Production of monoclonal antibodies by tobacco hairy roots

Abstract

Hairy roots of tobacco (Nicotiana tabacum) were used to produce full‐length murine lgG1 monoclonal antibody. The presence of heavy (γ) and light (κ) chains and fully assembled antibody was verified by Western blot analysis of root extracts. Antibody levels in the biomass and medium were quantified by ELISA based on detection of γ‐κ complexes. Antibody produced by hairy roots was fully functional as demonstrated in bacterial aggregation assays which confirmed bivalent antigen‐binding capacity. Eight antibody‐producing hairy root clones retained their ability to produce mouse immunoglobulin over a period of 19 months after transformation with Agrobacterium rhizogenes. For hairy roots grown in Gamborg's B5 medium, the maximum level of assembled antibody after 21‐day culture in shake flasks was 18 mg L−1 or 1.8% total soluble protein; up to 14% of the antibody was secreted into the medium. Antibody production by transgenic hairy roots had a negligible effect on growth compared with hairy roots of wild‐type tobacco. Antibody accumulation was growth associated with constant specific accumulation rate at the beginning of the culture; however, degradation of antibody was significant after 14 days and the amount of assembled antibody declined. Unlike hybridoma cultures, the time course of antibody accumulation by hairy roots showed a distinctive maximum very soon after the end of exponential growth. Total antibody levels were increased by addition of nitrate, polyvinylpyrrolidone, or gelatin to the medium. Polyvinylpyrrolidone and gelatin also markedly improved extracellular antibody concentrations; with these treatments, up to 43% of the antibody present was secreted into the medium. Antibody production was tested using hairy roots grown in an air‐driven bioreactor. The intracellular antibody content after 30‐day bioreactor culture was similar to that measured in shake flasks; however, the final extracellular antibody level was 1.7 times higher than the maximum measured in shake flasks. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 401–415, 1997.

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells

“…In the extracellular medium (intercellular fluid of leaves and culture medium of BY-2 cells), the yield was dramatically low. For instance, less than 200 lg/l antibody was found in the BY-2 culture medium while a yield of 1.99 mg/l was obtained for another antibody secreted from N. tabacum suspension cells derived from transgenic hairy roots (Sharp and Doran 1999;Wongsamuth and Doran 1997). A reason might be that different antibodies might behave differently regarding their transport out of the cell.…”

“…There are a few reports comparing antibody expression between transgenic leaves or hairy roots and cell suspensions derived from transgenic plants (De Neve et al 1993;Sharp and Doran 1999;Van Engelen et al 1994;Wongsamuth and Doran 1997). However, to our knowledge, there has been no report of a side-by-side comparison of the degradation, glycosylation and in situ localization of the same full size antibody expressed in both plant and suspension cells.…”

Hairy root biotechnology—indicative timeline to understand missing links and future outlook

“…At present, different categories of recombinant proteins like antibodies, cytokines, vaccines, and enzymes are known to be expressed and produced by HRCs of various plants (Obembe et al 2011;Georgiev et al 2012). The first ever report of HRC-based pharmaceutical protein production was of fulllength murine IgG1 antibody from tobacco hairy roots (Wongsamuth and Doran 1997). In comparison to other (bio) production platforms, the HRCs provide (1) assurance of climate-and contamination-free production of pharmacologically active proteins and (2) easy acetylation, phosphorylation, glycosylation, and other post-translational modifications because of their eukaryotic molecular organization.…”

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

“…Most research in this area has focused on the use of undifferentiated plant cultures such as cell suspensions rather than roots and shoots. Yet, hairy roots and shooty teratomas have been demonstrated to be effective host culture systems for recombinant protein synthesis (Wongsamuth and Doran, 1997;Sharp and Doran, 2001a,b;Jin et al, 2005), with potential advantages including greater long-term stability of transgene expression (Sharp and Doran, 2001b). Hairy roots are characterised by fast growth rates compared with whole plants and are more genetically stable than suspended cells (Aird et al, 1988;Flores, 1987).…”