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The Assessment of Flaxseed Protein Hydrolysates as Media Supplement for CHO Cell Growth and IgG Production

Background and Novelty Plant protein hydrolysates have a long history of being used as cell culture media supplement rich in non-animal peptides. Flaxseed oil cake, a byproduct of flaxseed oil extraction, contains around 30% proteins, and as such can be considered for the same purpose. The majority of high-value therapeutic proteins today are produced with CHO cells, preferably in animal component-free media. Therefore, this study aimed to screen for the effects of flaxseed protein hydrolysates, put as additives in chemically defined media (CDM), on the growth and IgG production of CHO cells. To our knowledge, this is the first report on using such hydrolysates as media supplements in CHO cell culture. Experimental approach Three different flaxseed protein hydrolysates (HX) were produced by three separate enzymatic digestions of proteins isolated from flaxseed oil cake. The enzymes used were microbial endopeptidases ; Alcalase (A), Neutrase (N), and Protamex (P). A portion of each HX was further processed for the size fractionation by centrifugal filter units (<10kDa and <1kDa). IgG- producing CHO cells were cultivated in CDM in 125 mL shake flasks. The initial assessment comprised of growing cells in media with our three hydrolysates: HA, HN, and HP at 0.5 g/L and 2 g/L. In the following assessment, the effects of only small peptides was to be tested, therefore the hydrolysate peptide fractions (HXF1 and HXF10) were supplemented to the media. In both assessments, the media without supplements served as a control condition. The cell density and viability were monitored by the trypan blue exclusion method with hemocytometer. The IgG titer was determined by HPLC using Protein A affinity chromatography column. The metabolite concentrations (glucose, lactate, ammonia and amino acids) were determined either by HPLC or using a spectrophotometer. Results and Discussion The media supplementation with HA, HN, and HP had no significant impact neither on viable cell number nor IgG production, comparatively to the control. Moreover, certain conditions had a negative effect on the cell growth and productivity. Considering this unfavourable outcome, together with the data from some earlier research, we decided to test only the smaller peptide fractions (HXF1 and HXF10). This new condition again had no positive effect on the cell growth, but at least it improved IgG production. Media supplementation with HNF1 and HNF10 at 0.5 g/L gave the highest IgG titer when compared to the control and all other conditions. Obtained results showed that adding low doses of small peptides in CDM can positively affect CHO productivity. More investigation is required to understand and standardize these effects.

CHO cells, hydrolysates, IgG, production

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