Zieliński Marcin, Romanik-Chruścielewska Agnieszka, Mikiewicz Diana, Łukasiewicz Natalia, Sokołowska Iwona, Antosik Jarosław, Sobolewska-Ruta Agnieszka, Bierczyńska-Krzysik Anna, Zaleski Piotr, Płucienniczak Andrzej
Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa, 02-516, Poland.
Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa, 02-516, Poland.
Protein Expr Purif. 2019 May;157:63-69. doi: 10.1016/j.pep.2019.02.002. Epub 2019 Feb 5.
The number of people with diabetes is estimated to be over 370 million, in 2030 it will increase to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%). According to the estimates of the International Diabetes Federation, the percentage of patients in the adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of pharmacotherapy in cases of extremely high hyperglycemia. A new bacterial host strain (Escherichia coli 20) was obtained at the Institute of Biotechnology and Antibiotics and a new pIBAINS expression vector was constructed that provides greater efficiency in the production of recombinant human insulin. In the IBA Bioengineering Department, successful attempts were made to produce recombinant human insulin on a laboratory and quarter-technical scale, and several batches were performed on a semi-technical scale. The production process has been divided into several stages: 1. biosynthesis of insulin in the fermenter, 2. isolation, purification and dissolution of inclusion bodies, 3. protein renaturation, 4. enzymatic reaction with trypsin, 5. multi-stage purification of insulin using low-pressure and HPLC techniques. At each stage of insulin production, qualitative and quantitative analyses were performed to confirm identity and purity. In particular, the molecular weight of insulin, the amount of insulin and the content of protein impurities were studied. The results of these experiments are presented in this work.
据估计，糖尿病患者人数超过3.7亿，到2030年将增至5.52亿。在波兰，糖尿病患者人数估计为350万（占9.1%）。根据国际糖尿病联合会的估计，在未来20年里，波兰成年人口中患者的比例将增至约11%。尽管胰岛素类似物已出现在药品市场上，但在极高血糖情况下，胰岛素给药仍是最有效的药物治疗方法。生物技术与抗生素研究所获得了一种新的细菌宿主菌株（大肠杆菌20），并构建了一种新的pIBAINS表达载体，该载体在重组人胰岛素生产中具有更高的效率。在IBA生物工程部门，已成功尝试在实验室和中试规模上生产重组人胰岛素，并在半工业规模上进行了几批生产。生产过程分为几个阶段：1. 在发酵罐中进行胰岛素的生物合成；2. 包涵体的分离、纯化和溶解；3. 蛋白质复性；4. 用胰蛋白酶进行酶促反应；5. 使用低压和高效液相色谱技术对胰岛素进行多阶段纯化。在胰岛素生产的每个阶段都进行了定性和定量分析，以确认其身份和纯度。特别是，研究了胰岛素的分子量、胰岛素的含量和蛋白质杂质的含量。这些实验的结果在本研究中呈现。
