Strategies for Enhancing Monoclonal Antibody Accumulation in Plant Cell and Organ Cultures

Abstract

Various strategies aimed at improving IgG(1) antibody accumulation in transgenic tobacco cell and organ cultures were tested. The form of tissue had a significant effect on antibody levels; shooty teratomas were less productive than hairy roots or suspended cells. Although there were several disadvantages associated with hairy roots compared with suspensions, such as slower growth, slower antibody production, and formation of a greater number of antibody fragments, the roots exhibited superior long-term culture stability. Antibody accumulation in hairy root cultures was improved by increasing the dissolved oxygen tension to 150% air saturation, indicating the need for effective oxygen transfer in root reactors used for antibody production. Preventing N-linked glycosylation using tunicamycin or inhibition of subsequent glycan processing by castanospermine reduced antibody accumulation in the biomass and/or medium in cell suspensions. Loss of antibody from the cultures after its secretion and release into the medium was identified as a major problem. This effect was minimized by inhibiting protein transport in the secretory pathway using Brefeldin A, resulting in antibody accumulation levels up to 2.7 times those in untreated cells. Strategies for protecting secreted antibody, such as addition of poly(vinylpyrrolidone) and periodic harvesting from the medium using hydroxyapatite resin, also increased antibody titers. The mechanisms responsible for the disappearance of antibody from plant culture media were not clearly identified; degradation by proteases and conformational modification of the antibody, such as formation of aggregates, provided an explanation for some but not all the phenomena observed. This work demonstrates that the manipulation and control of culture conditions and metabolic processes in plant tissue cultures can be used to improve the production of foreign proteins. However, loss of secreted antibody from plant culture medium is a significant problem that may limit the feasibility of using product recovery from the medium to reduce downstream processing costs relative to agricultural systems.

Expression of the antibody 14D9 in Nicotiana tabacum hairy roots

“…secretion of protein by using the KDEL signal peptide for retention in the ER, has been found to minimize antibody fragmentation and improve antibody accumulation in plant cells (Tsoi and Doran, 2002). Also proteases in the secretory pathway and in the apoplast are avoided (Sharp and Doran, 2001). In the culture medium the highest values of antibodies were about 1% of the amounts reached in the biomass for both clones (Figure 2).…”

“…Considering all the proteins that have been produced in plants, perhaps the most promising ones are monoclonal antibodies and antibody fragments. However, there have been few reports on antibody production by hairy root cultures (Wongsamuth and Doran, 1997;Sharp and Doran, 2001). The aim of this work is to study the expression of the 14D9 antibody in Nicotiana tabacum hairy root cultures.…”

Role of plant expression systems in antibody production for passive immunization

“…Antibodies have been produced in moss (Decker and Reski, 2008), microalgae , duckweed (Cox et al, 2006), plant cells (Hellwig et al, 2004), organ cultures (Hellwig et al, 2004, Sharp andDoran, 2001) and land plantsboth monocots (like maize) and dicots (like Arabidopsis, tobacco, potato, soybean, alfalfa) (De Muynck et al, 2010). All autotrophic plants have a relatively similar cellular machinery and glycosylation pattern, and most can be transformed by Agrobacterium-mediated transformation or by particle bombardment, two methods of which the former is preferred, as it tends to introduce a low T-DNA copy number in the plant genome (Cheng et al, 2004, Ko andKoprowski, 2005).…”

Transgenic Plants as Biofactories for the Production of Biopharmaceuticals: A Case Study of Human Placental Lactogen

“…Shooty teratomas are generated from transgenic seedlings co-cultured with A. tumefaciens strain T37. After coculture and Agrobacteria elimination, shoots are cultured in liquid medium (Sharp & Doran, 2001). …”