Influence of the KDEL signal, DMSO and mannitol on the production of the recombinant antibody 14D9 by long-term Nicotiana tabacum cell suspension culture

Abstract

We have established two transgenic cell suspension culture lines of Nicotiana tabacum that express the catalytic antibody 14D9 as a secretory product (sec-Ab) or as a KDEL-tagged product in the endoplasmic reticulum (Ab-KDEL), respectively. After 3 years of culture, the performance improved to a production level of 0.15 ± 0.03 lg ml -1 on the seventh day of culture for the sec-Ab line and 0.48 ± 0.05 lg ml -1 on the third day for Ab-KDEL line. Analysis of the effect of osmotic stress using mannitol (90 g l -1 ) as an osmolite revealed that there was a 12-fold increase in antibody yield (1.96 ± 0.20 lg ml -1 ) on the seventh day of culture in line sec-Ab and a fivefold increase (2.31 ± 0.18 lg ml -1 ) on the seventh day for line Ab-KDEL. The concentration of the antibody in the culture medium was not significant. Dimethyl sulfoxide used as a permeabilizing agent was not effective in increasing 14D9 yield, but it did cause distinctive cell damage at all concentrations tested.

Cis-regulatory elements used to control gene expression in plants

“…For instance, KDEL was applied to 14D9 mouse antibodies production in cell suspension cultures of N. tabacum. The level of accumulation increased fivefold in comparison to the wild type (López et al 2010). Similar studies on KDEL were focused on the 7Crp protein, a Japanese cedar pollen allergen.…”

“…KDEL signal directs proteins to the ER, where many proteins are accumulated in complexes, especially during the seeds maturation. KDEL presence provides protein protection from unfavorable cytoplasm environment inter alia proteases and oxidation or reduction processes (López et al 2010). Recombinant protein labelling with KDEL upon production allows to precisely determine the protein localization, ensures stability and facilitates purification (Ma and Wang 2012).…”

“…(Martinez et al 2011) Production of a phytase in B. napus (Peng et al 2006) Production of a mouse antibodies 14D9 in N. tabacum (López et al 2010) Production of a 7Crp vaccine against alergenes using O. sativa (Takaiwa et al 2009) …”

“…The wide implementation of novel methods depends on the economic benefits and public opinion. So far, plants have been modified to produce vaccines, hormones, enzymes and antibodies (Shaaltiel et al 2007;Rabindran et al 2009;López et al 2010;Sirko et al 2011;Ma and Wang 2012;Madanala et al 2015).…”

“…Furthermore, infection of the product by mammalian pathogens is avoided (Tekoah et al 2015). Unlike mammalian cells or microorganisms, use of plants allows a substantial increase in production scale due to product accumulation in given localization, primarily in the ER (López et al 2010;Martinez et al 2011), as well as by enlarging the culture volume or acreage. However, systems based on plants are not without disadvantages since they require longer production further protein transport and accumulation efficiency (Li et al 2012).…”