Investigation of sorption capacity of dye-affinity sorbents in the process of purification of factor VIII coagulation
Background. Chromatographic methods, in particular affinity chromatography, are the most effective in obtaining highly purified preparations of plasma proteins. The active triazine and vinyl sulfone dyes are the special group of ligands for affinity chromatograph. We found that the dye-affinity sorbents bind non-target to factor VIII (FVIII) proteins in the previous stages of the study. The sorption capacity of the affinity sorbent is defined as the maximum amount of sample (protein) that can bind to the column under certain conditions. Determining the adsorption limit or limiting capacity of the sorbent makes it possible to identify the feasibility of its use to extract a certain type of protein.
Objective. To study the sorption capacity of various dye-ligand affinity sorbents in the process of purification of FVIII.
Materials and methods. We used next sorbents: Diasorb-Procion Blue HB, Diasorb-Procion Gelb M4R and Diasorb-Procion Blue MXR. The cryoprecipitate was initial material. The total protein concentration was determined by the Bradford method, the activity of factors VIII – one-stage clotting method.
Results and discussion. Sorption of non-target proteins and FVIII activity were investigated after preparation of a number of dilutions of the initial solution of cryoprecipitate. Different concentrations of protein were applied per 1 cm3 of sorbent to select the optimal concentration and do not to oversaturate the column: I – 19.74±0.20 mg of protein/ml; II – 7.94±0.05 mg of protein/ml; III – 3.97±0.05 mg of protein/ml; IV – 1.96±0.04 mg of protein/ml. The maximum sorption capacity among the studied sorbents was 14.62±0.04 mg of protein / 1 cm3 for of sorbent Diasorb-Procion Blue HB. It was found that to achieve maximum purification of FVIII (highest specific activity), the optimal concentration of protein to 1 ml of sorbent should be in the range of 4-8 mg of protein / 1 cm3 of sorbent. The highest degree of purification for these sorbents was 19.65 times at an initial protein concentration of about 4 mg protein/ml (p≤0.01).
Conclusions. The sorption capacity of sorbents was calculated. It was demonstrated that the maximum sorption capacity is approximately 15 (14.62±0.04) mg of protein / 1 cm3 of sorbent.
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