Optimizing interleukin-6 and 8 expression, clarification and purification in plant cell packs and plants for application in advanced therapy medicinal products and cellular agriculture

Journal of Biotechnology, vol. 390, pp. 1–12

Abstract

Healthcare and nutrition are facing a paradigm shift in light of advanced therapy medicinal products (ATMPs) and cellular agriculture options respectively. Both options heavily rely on some sort of animal cell culture, e.g. autologous stem cells. These cultures require various growth factors, such as interleukin-6 and 8 (IL-6/8), in a pure, safe and sustainable form that can be provided in a scalable manner. Plants seem well suited for this task because purification of small proteins can be readily achieved by membrane separation, human/animal pathogens do not replicate in plants and production can be scaled up using in-door farming or agricultural practices. Here, we illustrate this capacity by first optimizing the codon usage of IL-6/8 for translation in Nicotiana spp., as well as testing the effect of untranslated regions and product targeting to different sub-cellular compartments on expression in a high-throughput plant cell pack (PCP) assay. In the chloroplast, IL-6 accumulated up to 6.9±3.8 (SD, n=2) and 14.4±7.4 mg kg-1 (SD, n=5) were observed in case of IL-8. When transferring IL-8 expression into whole plants, accumulation was 12.3±1.5 mg kg-1 (SD, n=3). After extraction and clarification, IL-8 was purified using a two-stage process consisting of an ultrafiltration/diafiltration step with 100 kDa and 10 kDa cut off membranes followed by an IMAC polishing step. The purity, yield and recovery were 97.8%, 6.6 mg kg-1 and 38%, respectively. We evaluated the ability of the proposed purification process to remove endotoxins to ensure the compatibility of plant-made growth factors with cell culture.

Authors 2

1. Patrick Opdensteinen Aachen Fraunhofer Institute for Molecular Biology and Applied Ecology IME Institute for Molecular Biotechnology

   RWTH Aachen University · Fraunhofer Institute for Molecular Biology and Applied Ecology

   Affiliation as printed

   Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen 52074, Germany; Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, Aachen 52074, Germany

   Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany

   Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, 52074 Aachen, Germany

2. Johannes F. Buyel corresponding

   BOKU University

   Affiliation as printed

   University of Natural Resources and Life Sciences, Vienna (BOKU), Department of Biotechnology (DBT), Institute of Bioprocess Science and Engineering (IBSE), Muthgasse 18, Vienna A-1190, Austria. Electronic address: johannes.buyel@rwth-aachen.de

   University of Natural Resources and Life Sciences, Vienna (BOKU), Department of Biotechnology (DBT), Institute of Bioprocess Science and Engineering (IBSE), Muthgasse 18, A-1190 Vienna, Austria