Plant production of a tumour-targeting human IgG. Considerations on expression strategies, glycosylation profile and in planta proteolysis

Abstract

Compared to other expression platforms, plants present several advantages for the expression of recombinant antibodies such as the ability to assemble multimers, the possibility to achieve posttranslational modifications similar to those found in mammalian cells, low management costs and ease of scalability. Despite these advantages, some drawbacks need to be addressed. One of the limits of the plant expression systems, is represented by downstream purification steps, in which many factors, such as low antibody concentration, extraction methodologies and purification strategy, could influence the effectiveness of the purification. Another important issue to be addressed in the use of plants for antibody expression, is represented by plant glycosylation. The considerable differences between the sugar moieties harbored by proteins expressed in plants and mammals, represents a major drawback in the application of the plant-derived immunoglobulins in human therapy since the possible immunogenicity of these sugar moieties. An additional drawback of recombinant antibodies obtained from plants is represented by the final recovery yield, that can be, in some cases, reduced by the unintended proteolytic process taking place in the plant cell machinery. Particularly, in the case of antibody expression, as reported in many studies, degradation could produce a protein reduction of about 90 % of the final purified product. In this work, we described the expression of the human recombinant antitumoral antibody H10 in Nicotiana benthamiana using a transient expression system based on vacuum agroinfiltration, focusing our research efforts on the above mentioned issues: extraction and purification strategies, glycosylation profile and proteolysis. The first part of the work emphasized that the application of different extraction methodologies was able to significantly affect the integrity and yield of the IgG H10 antibody purified from agroinfiltrated N. benthamiana leaves. Based on the data collected, we optimized a pilot-scale purification method, using a two-step purification protocol from 250 g of fresh agroinfiltrated leaves obtaining an average yield of 40 mg per Kg fresh weight of intact IgG with 99.4 % purity, with endotoxin levels < 1 EU/ml, free of phenolic, alkaloid compounds. In the second part, the glycosylation pattern of plant produced H10 was modulated using three different strategies in order to study the effect of the different glycosylation profiles on antibody quality and plant expression yield. Finally, the proteolysis of the plant-derived IgG H10 was studied in order to analyze in planta antibody degradation profile and to identify specific cleavage sites of the immunoglobulin. Furthermore, an in silico analysis permitted the identification of proteases responsible of the cleavage. Data herein represent valid a contribution in the field of plant-made antibodies, and intend to provide new information in the molecular pharming area. Our research efforts aimed to confirm the possibility to over-express immunoglobulins with a “human like” glycosylation profile in N. benthamiana plants, reducing the unintended plant proteolysis to increase the final yield of intact IgG.