Glycoengineering of Interferon-b 1a Improves Its Biophysical and Pharmacokinetic Properties Kyoung Song 1,3 , In-Soo Yoon 4 , Nam Ah Kim 5 , Dong-Hwan Kim 1 , Jongmin Lee 6 , Hee Jung Lee 3 , Saehyung Lee 1 , Sunghyun Choi 3 , Min-Koo Choi 7 , Ha Hyung Kim 8 , Seong Hoon Jeong 5 , Woo Sung Son 9 , Dae-Duk Kim 1 , Young Kee Shin 1,2 * 1 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea, 2 Institutes of Entrepreneurial BioConvergence, Seoul National University, Seoul, Republic of Korea, 3 Research Institute of Reference Biolabs. Inc., Seoul, Republic of Korea, 4 College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Jeonnam, Republic of Korea, 5 College of Pharmacy, Dongguk University-Seoul, Gyeonggi-do, Republic of Korea, 6 PanGen Biotech Inc., Gyeonggi-do, Republic of Korea, 7 College of Pharmacy, DanKook University, Chungnam, Republic of Korea, 8 Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea, 9 College of Pharmacy, CHA University, Gyeonggi-do, Republic of Korea Abstract The purpose of this study was to develop a biobetter version of recombinant human interferon-b 1a (rhIFN-b 1a) to improve its biophysical properties, such as aggregation, production and stability, and pharmacokinetic properties without jeopardizing its activity. To achieve this, we introduced additional glycosylation into rhIFN-b 1a via site-directed mutagenesis. Glycoengineering of rhIFN-b 1a resulted in a new molecular entity, termed R27T, which was defined as a rhIFN-b mutein with two N-glycosylation sites at 80 th (original site) and at an additional 25 th amino acid due to a mutation of Thr for Arg at position 27 th of rhIFN-b 1a. Glycoengineering had no effect on rhIFN-b ligand-receptor binding, as no loss of specific activity was observed. R27T showed improved stability and had a reduced propensity for aggregation and an increased half-life. Therefore, hyperglycosylated rhIFN-b could be a biobetter version of rhIFN-b 1a with a potential for use as a drug against multiple sclerosis. Citation: Song K, Yoon I-S, Kim NA, Kim D-H, Lee J, et al. (2014) Glycoengineering of Interferon-b 1a Improves Its Biophysical and Pharmacokinetic Properties. PLoS ONE 9(5): e96967. doi:10.1371/journal.pone.0096967 Editor: Michael Massiah, George Washington University, United States of America Received January 21, 2014; Accepted February 23, 2014; Published May 23, 2014 Copyright: ß 2014 Song et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by Basic Science Research Program through the National Research Foundation (NRF)(NRF-2012R1A1A1043288, URL https://www.nrf.re.kr/nrf_tot_cms/index.jsp?pmi-sso-return2 = none) and Project through Korea Drug Development Fund (20100030032, URL http://www.kddf. org/Main/) of Korea funded by the Ministry of Education, Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Kyoung Song, Dae-Duk Kim, and Young Kee Shin currently hold stock in Reference Biolabs, Inc. This submission is related to Patent 10- 0781666, USA 8,101,716 B2, 4637913, ZL 2005 8 0045291.5, 1809661, TU201108302TA, 3075773, PU110085EP and DK/EP1809661 in which inventor included Young Kee Shin, Kyoung Song, Joung Mim Lee. Reference Biolabs, Inc. is currently developing the R27T product. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: ykeeshin@snu.ac.kr Introduction Multiple sclerosis (MS) is a chronic neurodegenerative disease affecting the brain and spinal cord, leading to symptoms, including blurred vision, muscle weakness, trouble with mobility and balance, cognitive and memory problems, and sensory distur- bances [1–3]. It typically occurs between the ages of 20 to 50, is more common in women than in men, and has a variable course [3,4]. As gold standard MS therapeutics, recombinant human interferon-b (rhIFN-b) products are widely used as a first-line treatment and have had a good long-term safety record over the last few decades [5,6]. Although rhIFN-b is currently competitive with newer oral medicines that provide improved compliance and tolerance, it is still uncertain whether injectable treatments can be completely replaced by oral drugs, at least until oral drugs demonstrate good long-term safety records [6,7]. This is partic- ularly important because drug safety is one of the biggest issues in MS therapeutics, as MS is not a life-threatening disease but a life- long disease (since it occurs at an early age) [4,6]. Therefore, the production of biobetter versions of rhIFN-b would fulfill consid- erable unmet needs in the MS therapy, both with respect to considerations within the pharmaceutical industry, such as biophysical stability and low production costs, and in medicine, such as fewer side effects, longer dosing intervals and route of administration. Protein modification with polyethylene glycol (PEG) or oligo- saccharide moieties is an approach often used to improve bioactive proteins, especially in regard to physical and thermal stability, increased solubility, protection against enzymatic digestion, increased circulating half-life, and in some cases, decreased immunogenicity [8–10]. Notably, next generation rhIFN-b therapeutics has been created using Fc fusions or PEGylation of rhIFN-b at its N- or C-terminal region, or at cysteine residues. PEGylated-Avonex is currently undergoing phase III clinical trials [11]. However, production problems with rhIFN-b 1a still remain a concern for the development of PEG-rhIFN-b 1a, because of rhIFN-b 1a aggregation, and the increased production cost associated with PEGylation. Unlike PEGylation, glycoengineering requires no additional manipulation processes after the construction of a relevant cell line because glycosylation is a natural protein modification within mammalian cells. In addition, it is well known that glycosylation is PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e96967