Biotechnology and Bioprocess Engineering 2022; 27(2): 244-252  
Differences in Proportion of N-acetyllactosamine and O-acetylated Sialic Acid Have No Significant Effect on the Pharmacokinetics and Biological Activity of Darbepoetin Alfa
Won Jeong Lee, Hookeun Oh, Inseong Choi, Keunho Lee, Hyunwoo Shin, Yoon Jung Lee, Jeehye Park, Yoo Hee Yang, Gyong-Sik Ha, Dong-Eok Lee, Kang-Yell Choi, and Eunyoung Choi
Won Jeong Lee, Hookeun Oh, Yoon Jung Lee, Jeehye Park, Yoo Hee Yang, Gyong-Sik Ha, Dong-Eok Lee, Eunyoung Choi*
Biopharmaceutical Research Center, HK inno.N, Icheon 17389, Korea
Tel: +82-2-6477-0162
E-mail: eunyoung.choi@inno-n.com
Won Jeong Lee
Graduate Program in Biomaterials Science and Engineering, Yonsei University, Seoul 03722, Korea
Inseong Choi
Pharmaceutical Analysis Center, HK inno.N, Icheon 17389, Korea
Keunho Lee, Hyunwoo Shin
Drug Evaluation Cente, HK inno.N, Icheon 17389, Korea
Kang-Yell Choi*
The Division of Life Sciences, the Graduate School, Yonsei University, Seoul 03722, Korea
Tel: +82-2-2123-6592
E-mail: kychoi@yonsei.ac.kr
Received: February 16, 2021; Revised: March 15, 2021; Accepted: April 4, 2021; Published online: April 30, 2022.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Darbepoetin alfa is used to treat anemia in patients with chronic renal failure and is a therapeutic glycoprotein with five N-glycosylation sites and one Oglycosylation site. Since the glycosylation of therapeutic proteins is known to affect pharmacokinetics and biological activity, it was wondered how the certain glycosylation structure of darbepoetin alfa affects pharmacokinetics and biological activity. To investigate the effects of glycosylation structures, several darbepoetin alfa samples were generated through the processes slightly modified from HK-DPO production process. Analysis of N-linked glycosylation profiles showed that darbepoetin alfa samples have different proportions of N-acetyllactosamine and O-acetylated sialic acids. Since these structures were known to affect the stability and efficacy of glycoproteins, we evaluated the pharmacokinetics and biological activities of darbepoetin alfa samples. As a result of pharmacokinetics, the time course of the concentration in plasma was similar to that of NESP®, commercially available darbepoetin alfa. Also, the relative AUClast ratio (sample/reference) was ranged from 84% to 114%, which indicated they did not have meaningful differences. For in vitro biological activity, a cell proliferation test was conducted in the EPO-dependent F36E cell-line. An in vivo biological activity test was performed with EP-based assay using B6D2F1 female mice. As a result, the biological activities of each samples were similar to those of reference drug, NESP®. In conclusion, different proportions of N-acetyllactosamine and O-acetylation of sialic acid in darbepoetin alfa did not show any meaningful effects on pharmacokinetics and biological activities.
Keywords: darbepoetin alfa, N-acetyllactosamine, O-acetylated sialic acid, pharmacokinetics, biological activity


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