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.
Highlights
Monoclonal antibodies have dramatically transformed how we approach the treatment of a myriad of diseases [1]
The temporal expression pattern of Hydrophobin-Protein A fusion protein (HPA) is similar to that of other proteins using MagnICON vectors [21], increasing up until 8–10 days post infiltration (DPI), where the protein level peaked at 218.7–272.9 μg/g fresh leaf weight (FLW) (Figure 1)
We built upon the novel concept of utilizing a bifunctional fusion protein to capture a Monoclonal antibodies (mAbs) in an aqueous two-phase separation (ATPS) to reduce downstream costs of purifying mAbs from plants
Summary
Monoclonal antibodies (mAbs) have dramatically transformed how we approach the treatment of a myriad of diseases [1]. The presence of these contaminating proteins and compounds can be reduced or eliminated by combining multiple and varying chromatography steps, prolonging the longevity of the Protein A resin of the initial capture step remains a challenge This calls for the development of alternative strategies that can effectively enrich mAbs from plant extracts at a low cost. We utilized the same bifunctional protein in a water-surfactant two-phase system to serve as an initial capture step in purifying plant-made mAbs. To achieve this, we transiently expressed two protein molecules separately in Nicotiana benthamiana: an anti-West Nile virus (WNV) mAb, E16, and the bifunctional fusion protein (HPA) described above, containing the antibody-binding domain of Protein A and the hydrophobin tag. ATPS based on plant-produced HPA in unpurified extract has potential to change the way that plant-made mAb purification is approached
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