Investigation To Conversion Process Of Recombinant Insulin Precursors To Human Insulin And Insulin Detemir

Insulin and its analogues are the most effective drugs to treat diabetes directly. Developing insulin and new analogs those are safe, effective and easy-to-use has been a hotspot of biological drug development. Based on a Pichia pastoris strain previously constructed in our laboratory that could efficiently express insulin precursor, the following studies were conducted:large scale fermentation of this strain, molecule structure and cleavage order of the insulin precursor, digestion and transpeptidation of insulin precursor to form desB30(human insulin with deletion of threonineB30) and human insulin, and selective one-step acylation of free ε-amino group of B29in desB30. The work would help to build the practical process for the dustrial productions of human insulin and insulin detemir.The large-scale fermentation process of Pichia pastoris for the production of insulin precursor was established. A capillary gas chromatography was used to monitor the methanol concentration in fermentation broth, with short measurement time, responsive signal and low error not more than1%. Combining with DO spike method, the precise control of methanol feeding in fermentation process was achieved. The feed and induction techniques were successfully scaled up to300L fermentor, and the insulin precursor expression in the system could be more than3.0g/L, comparable to that in small pilot scale. At the same time, a preliminary purification process was set up to purify the insulin precursor using CM-Sepharose FF ion-exchange chromatography, by which the insulin precursor was isolated from the fermentation supernatant rich in pigments, with the purity of88%and the protein recovery ratio of more than95%. This served as the foundation for the structure analysis of insulin precursor and the establishment of the digestion, transpeptidation and acylation process of insulin precursor.Insulin precursor expressed in Pichia pastoris is a single-chain protein with a spacer peptide (EEAEAEAEPK) localized at its N-terminal. The heterogeneity phenomenon on the N-terminal of insulin precursor produced by Pichia pastoris was found. The phenomenon was presumed to be caused by the following reasons. To increase the insulin precursor expression, a synthetic spacer peptide sequence (EEAEAEAEPK) was introduced into the N-terminus of target protein when the Pichia pastoris was constructed. Because the low specificity of a dipeptidyl aminopeptidase A encoded by the STE13gene in Pichia pastoris towards the restriction sites, the N-terminus of the insulin precursor was digested at several positions to obtain various kinds of heterologous products. At the same time, it was confirmed that the degradation of C-terminal of insulin precursor did not occur during the fermentation process.In trypsination, the digestion order of three restriction sites on the single-chain insulin precursor protein was sorted out. It was found by HPLC and LC-MS analyses that the spacer peptide fragment on insulin precursor was rapidly removed by trypsin to generate a single chain insulin precursor, subsequently the peptide bond behind the connecting peptide AAK on the single-chain insulin was digested to generate double-chain insulin precursor, and finally, the linker peptide AAK was removed from double-chain product to form the insulin desB30. However the three cleavage steps varied on the speed greatly. The first step in digestion was fast, the second step was relatively slow and the third step was so slow that only80%of the double-chain insulin precursor could be transformed into desB30after overnight digestion. Based on the three-dimensional structure and the properties, insulin molecules were easy to form dimers, and the cleavage sequence and digestion rate were identified to be related to the dimerization and polymerization. If the polarity of the solution was reduced to form a hydrophobic environment with a low concentration of organic solvent, the dimer content of insulin precursors and double-chain insulin precursors was significantly decreased from far-UV circular dichroism measurement. When the digestion was carried out in the solution with a low concentration of organic solvent, the conversion ratio of insulin precursor to desB30increased from76.8%to95.6%, which also indirectly confirmed the steric hindrance of dimmers of the double-chain insulin precursors was the main cause for the low conversion ratio.A two-step transpeptidation process was established to convert insulin precursor to human insulin ester. In one-step transpeptidation process used previously, insulin precursor had to be digested three times in hydrophobic environment to generate desB30and then followed by an enzymatic catalyzed coupling of threonine ester to the terminal lysineB29residue of desB30to generate human insulin ester. The low cleavage conversion of insulin precursor to desB30in this environment resulted in a total transpeptidation conversion of only43.9%. Actually, under the effect of trypsin, cleavage and coupling was a reversible reaction. The cleavage reaction hydrolyzed the peptide bond, while the coupling reaction connected the peptide bond. However, in this reversible reaction, the optimal conditions for each reaction were greatly different. In the present study, the two reactions were conducted in separate processes, and the one-step transpeptidation was replaced by a two-step transpeptidation to convert human insulin precursor into human insulin ester. Thus, two reactions could be carried out under their respective optimum conditions. As results, the final transpeptidation conversion was nearly doubled with the reaction time shortened to one tenth and the usages of threonine ester and trypsin reduced by half and a quarter, respectively. Less byproducts, high overall conversion and low cost made competitive the large-scale production of recombinant human insulin by Pichia pastoris.Furthermore, a simple and efficient production process was established to prepare insulin detemir by selective one-step acylation of free s-amino group of B29in desB30. As the critical step for preparing insulin detemir, the conversion ratio of desB30to insulin determir in acylation reaction would greatly influence the cost of production. The protective acylation process used by Novo Nordisk was of multi-steps and low-yield, causing high production cost. In the work, the selective acylation of ε-amino group of lysineB29in desB30was established without protecting α-amino group on N-terminus under alkaline conditions, and the conversion ratio of single-acylated product was more than80%. After purified by SOURCE30 RPC, the purity of final product was as high as98%, which met the purity requirements of the insulin products. The molecular weight, acylation sites, biological activity, and pharmacodynamics of final aimed product were comprehensively tested, and the results showed that the product prepared by selective acylation were the same as Novo Nordisk Levemir(?). The insulin detemir preparation process established in the work could serve as the basis for further development of pharmaceutical. Furthermore, in order to reduce the cost of the acylation reaction, very expensive myristic acid N-hydroxysuccinimide ester was produced by using the cheap myristic acid in this study and the purity of the prepared activated ester met the requirements. The selective acylation of desB30and the preparation of activated ester would significantly reduce the cost of insulin detemir, which laid a solid foundation for industrial production in future.