Biotechnology and Bioprocess Engineering 2020; 25(1): 117-125  
A Liquid Formulation of a Long-acting Erythropoietin Conjugate
Hyung-Kyu Lim1,2, Sung Hee Hong2, Sung Min Bae2, In Young Choi2,*, and Ha Hyung Kim1,*
1Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, Seoul 06974, Korea
2Hanmi Research Center, Hanmi Pharm. Co. Ltd., Hwaseong 18469, Korea
Correspondence to: Ha Hyung Kim*
Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, Seoul 06974, Korea
Tel: +82-2-820-5612; Fax: +82-2-823-5612
E-mail: hahyung@cau.ac.kr
In Young Choi*
Hanmi Research Center, Hanmi Pharm. Co. Ltd., Hwaseong 18469, Korea
Tel: +82-31-371-5141; Fax: +82-31-371-5128
E-mail: iychoi@hanmi.co.kr
Received: May 23, 2019; Revised: August 12, 2019; Accepted: September 22, 2019; Published online: February 29, 2020.
© 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
Erythropoietin (EPO) is a glycoprotein that acts as a cytokine for erythrocyte precursors in the bone marrow, thus, being regulating erythropoiesis. Since polypeptides such as EPO are less stable, a breakthrough for protein drugs is needed. To avoid these limitations, a long-acting conjugate was developed in this study, where we aimed to formulate a long-acting EPO conjugate consists of EPO linked to the immunoglobulin Fc fragment. Here we performed RP-HPLC and SE-HPLC analyses and biological activity assays after buffer exchange under various conditions for the development of liquid stabilizer formulations of a long-acting EPO conjugate and to find conditions that its maintain purity and activity. First, the ability of various stabilizers to stabilize the long-acting EPO conjugate was analyzed, and it was confirmed that a specific stabilizer (polysorbate, mannitol, and sodium chloride) maintained the long-acting EPO conjugate. In addition, following long-term storage stability, it was confirmed that the liquid formulation kept the long-acting EPO conjugate stable at 4ºC for 12 months. Even without neutral amino acids, the liquid formulation from this study can provide excellent storage stability for EPO, making it more economically beneficial than other stabilizers and lyophilizing agents.
Keywords: long-acting erythropoietin conjugate, liquid formulation, stabilizer, high performance liquid chromatography


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