琼脂糖亲水相互作用色谱在多肽分离纯化中的应用
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摘要
多肽具有分子量小、结构简单、易于合成、易于被机体吸收、给药途径多样化等特点,成为新药研发的热点。其生物功能多样,涉及抗菌、抗病毒、抗氧化、抗肿瘤、神经调节、免疫调节等。化学合成多肽在合成过程中形成多种结构类似物,天然来源的多肽包含多种杂质,目前多肽的分离纯化技术有限,以反相色谱为主,不能满足多肽药物快速发展的需要。
本论文提出以琼脂糖凝胶作为亲水相互作用色谱介质,应用于多肽的分离纯化,实现琼脂糖凝胶应用领域和多肽分离纯化方法的拓展。首先采用12%浓度的高交联度琼脂糖凝胶介质Superose 12分离了人工合成的甘氨酸二肽和甘氨酸三肽,考察了该介质对多肽的基本骨架结构(肽键和末端可电离基团)产生吸附的一般规律。随后将Superose12介质应用于蛋白质水解多肽的分离纯化,初步建立了Superose 12亲水相互作用色谱的多肽保留预测模型。最后将包括Superose 12介质在内的四种粒径和配基不同的琼脂糖凝胶介质应用于固相合成缩宫素粗品的纯化,对不同介质的分离效果进行了对比。主要结论包括：
1.采用Superose 12琼脂糖凝胶介质分离了人工合成的甘氨酸二肽和甘氨酸三肽。在乙腈-水流动相条件下,介质对甘氨酸寡肽形成了吸附保留,并将甘氨酸二肽与甘氨酸三肽完全分开,甘氨酸寡肽的保留体积随流动相中乙腈含量的增加而增加,流动相中添加尿素或中性盐削弱了吸附作用,流动相的pH值从3-12升高的过程中保留减弱,证明介质与甘氨酸寡肽之间形成的是一种氢键吸附与静电相互作用混和模式的吸附,采用特征点洗脱法获得了甘氨酸寡肽的动态吸附等温线,结合吸附能分布计算的结果,证明介质与甘氨酸寡肽之间相互作用存在轻度不均一性,用Toth模型能够很好地拟合获得的等温吸附数据。
2.采用Superose 12琼脂糖凝胶介质分离了蛋白水解多肽。在含水量逐渐增加的乙腈-水梯度洗脱条件下,溶菌酶水解多肽、核糖核酸酶A水解多肽和胰蛋白酶自身水解多肽经Superose 12介质分离,各获得了10～20个洗脱峰,通过质谱氨基酸序列分析,从三个蛋白水解多肽中总共识别并收集到了47个多肽片段。以20种氨基酸和肽键的保留相关系数为未知变量,建立了Superose 12介质的多肽保留预测模型,并根据47个多肽片段的色谱保留数据求解了未知变量,推断侧链带有可电离基团、酚羟基或酰胺基团的氨基酸残基能够增强保留。
3.采用Superose 12、Superose Prototype、Sepharose HP-β-CD和Novarose SE-100/40-Tris四种琼脂糖凝胶介质分离纯化了固相合成缩宫素粗品。平均粒径为10μm的Superose12介质(氢键受体型),在优化的乙腈-水-乙酸梯度洗脱条件下,将缩宫素粗品的峰面积百分比纯度由56.7%提高到92.8%,纯化倍数为1.64倍,回收率78.7%,且样品载量高,超过0.34 mg/ml凝胶；平均粒径为30μm的Superose Prototype介质(氢键受体型)和Sepharose HPβ-CD介质(混合型),洗脱谱图和分离效果与Superose12介质相近,适合工业放大；而Novarose SE-100/40 Tris介质(氢键供体型,40μm平均粒径)在乙腈-水流动相条件下不能对缩宫素形成有效的吸附保留。
Great attention has been paid to peptides in the field of new drugs development in the past few years due to the special properties of peptides, including lower molecular weight than that of proteins, simple structure, easy to be synthesized and uptaken by the organism and the diversity of administration route. Peptides have a lot of biological functions, such as antibiosis, antivirus, antioxidation, antitumor, neuroregulation and immunoregulation. Some structural analogues are formed during the process of chemical synthesis of peptides, and natural derived peptide crudes also contain a variety of impurities. At present, the items of available separation and purification methods of peptide are very limited and mainly based on reversed phase chromatography. Those methods can not meet the demands of quick developing market of peptide drugs.
In the present thesis, agarose gels were introduced into the field of separation and purification of peptides as hydrophilic interaction chromatographic media. The purpose is to expand the application field of agarose gel and the method of peptide separation. First, the highly cross-linked 12% agarose gel Superose 12 was used to separate synthetic glycine dipeptide and tripeptide. The general rules of the formation of adsorption between Superose 12 and the framework of peptides (peptide bond and terminal charged groups) were investigated. Second, Superose 12 was introduced into separation and purification of peptides from protein hydrolysis. A preliminary prediction model for peptide retention time of Superose 12-hydrophilic interaction chromatography was established. Finally, four types of agarose gels with different partical sizes and ligands were introduced to separate synthetic oxytocin crude, including Superose 12. The separation results with different media were compared. The main conclusions were as follows:
1. Synthetic glycine dipeptide and tripeptide were separated by Superose 12, a agarose gel medium. Glycine oligopeptides can be retardated efficiently on Superose 12 when mobile phase of acetonitrile-water was used. The dipeptide and tripeptide were completely separated from each other. The retention volumes of glycine oligopeptide increased with the fraction of acetonitrile in the mobile phase and decreased with pH value of the mobile phase. The retention volume was reduced when urea or sodium chloride was added into the mobile phase. These results indicated that the adsorption mechanism between Superose 12 and glycine oligopeptides could be a mixed-mode of hydrogen bond adsorption and electrostatic interaction. Dynamic adsorption isotherms were determined by the method of elution by characteristic points. Together with the calculation results of adsorption energy distribution, it was suggested that the interactions between the Superose 12 medium and glycine oligopeptides were slightly heterogonous and the isotherm adsorption data could be fitted by the Toth model excellently.
2. Peptides from protein hydrolysis were separated by Superose 12 medium. Lysozyme hydrolysate, ribonuclease A hydrolysate and peptides from trypsin autodigestion were separated with decreased acetonitrile gradients, respectively. Ten to twenty peaks were appeared per run. According to the results of amino acid sequence analysis by mass spectra, forty seven peptides were recognized and collected from three hydrolysates. A prediction model of peptide retention time on Superose 12 column was established based on the retention coefficients corresponding to 20 amino acids and peptide bond. According to the retention datas of 47 peptides, the coefficients as unknown variables were solved. It can be demonstrated that amino acid residues with ionogen, phenolic hydroxyl group or amide group could have a positive contribution to the retention of peptides.
3. Synthetic oxytocin crude was separated by four types of agarose gel media including Superose 12, Superose Prototype, Sepharose HP-β-CD and Novarose SE-100/40-Tris. Using an optimized gradient elution method with acetonitrile-water-acetic acid sequentially, the purity of oxytocin crude increased from 56.7% to 92.8% after separation on Superose 12 column (10μm particle size, hydrogen bond acceptor type). The purification folds were 1.64 and the recovery rate of oxytocin was 78.7%. The sample capacity of Superose 12 was very high and exceeded 0.34 mg oxytocin per ml gel. The elution profiles and separation results of Superose Prototype (hydrogen bond acceptor type) and Sepharose HPβ-CD (mixed type) media were similar to that of Superose 12. These media with 30μm particle size could be used to scale up. However, oxytocin could not be retardated effectively by Novarose SE-100/40 Tris (hydrogen bond donor type) medium under the acetonitrile-water mobile phase condition.
本论文提出以琼脂糖凝胶作为亲水相互作用色谱介质,应用于多肽的分离纯化,实现琼脂糖凝胶应用领域和多肽分离纯化方法的拓展。首先采用12%浓度的高交联度琼脂糖凝胶介质Superose 12分离了人工合成的甘氨酸二肽和甘氨酸三肽,考察了该介质对多肽的基本骨架结构(肽键和末端可电离基团)产生吸附的一般规律。随后将Superose12介质应用于蛋白质水解多肽的分离纯化,初步建立了Superose 12亲水相互作用色谱的多肽保留预测模型。最后将包括Superose 12介质在内的四种粒径和配基不同的琼脂糖凝胶介质应用于固相合成缩宫素粗品的纯化,对不同介质的分离效果进行了对比。主要结论包括：
1.采用Superose 12琼脂糖凝胶介质分离了人工合成的甘氨酸二肽和甘氨酸三肽。在乙腈-水流动相条件下,介质对甘氨酸寡肽形成了吸附保留,并将甘氨酸二肽与甘氨酸三肽完全分开,甘氨酸寡肽的保留体积随流动相中乙腈含量的增加而增加,流动相中添加尿素或中性盐削弱了吸附作用,流动相的pH值从3-12升高的过程中保留减弱,证明介质与甘氨酸寡肽之间形成的是一种氢键吸附与静电相互作用混和模式的吸附,采用特征点洗脱法获得了甘氨酸寡肽的动态吸附等温线,结合吸附能分布计算的结果,证明介质与甘氨酸寡肽之间相互作用存在轻度不均一性,用Toth模型能够很好地拟合获得的等温吸附数据。
2.采用Superose 12琼脂糖凝胶介质分离了蛋白水解多肽。在含水量逐渐增加的乙腈-水梯度洗脱条件下,溶菌酶水解多肽、核糖核酸酶A水解多肽和胰蛋白酶自身水解多肽经Superose 12介质分离,各获得了10～20个洗脱峰,通过质谱氨基酸序列分析,从三个蛋白水解多肽中总共识别并收集到了47个多肽片段。以20种氨基酸和肽键的保留相关系数为未知变量,建立了Superose 12介质的多肽保留预测模型,并根据47个多肽片段的色谱保留数据求解了未知变量,推断侧链带有可电离基团、酚羟基或酰胺基团的氨基酸残基能够增强保留。
3.采用Superose 12、Superose Prototype、Sepharose HP-β-CD和Novarose SE-100/40-Tris四种琼脂糖凝胶介质分离纯化了固相合成缩宫素粗品。平均粒径为10μm的Superose12介质(氢键受体型),在优化的乙腈-水-乙酸梯度洗脱条件下,将缩宫素粗品的峰面积百分比纯度由56.7%提高到92.8%,纯化倍数为1.64倍,回收率78.7%,且样品载量高,超过0.34 mg/ml凝胶；平均粒径为30μm的Superose Prototype介质(氢键受体型)和Sepharose HPβ-CD介质(混合型),洗脱谱图和分离效果与Superose12介质相近,适合工业放大；而Novarose SE-100/40 Tris介质(氢键供体型,40μm平均粒径)在乙腈-水流动相条件下不能对缩宫素形成有效的吸附保留。
Great attention has been paid to peptides in the field of new drugs development in the past few years due to the special properties of peptides, including lower molecular weight than that of proteins, simple structure, easy to be synthesized and uptaken by the organism and the diversity of administration route. Peptides have a lot of biological functions, such as antibiosis, antivirus, antioxidation, antitumor, neuroregulation and immunoregulation. Some structural analogues are formed during the process of chemical synthesis of peptides, and natural derived peptide crudes also contain a variety of impurities. At present, the items of available separation and purification methods of peptide are very limited and mainly based on reversed phase chromatography. Those methods can not meet the demands of quick developing market of peptide drugs.
In the present thesis, agarose gels were introduced into the field of separation and purification of peptides as hydrophilic interaction chromatographic media. The purpose is to expand the application field of agarose gel and the method of peptide separation. First, the highly cross-linked 12% agarose gel Superose 12 was used to separate synthetic glycine dipeptide and tripeptide. The general rules of the formation of adsorption between Superose 12 and the framework of peptides (peptide bond and terminal charged groups) were investigated. Second, Superose 12 was introduced into separation and purification of peptides from protein hydrolysis. A preliminary prediction model for peptide retention time of Superose 12-hydrophilic interaction chromatography was established. Finally, four types of agarose gels with different partical sizes and ligands were introduced to separate synthetic oxytocin crude, including Superose 12. The separation results with different media were compared. The main conclusions were as follows:
1. Synthetic glycine dipeptide and tripeptide were separated by Superose 12, a agarose gel medium. Glycine oligopeptides can be retardated efficiently on Superose 12 when mobile phase of acetonitrile-water was used. The dipeptide and tripeptide were completely separated from each other. The retention volumes of glycine oligopeptide increased with the fraction of acetonitrile in the mobile phase and decreased with pH value of the mobile phase. The retention volume was reduced when urea or sodium chloride was added into the mobile phase. These results indicated that the adsorption mechanism between Superose 12 and glycine oligopeptides could be a mixed-mode of hydrogen bond adsorption and electrostatic interaction. Dynamic adsorption isotherms were determined by the method of elution by characteristic points. Together with the calculation results of adsorption energy distribution, it was suggested that the interactions between the Superose 12 medium and glycine oligopeptides were slightly heterogonous and the isotherm adsorption data could be fitted by the Toth model excellently.
2. Peptides from protein hydrolysis were separated by Superose 12 medium. Lysozyme hydrolysate, ribonuclease A hydrolysate and peptides from trypsin autodigestion were separated with decreased acetonitrile gradients, respectively. Ten to twenty peaks were appeared per run. According to the results of amino acid sequence analysis by mass spectra, forty seven peptides were recognized and collected from three hydrolysates. A prediction model of peptide retention time on Superose 12 column was established based on the retention coefficients corresponding to 20 amino acids and peptide bond. According to the retention datas of 47 peptides, the coefficients as unknown variables were solved. It can be demonstrated that amino acid residues with ionogen, phenolic hydroxyl group or amide group could have a positive contribution to the retention of peptides.
3. Synthetic oxytocin crude was separated by four types of agarose gel media including Superose 12, Superose Prototype, Sepharose HP-β-CD and Novarose SE-100/40-Tris. Using an optimized gradient elution method with acetonitrile-water-acetic acid sequentially, the purity of oxytocin crude increased from 56.7% to 92.8% after separation on Superose 12 column (10μm particle size, hydrogen bond acceptor type). The purification folds were 1.64 and the recovery rate of oxytocin was 78.7%. The sample capacity of Superose 12 was very high and exceeded 0.34 mg oxytocin per ml gel. The elution profiles and separation results of Superose Prototype (hydrogen bond acceptor type) and Sepharose HPβ-CD (mixed type) media were similar to that of Superose 12. These media with 30μm particle size could be used to scale up. However, oxytocin could not be retardated effectively by Novarose SE-100/40 Tris (hydrogen bond donor type) medium under the acetonitrile-water mobile phase condition.
引文
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