

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