Purification of plant-derived anti-virus mAb through optimized pH conditions for coupling between protein A and epoxy-activated beads

Ilchan Song; Yang Joo Kang; Su-Lim Choi; Dalmuri Han; Deuk-Su Kim; Hae Kyung Lee; Joon-Chul Lee; Jeanho Park; Do-Sun Kim; Kisung Ko

doi:10.7717/peerj.6828

Purification of plant-derived anti-virus mAb through optimized pH conditions for coupling between protein A and epoxy-activated beads

PeerJ. 2019 May 21:7:e6828. doi: 10.7717/peerj.6828. eCollection 2019.

Authors

Ilchan Song^#¹, Yang Joo Kang^#¹, Su-Lim Choi², Dalmuri Han³, Deuk-Su Kim¹, Hae Kyung Lee³, Joon-Chul Lee⁴, Jeanho Park⁴, Do-Sun Kim⁵, Kisung Ko¹

Affiliations

¹ Department of Medicine, College of Medicine, Chung-Ang University, Seoul, South Korea.
² Protein Purification Laboratory, Biotech R&D Center, Amicogen, Jinju, South Korea.
³ Division of Bacterial Disease Research, Korea Centers for Disease Control and Prevention, Osong, South Korea.
⁴ Research Institute of Industrial Technology Convergence, Korea Institute of Industrial Technology, Ansan, South Korea.
⁵ Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun, South Korea.

^# Contributed equally.

Abstract

The main goal of this research was to determine optimum pH conditions for coupling between protein A and epoxy-activated Sepharose beads for purification of monoclonal antibodies (mAbs) expressed in plants. To confirm the effect of pH conditions on purification efficacy, epoxy-activated agarose beads were coupled to protein A under the pH conditions of 8.5, 9.5, 10.5, and 11.5 (8.5R, 9.5R, 10.5R, and 11.5R, respectively). A total of 300 g of fresh leaf tissue of transgenic Arabidopsis expressing human anti-rabies mAb (mAb^P) SO57 were harvested to isolate the total soluble protein (TSP). An equal amount of TSP solution was applied to five resin groups including commercial protein A resin (GR) as a positive control. The modified 8.5R, 9.5R, 10.5R, and 11.5R showed delayed elution timing compared to the GR control resin. Nano-drop analysis showed that the total amount of purified mAb^PSO57 mAbs from 60 g of fresh leaf mass were not significantly different among 8.5R (400 μg), 9.5R (360 μg), 10.5R (380 μg), and GR (350 μg). The 11.5R (25 μg) had the least mAb^PSO57. SDS-PAGE analysis showed that the purity of mAb^PSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAb^PSO57 from all the five different resins including the positive control resin were similar. Taken together, both pH 8.5 and 10.5 coupling conditions with high recovery rate should be optimized for purification of mAb^PSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system.

Keywords: Antibody; Epoxy-activated beads; Protein A; Purification; pH condition.

Grants and funding

This research was supported by a grant (Code#PJ0134372019) from the Korean Rural Development Administration, by the Technology Innovation Program (10079457) funded by the Ministry of Trade, Industry & Energy (MI, Korea), and the Basic Science Research Program through NRF funded by the Ministry of Education (NRF-2016R1A6A3A11930180). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.