Fast Purification Of Insulin From Bovine Pancreas Extract By Two-dimensional Liquid Chromatography Using A Single Column

Insulin is the specific medicine for diabetes therapy. Clinic insulin sources from recombinant human insulin, semi-synthetic human insulin and animal insulin. No matter which method is used, a series of separation and purification procedures are needed to acquire highly pure (>99%) insulin. Two-and multi-dimensional liquid chromatography (2D-LC and mD-LC) show broad biopharmaceutical application prospects for their strong separation power. High yield, short processing technology time and easily maintaining bioactivity of proteins are required in those procedures. Conventional 2D-LC and mD-LC need two or more chromatographic columns. While 2D-LC using a single column (2D-LC-1C) is a special mixed-mode chromatography (MMC) which has some special advantages. Compared with single mode columns, the two-dimensional column (2D column) applied in 2D-LC-1C could work in two different modes and shows higher separation power and higher selectivity, and it can save the consuming of packing materials as well. Therefore, it has unique advantages in both analytical and preparative chromatography. In view of the physicochemical property of insulin, we preliminarily established a simplest on-line 2D-LC system without fractions collection, reinjection and extra buffer exchange systems in this research. We got highly pure insulin from bovine pancreas extract by applying the novel method, which laid the foundation for further industrialization.The thesis includes the following four sections:1. Review:The development history, characteristics, sorts and combinations of MMC, explanations for its mechanisms, and its applications on biomacromolecules were introduced and reviewed in detail.38 references were cited,50% of which were reported in the last 3 years.2. Experimental methods:all the equipments, reagents and operations applied in this experiment are included.3. We optimized the chromatographic conditions for the separation of insulin on a SAX/HIC (Strong-anion Exchange/Hydrophobic Interaction Chromatography) 2D column whose ligand contains 3 ethyl, a quaternary ammonium and a hydroxyl. Under the optimum condition, we obtained insulin with high purity from bovine pancreas extract via off-line SAX-HIC 2D-LC-1C. While the purity is not enough.4. Based on the last chapter, we selected another SAX/HIC 2D column whose ligand contains 3 n-octyl, a quaternary ammonium and a hydroxyl to improve the purity of insulin. Two mobile phases were found for both SAX and HIC modes in which insulin could not be eluted under gradient elution while other standard proteins could be eluted, so it has excellent selectivity over insulin. Insulin was eluted in the last dimension by RPLC and the product was further purified as well. Therefore, the on-line collection, buffer exchange and re-injection systems and corresponding operations needed in conventional mD-LC could be omitted and thus a simplest mD-LC system was preliminarily established. We can get highly pure insulin in 17-32 min by applying this novel system to purify insulin from bovine pancreas extract by means of SAX-RPLC, HIC-RPLC and SAX-HIC-RPLC modes, and mass recovery is between 85.4% and 120.6%.