Hydrophobin-Protein A Fusion Protein Produced in Plants Efficiently Purified an Anti-West Nile Virus Monoclonal Antibody from Plant Extracts via Aqueous Two-Phase Separation

Abstract

The development of monoclonal antibodies (mAbs) has provided vast opportunities to treat a wide range of diseases from cancer to viral infections. While plant-based production of mAbs has effectively lowered the upstream cost of mAb production compared to mammalian cell cultures, further optimization of downstream processing, especially in extending the longevity of Protein A resin by an effective bulk separation step, will further reduce the overall prohibitive cost of mAb production. In this study, we explored the feasibility of using aqueous two-phase separation (ATPS) in capturing and separating plant-made mAbs from host proteins. Our results demonstrated that an anti-West Nile virus mAb (E16) was efficiently separated from most plant host proteins by a single ATPS step, comprising the mixing of plant extracts containing Hydrophobin-Protein A fusion protein (HPA) and E16 and the subsequent incubation with an inexpensive detergent. This simple ATPS step yielded a highly enriched E16 mAb preparation with a recovery rate comparable to that of Protein A chromatography. The ATPS-enriched E16 retained its structural integrity and was fully functional in binding its target antigen. Notably, HPA-based ATPS was also effective in enriching E16 from plant host proteins when both HPA and E16 were produced in the same leaves, supporting the potential of further streamlining the downstream purification process. Thus, ATPS based on plant-produced HPA in unpurified extract is a cost-effective yet efficient initial capture step for purifying plant-made mAbs, which may significantly impact the approach of mAb purification.

Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice

“…The DNA construct of HBcAg-wDIII was then cloned into the TMV-based expression vector pIC11599 of the MagnICON system [ 13 ]. The MagnICON vectors were chosen because they have been demonstrated to drive high-level accumulation of recombinant proteins in N. benthamiana plants [ 14 , 15 , 16 , 17 ].…”

Affinity sedimentation and magnetic separation with plant-made immunosorbent nanoparticles for therapeutic protein purification

“…A range of biotic technologies beyond virus-based nanomaterials have been developed and studied for biopharmaceutical purification (Dias and Roque, 2016; Mahmoodi et al , 2019). These can be classified by utility as fusion tags (e.g., inteins (Belfort et al , 1999), carbohydrate binding modules (Shoseyov et al , 2006)), thermo-responsive biopolymers (e.g., elastin-like polypeptides (Sheth, Bhut, et al , 2014)), and hydrophobic nanoparticles (e.g., polyhydroxyalkanoates (Banki et al , 2005), oleosins (Bhatla et al , 2010), hydrophobins (Jugler et al , 2020)). Fusion tags have by and large been the most widely adopted biotic purification technology for the accessibility they present to early-stage research labs.…”

Evaluating the cost of pharmaceutical purification for a long-duration space exploration medical foundry

“…The simplicity of production, reagents that can be produced using self-replicating organisms, and potential recyclability of spent consumables are significant advantages of biological purification technology for space or other limited resource applications. Examples of primary biological technologies include fusion tags 20 , stimuli-responsive biopolymers 21 , hydrophobic nanoparticles 22 , and plant virus nanoparticles 23,24 .…”