Separation Characteristics of Biopolymers by High-Performance Membrane Chromatography

초록

Variants of biopolymers are generated during downstream processing by deamidation, oxidation, proteolysis, nicking and aggregation. The fraction of degradation products increases with residence time, so shorter process times can net higher recoveries and product purity. From this light, high-performance membrane chromatography (HPMC) is a useful separation method. To achieve the separation under gradient conditions, multiple steps of adsorption-desorption process are needed and therefore conventional chromatographic columns with long and narrow layer of separation material were considered as a necessary tool for achieving this effect. Recently, CIM (Convective Interaction Media)disk shaped chromatographic columns are shortest, having a length of only 3mm. They were mainly developed for gradient separation of lager biopolymers. Separation mechanism was anion-exchange, and the stationary phase was anion CIM DEAE disk(16×3 mm). In this work, biopolymers were separated by HPMC, a combined system of chromatography and membrane. The samples used in this experiment were oligodeoxynucleotides of 8, 10, 12, 14, 15, 16 mers and proteins of myoglobin, conalbumin and soybean trypsin inhibitor. Two types of mobile phase were utilized, buffer A (20mM Tris-HCI, pH 7.4) and buffer B (buffer A + 1M NaCl). As the amount of NaCl dissolved in the mobile phase linearly increased, the retention time was shorter, which enabled the gradient elution mode. Based on the number of theoretical plates and resolution of the samples with different conditions of mobile phases, the optimum mobile phase and operating condition were experimentally obtained. In this experimental condition, the biopolymers of oligodeoxynucleotides or proteins could be separated within 2 min at 4-6 ml/min of mobile phase flow rate.