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

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

doi:10.7717/peerj.6828

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 (mAbP) 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 mAbPSO57 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 mAbPSO57. SDS–PAGE analysis showed that the purity of mAbPSO57 was not significantly different among the five groups. Rapid fluorescent focus inhibition tests revealed that virus-neutralizing efficacies of purified mAbPSO57 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 mAbPSO57 from transgenic Arabidopsis plant, which will eventually reduce down-stream cost required for mAb production using the plant system.

Highlights

Plants have enormous potential as bioreactors for the large-scale production of therapeutic reagents such as recombinant vaccines and antibodies (Kang et al, 2017; Kim et al, 2016; Lim et al, 2014)
Protein A should be properly coupled to a matrix such as agarose beads, as the orientation of protein A when coupled to the bead affects the holding capacity of the monoclonal antibodies (mAbs) and can affect purification efficiency (Groher & Suess, 2016; Welch et al, 2017; Zhang, Duan & Zeng, 2017)
The expected polymerase chain reaction (PCR) heavy chain (HC) and light chain (LC) gene bands were observed in all T1 Arabidopsis plants (Fig. 1B)

Summary

Introduction

Plants have enormous potential as bioreactors for the large-scale production of therapeutic reagents such as recombinant vaccines and antibodies (Kang et al, 2017; Kim et al, 2016; Lim et al, 2014). Plant leaf extracts contain an array of indigenous proteins and other water-soluble cell components such as DNA, chlorophyll and other pigments, alkaloids, phenolics, polysaccharides, and proteases (Wilken & Nikolov, 2012; Zhang et al, 2015). These components can affect the quality and yield of final purified protein or reduce purification efficiency because of resin or membrane fouling (Jha et al, 2016; Moussavou et al, 2015). The main goal of the present research was to determine coupling pH conditions between protein A and epoxy-activated agarose beads that could efficiently purify anti-rabies monoclonal antibodies (mAbs) derived from transgenic Arabidopsis plants. The purified plant-derived mAbs (mAbPs) from four different resins and a commercially available protein A agarose resin as a positive control were compared for purification efficiency, purity, and neutralizing activity

Objectives

Methods

Results

Discussion

Conclusion