Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120-and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.

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.

No abstract

No abstract