重组胰岛素前体转化成人胰岛素和地特胰岛素的工艺研究
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摘要
胰岛素及其类似物是治疗糖尿病最直接和最有效的药物,开发安全有效、使用方便的胰岛素及新型类似物一直是生物药物开发的一个热点。每日只需注射一次便可稳定控制血糖的地特胰岛素目前已成为长效胰岛素药物中的优秀代表。本文利用实验室以前构建的高效表达胰岛素前体的毕赤酵母菌株,从建立菌株的大规模发酵工艺、确立重组胰岛素前体的分子结构及酶切顺序、提高胰岛素前体转肽生成胰岛素酯的转化率、建立和优化地特胰岛素的酰化、分离工艺和药效学研究等几个方面进行了深入研究,为原型胰岛素和地特胰岛素的工业化制备初步建立了一套简单可行的生产工艺。
首先,建立了重组毕赤酵母生产胰岛素前体的大规模发酵工艺。利用毛细管气相色谱柱建立了发酵液甲醇浓度快速检测的方法,用于精确控制罐发酵过程中的甲醇流加。在毕赤酵母小试发酵工艺的基础上,成功建立了300L规模的胰岛素前体发酵甘油流加培养及甲醇流加诱导表达的生产工艺,胰岛素前体的产量超过3.0g/L,与小试规模相当。同时,利用CM-Sepharose FF离子交换层析建立了简单有效的胰岛素前体的初步纯化工艺,胰岛素前体从富含色素的发酵上清中得到较好的浓缩和提纯,一步纯化胰岛素前体的纯度达到88%,目的蛋白回收率达到95%,这为后续胰岛素前体的结构分析及酶切、偶联、酰化等工艺的建立和优化奠定了基础。
其次,发现了毕赤酵母表达的胰岛素前体N末端的不均一性现象并分析了原因。本文通过质谱和N末端测序证实,利用重组毕赤酵母发酵得到的胰岛素前体是一种N末端不均一的单链融合蛋白。实际上,在构建重组菌株时,为增加胰岛素前体的表达量,在目的蛋白的N末端引入了一段人工合成间隔肽序列(EEAEAEAEPK).间隔肽的存在成功实现了目的蛋白的高表达,但由于毕赤酵母自身产生的二肽氨肽酶A的酶活性不足和(或)专一性差,造成了胰岛素前体蛋白的N末端不均一性。同时实验也间接证实胰岛素前体在发酵过程没有发生C末端降解现象。
然后,证实了胰岛素前体单链蛋白上的三个酶切位点在酶切时存在先后顺序。酶切过程不同酶切时间样品采用HPLC和LC-MS的分析方法,发现胰蛋白酶首先将胰岛素前体的间隔肽片断迅速去除,生成单链胰岛素前体。随后,单链胰岛素在连接肽AAK的后面被进一步酶切,生成双链胰岛素前体。最后,该双链产物再经一次酶切去除连接肽,生成了B链缺少第30位苏氨酸的缺苏胰岛素产物(desB30)。这三步的酶切速率差别很大,第一步酶切速率很快,第二步相对较慢,第三步则不能完全反应,即使过夜酶切还有近20%的双链胰岛素前体无法转变为desB30。根据胰岛素的立体结构特点和容易生成聚体的特点,对造成这种酶切顺序和酶切速率差异的原因进行了分析。通过圆二色谱实验证实,在含有低浓度有机溶剂的溶液环境中，明显降低了胰岛素前体聚体的含量,同时在此环境下进行胰岛素前体酶切,desB30的收率由76.8%提高到95.6%,间接证实胰岛素前体形成的聚体空间结构是造成desB30收率不能进一步提高的原因。结合胰岛素前体的纯化工艺,提出在反相纯化收集液中进行酶切的新工艺,胰岛素前体酶切生成desB30的转化率提高了20%,未发现有其他酶切副产物产生的现象。
再者,建立了胰岛素前体两步法高效转肽生成原型人胰岛素的工艺。胰岛素前体采用一步法酶切转肽生成人胰岛素时,需要在高浓度的有机环境中经三次酶切生成desB30,然后再偶联生成重组人胰岛素酯。由于该转肽环境下的酶切效率不足而导致desB30生成受限,致使最高转化率仅为43.9%。胰蛋白酶作用下的酶切与偶联反应,实际是一对可逆的反应,即酶切为肽键的断裂,偶联为肽键的形成。然而,这对可逆反应各自的最优的反应条件却相差很大。本研究将酶切和偶联两个过程分开,采用了两步法代替现有的一步法将人胰岛素前体转化成人胰岛素酯,两个反应分别在各自的最优的条件下进行反应,最终转化率提高近一倍,转肽的反应时间缩短为原来的十分之一,活化酯用量减少一半,胰蛋白酶的用量约为原来的四分之一,副产物少,整体收率更高,成本更低。这为规模化、工业化利用毕赤酵母表达生产重组人胰岛素提供了一种简捷、高效的方法。
最后,建立了desB30选择性酰化制备地特胰岛素简单高效的生产工艺。制备地特胰岛素的关键工艺步骤是酰化反应,这步反应的收率直接决定了生产的成本。诺和诺德公司采用的是保护性酰化工艺,该工艺步骤多,收率低,生产成本较高。本研究建立了未保护N末端α氨基碱性条件下的选择性酰化B29位赖氨酸ε氨基的工艺,目的单酰化产物收率达到80%以上：酰化反应产物利用一步的SOURCE30RPC反相纯化工艺,目的产物的纯度超过98%,达到了胰岛素制品的纯度要求。对制备得到的酰化目的产物的分子量、酰化位点、生物活性和药效动力学进行了全面检测,实验证明本研究制备得到的酰化产物与对照品一致,说明的酰化胰岛素制备工艺是可行的,初步具备了进一步开展药学和临床前实验的基础。另外,为了进一步降低酰化反应的成本,建立了昂贵的肉豆蔻酸丁二酰亚胺酯的生产工艺,得到了纯度和结构符合要求的活化酯。选择性酰化工艺和活化酯自制成功,将大幅度降低地特胰岛素的生产成本,为将来的工业化制备奠定了坚实的基础。
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.
首先,建立了重组毕赤酵母生产胰岛素前体的大规模发酵工艺。利用毛细管气相色谱柱建立了发酵液甲醇浓度快速检测的方法,用于精确控制罐发酵过程中的甲醇流加。在毕赤酵母小试发酵工艺的基础上,成功建立了300L规模的胰岛素前体发酵甘油流加培养及甲醇流加诱导表达的生产工艺,胰岛素前体的产量超过3.0g/L,与小试规模相当。同时,利用CM-Sepharose FF离子交换层析建立了简单有效的胰岛素前体的初步纯化工艺,胰岛素前体从富含色素的发酵上清中得到较好的浓缩和提纯,一步纯化胰岛素前体的纯度达到88%,目的蛋白回收率达到95%,这为后续胰岛素前体的结构分析及酶切、偶联、酰化等工艺的建立和优化奠定了基础。
其次,发现了毕赤酵母表达的胰岛素前体N末端的不均一性现象并分析了原因。本文通过质谱和N末端测序证实,利用重组毕赤酵母发酵得到的胰岛素前体是一种N末端不均一的单链融合蛋白。实际上,在构建重组菌株时,为增加胰岛素前体的表达量,在目的蛋白的N末端引入了一段人工合成间隔肽序列(EEAEAEAEPK).间隔肽的存在成功实现了目的蛋白的高表达,但由于毕赤酵母自身产生的二肽氨肽酶A的酶活性不足和(或)专一性差,造成了胰岛素前体蛋白的N末端不均一性。同时实验也间接证实胰岛素前体在发酵过程没有发生C末端降解现象。
然后,证实了胰岛素前体单链蛋白上的三个酶切位点在酶切时存在先后顺序。酶切过程不同酶切时间样品采用HPLC和LC-MS的分析方法,发现胰蛋白酶首先将胰岛素前体的间隔肽片断迅速去除,生成单链胰岛素前体。随后,单链胰岛素在连接肽AAK的后面被进一步酶切,生成双链胰岛素前体。最后,该双链产物再经一次酶切去除连接肽,生成了B链缺少第30位苏氨酸的缺苏胰岛素产物(desB30)。这三步的酶切速率差别很大,第一步酶切速率很快,第二步相对较慢,第三步则不能完全反应,即使过夜酶切还有近20%的双链胰岛素前体无法转变为desB30。根据胰岛素的立体结构特点和容易生成聚体的特点,对造成这种酶切顺序和酶切速率差异的原因进行了分析。通过圆二色谱实验证实,在含有低浓度有机溶剂的溶液环境中，明显降低了胰岛素前体聚体的含量,同时在此环境下进行胰岛素前体酶切,desB30的收率由76.8%提高到95.6%,间接证实胰岛素前体形成的聚体空间结构是造成desB30收率不能进一步提高的原因。结合胰岛素前体的纯化工艺,提出在反相纯化收集液中进行酶切的新工艺,胰岛素前体酶切生成desB30的转化率提高了20%,未发现有其他酶切副产物产生的现象。
再者,建立了胰岛素前体两步法高效转肽生成原型人胰岛素的工艺。胰岛素前体采用一步法酶切转肽生成人胰岛素时,需要在高浓度的有机环境中经三次酶切生成desB30,然后再偶联生成重组人胰岛素酯。由于该转肽环境下的酶切效率不足而导致desB30生成受限,致使最高转化率仅为43.9%。胰蛋白酶作用下的酶切与偶联反应,实际是一对可逆的反应,即酶切为肽键的断裂,偶联为肽键的形成。然而,这对可逆反应各自的最优的反应条件却相差很大。本研究将酶切和偶联两个过程分开,采用了两步法代替现有的一步法将人胰岛素前体转化成人胰岛素酯,两个反应分别在各自的最优的条件下进行反应,最终转化率提高近一倍,转肽的反应时间缩短为原来的十分之一,活化酯用量减少一半,胰蛋白酶的用量约为原来的四分之一,副产物少,整体收率更高,成本更低。这为规模化、工业化利用毕赤酵母表达生产重组人胰岛素提供了一种简捷、高效的方法。
最后,建立了desB30选择性酰化制备地特胰岛素简单高效的生产工艺。制备地特胰岛素的关键工艺步骤是酰化反应,这步反应的收率直接决定了生产的成本。诺和诺德公司采用的是保护性酰化工艺,该工艺步骤多,收率低,生产成本较高。本研究建立了未保护N末端α氨基碱性条件下的选择性酰化B29位赖氨酸ε氨基的工艺,目的单酰化产物收率达到80%以上：酰化反应产物利用一步的SOURCE30RPC反相纯化工艺,目的产物的纯度超过98%,达到了胰岛素制品的纯度要求。对制备得到的酰化目的产物的分子量、酰化位点、生物活性和药效动力学进行了全面检测,实验证明本研究制备得到的酰化产物与对照品一致,说明的酰化胰岛素制备工艺是可行的,初步具备了进一步开展药学和临床前实验的基础。另外,为了进一步降低酰化反应的成本,建立了昂贵的肉豆蔻酸丁二酰亚胺酯的生产工艺,得到了纯度和结构符合要求的活化酯。选择性酰化工艺和活化酯自制成功,将大幅度降低地特胰岛素的生产成本,为将来的工业化制备奠定了坚实的基础。
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.
引文
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