Study On The Separation Medium And Condition Of The Preparation Of Insulin

Diabetes mellitus is a metabolic disease characterized by hyperglycemia.There are 110 million diabetic patients in China.20% to 30% of them need insulin injection to control blood sugar.Therapeutic insulin is produced predominantly with genetic engineering technology and purified by a series of operations.Chromatography is the most widely used one.The core of chromatography column is the chromatographic stationary phase,so the choice of chromatography packing is the critical of protein preparation separation process study.There are several separation modes of preparative liquid chromatography base on the different packing materials,the most common ones are reversed-phase high performance liquid chromatography(RPLC),ion exchange chromatography(IEC),hydrophobic interaction chromatography(HIC),affinity chromatography(AFC).The suitable mode can be selected according to the characters of the protein molecule,such as size and shape,special structure,electric charge and hydrophobicity etc.,as well as the source and separation purpose.The separation and purification of the whole protein at the preparation scale has the invaluable value for accelerating the downstream industrialization of bioengineering and solving the problem of sample preparation & separation in proteomics research.Therefore,it is necessary to study the suitability of chromatographic packing materials and the effect on the preparation & separation process caused by different column equipment and different process conditions.In this paper,we will combine with the author’s work to study the effects of different packing materials,chromatographic column equipment and process conditions on the preparative chromatography separation process of insulin,and come to the conclusion.(1)The separation efficiency of four types of fillers was compared.Two of them(particle size of 10 u M and pore size of 100 ?)could be re-used up to 120 times,and tolerant to alkali washing up to 10 times.The percentage of qualified production could reach 86.62%.These two types of fillers could significantly reduce the cost of insulin production.(2)The effects of two distributors and various heights of column bed on chromatographic results were studied.Stainless steel sieve plate and column bed height of 25-30 cm is an optimal combination for insulation purification.82.01% of insulin purified with this combination met approval standard.(3)The effects of salt type,salt concentration,p H value,acid type,isopropanol concentration and temperature on ion exchange and reverse phase chromatography were studied.The purification effect of low-cost strong electrolyte salt was better than that of high-valent salt.The peak tailing removal effect of TFA was better than that of phosphoric acid.The separation effect could be improved with high p H values(higher than p I value)and high temperature.However,temperature > 25℃ and p H > 8.5 could lead to degradation of insulin.