Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures

Abstract

Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L(-1) in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…Investigation of these factors has been reviewed previously [55]. Further evidence for the destruction of secreted proteins by medium proteases and adsorptive losses from plant suspension cultures in glass, plastic and stainless steel vessels has since been reported [25,38,59,79,80]. Adsorption is a major factor affecting protein stability in dilute solutions; however, the interaction of proteins with solid surfaces and particles can cause other undesirable responses such as protein aggregation and oxidation [81].…”

“…Additional techniques based on the optimisation of medium conditions such as pH and rapid or semi/ continuous harvesting of product have also been successful [38,55,82]. The benefits of using hydrophilic/hydrophobic and charged surfaces such as glass, plastic and stainless steel to suppress adsorption depend on the protein characteristics and medium properties [80,83]. As relatively large molecules with low internal stability [84], IgG antibodies are known to be particularly susceptible to adsorption onto all surfaces irrespective of any electrostatic interactions [84,85].…”

Elucidating Rice Cell Metabolism under Flooding and Drought Stresses Using Flux-Based Modeling and Analysis

“…On the other hand, cells supplemented with Suc under anoxia showed a delayed increase in pH of the medium (Fig. 1B), possibly suggesting that the external invertase activity is dependent upon the presence of oxygen (Kwon et al, 2012). To explain this phenomenon clearly, further studies are required where the correlations among cell growth, invertase activity, and energy consumption must be investigated thoroughly.…”

Improving Pharmaceutical Protein Production in Oryza sativa

“…Thus, disposable, smaller volume, bioreactors using plastic containers have become increasingly more popular in animal suspension cells [110]. In addition, there have been studies indicating that protein loss involved in suspension cell cultures may be associated with adsorptive properties of glassware, indicating that material used as bioreactor tank may also play important roles in maximizing protein yield [111]. An air-lift bioreactor using a 5-liter plastic container has been utilized to produce mouse GM-CSF in rice cells [25].…”