Molecular farming of tobacco plants provides a viable option over traditional microbial and mammalian cell culture technologies for production of recombinant proteins, allowing for cost effective, highly scalable and safe production of recombinant therapeutic proteins. The current work is focused on production, purification and N-linked glycosylation pattern of recombinant anthrax receptor fusion protein (CMG2-Fc), transiently expressed in Nicotiana benthamiana. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). Fc-fusion proteins have been intensely investigated for their effectiveness to control a range of pathologies, with several remarkable recent successes coming to market. These promising outcomes have stimulated the development of novel approaches to improve their efficacy and safety, however the influence of bioprocessing conditions on the glycosylation of the Fc-fusion protein is being investigated to improve drug efficacy. In this work Agrobacterium tumefaciens harboring the CMG2-Fc gene under the control of the constitutive CaMV 35S promoter system is delivered into Nicotiana benthamiana plants via vacuum agroinfiltration. A viral RNA gene silencing suppressor (P19) is co-expressed to improve CMG2-Fc production. Purification of target molecule from the leafy biomass was starts with liquid nitrogen extraction using PBS buffer followed by sequential filtration steps. A microfiltration step performed with a 0.22 µm filter followed by ultrafiltration with a 30 kDa MWCO filter was used to prepare the extract for Protein A affinity chromatography. The purity and quantity of the target protein was assessed using SDS-PAGE and ELISA respectively, and its molecular weight was confirmed with Western blot. Site specific glycoform analyses are performed using UPLC system coupled to a triple quadrupole mass spectrometer at each step in the purification process to determine how downstream bioprocessing operations impact glycosylation.

Influence of downstream processing on the glycosylation of recombinant anthrax receptor fusion protein transiently expressed in <i>Nicotiana benthamiana</i>

251st National Meeting (2016)

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Influence of downstream processing on the glycosylation of recombinant anthrax receptor fusion protein transiently expressed in Nicotiana benthamiana