双水相萃取分离免疫球蛋白和单克隆抗体研究
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摘要
抗体广泛用于疾病治疗、医疗诊断和免疫分离,具有广阔的市场需求和发展前景。抗体主要从动物血液、腹水和细胞培养液中分离得到,尤其是动物细胞培养制备单克隆抗体,已实现规模化生产。然而,目前抗体分离过程的成本仍旧较高,成为抗体产业发展的一个瓶颈,开发经济高效的抗体分离新方法,具有重要意义。双水相萃取具有生物相容性好、分离条件温和、成本相对较低、易于放大等优点,本文针对含人血清白蛋白(HSA)料液中分离免疫球蛋白G (IgG)和细胞培养液中分离单克隆抗体两类典型的抗体分离过程,探讨双水相萃取分离抗体的可行性。主要内容如下：
(1)从含有HSA的混合蛋白中双水相萃取分离IgG。比较分析了不同类型双水相系统中IgG的分配情况,选择聚乙二醇(PEG)/羟丙基淀粉(HPS)为合适的双水相系统。考察了PEG浓度、HPS浓度、NaCl浓度和pH对IgG和HSA在PEG/HPS系统中分配的影响,以IgG上相收率(Ytop)和纯化因子(PF)为响应值,利用响应面法评价了四个因素的显著性,并优化了萃取条件,确定最佳分离条件是12%PEG、18%HPS、10%NaCl和pH8.0, Ytop为99.2%,PF为5.28。进一步考察了PEG/磷酸盐双水相系统反萃取IgG,确定反萃取最适条件为磷酸盐(pH7)浓度10%,第一步萃取的上相量与磷酸盐母液(40%,w/w)的质量比为1.6：1。经萃取和反萃取两步分离,IgG收率为84.0%,纯化因子为5.73,实现了从含HSA料液中高效分离IgG。
(2)探讨了NaCl对IgG在双水相系统中分配和溶解性影响的作用机制。首先考察了NaCl对kG分配的影响机制,发现添加NaCl造成PEG/HPS双水相系统相图的双节点曲线向偏离坐标原点方向移动,两相间的疏水差异性增大；利用荧光法测定了NaCl对IgG疏水性的影响,发现随着NaCl浓度增大,IgG分子疏水性增大,这正是IgG选择性分配至上相的主要原因。其次考察了添加NaCl对IgG溶解性的影响,发现添加NaCl加剧了IgG在(NH4)2SO4溶液中沉淀析出,却增大了IgG在PEG溶液中的溶解性,解释了PEG/(NH4)2SO4和PEG/HPS双水相系统中IgG收率的差异。进一步采用等温滴定量热法分析了NaCl对IgG-PEG间相互作用的影响,发现随NaCl浓度增大,IgG-PEG间的相互作用方式发生了改变,高NaCl浓度下IgG-PEG间疏水相互作用增强,明确了添加NaCl增大IgG在PEG溶液中溶解性的内在原因,并提出了NaCl影响的可能机制。
(3)采用两步双水相萃取法从中国仓鼠卵巢(CHO)细胞培养液中分离单克隆抗体MAB。考察了PEG/HPS双水相系统萃取分离MAB,优化了NaCl添加量、pH和料液添加量,确定了最佳条件为12%PEG、18%HPS、15%NaCl、pH6.0和6%CHO细胞培养液,MAB收率和纯度达96.7%和96.0%。采用PEG/磷酸盐双水相系统实现反萃取,得到合适的分离条件为系统中磷酸盐浓度为8%,第一步萃取的上相量与磷酸盐母液的质量比为2.5：1。经两步萃取,MAB收率和纯度分别为86.8±1.0%和97.6±0.5%。比较了双水相萃取和Protein A亲和层析,发现二者得到的MAB纯度相近,表明双水相萃取可作为Protein A亲和层析的潜在替代方法,用于从细胞培养液中高效分离单克隆抗体。
(4)探讨了混合模式配基双水相系统分离抗体的可行性。首先实现了PEG分子与配基的高效偶联,制备了系列配基-PEG。采用环氧氯丙烷法活化PEG,环氧基接入量可达到460μmol·g-1PEG;再将活化PEG与配基偶联,配基接入率达到90%以上。考察了混合模式配基对IgG在双水相系统中分配的影响,包括疏水型配基(MMI-PEG)和荷电配基(MBA-PEG、AHNSA-PEG、MBIA-PEG和MBIS-PEG)。比较分析后发现巯基咪唑苯甲酸(MBIA)较合适,当pH5.0时,添加MBIA-PEG可使IgG富集于上相而大部分HSA分配在下相中。结果表明,混合模式配基双水相系统分离IgG具有一定可行性,相关系统研究还需进一步开展。
综上所述,本文探讨了双水相萃取分离IgG和单克隆抗体,为了提高分离效率,采取了两个策略：(1)添加中性盐,在聚合物／聚合物双水相系统中添加NaCl,通过影响相平衡和抗体疏水特性,促进抗体从下相转移分配入上相；(2)引入混合模式配基,PEG分子与功能配基偶联,通过配基与抗体发生相互作用,吸引抗体进入上相,实现与杂蛋白分离。本文证实了两种方法的有效性,并分析了相关机制,为抗体分离新方法的开发提供了新思路。
Antibodies have been widely used for the therapeutic drug, diagnostic reagent and immunoaffinity separation, which has a broad market demand and prospects. Antibodies are normally separated from animal blood, ascites and mammalian cell culture. The manufacturing of monoclonal antibody from mammalian cell culture has been applied in large-scale production. As we know, the high-cost downstream process for antibody production has become one bottleneck of antibody industry. It is of great importance to develop new techniques with low cost and high efficiency for antibody separation. As new bioseparation technology, aqueous two-phase extraction (ATPE) has some advantages such as good biocompatibility, mild condition, relative low cost, easy to scale-up and so on. In this thesis, the feasibility of antibody separation with ATPE was investigated, which aimed at two typical processes of the separation of immunoglobulin G (IgG) from human serum albumin (HSA) containing feedstock and the purification of monoclonal antibody from mammalian cell culture broth.
Main contents are summerized as follows:
(1) Aqueous two-phase system (ATPS) was studied for the extraction of IgG from HSA containing feedstock. The partition behaviors of IgG in different types of ATPSs were compared firstly, and polyethylene glycol (PEG)/hydroxypropyl starch (HPS) ATPS was chosen as the suitable system. The effects of the concentrations of PEG, HPS and NaCl and pH on the partition of IgG and HSA in PEG/HPS ATPS were investigated. With the yield of IgG in top phase (Ytop) and the purification factor (PF) as the objective parameters, the response surface methodology (RSM) was used to evaluate the effects of some key factors and the separation conditions were optimized. The optimal conditions were obtained as12%(w/w) PEG4000,18%(w/w) HPS and10%(w/w) NaCl at pH8.0, and Ytop was99.2%with PF of5.28. The back extraction of IgG with PEG/phosphate ATPS were studied, the optimal conditions were10%phosphate (pH7) and the mass ratio of top phase of the first extraction to phosphate stock solution (40%w/w) of1.6:1. After two steps of ATPE, the total yield of IgG was84.3%with PF of5.73. The results demonstrated that IgG could be efficiently separated from HSA containing feedstock with suitable ATPE process.
(2) The mechanisms of NaCl effects on the partition and solubility of IgG in ATPS were investigated. It was found that the addition of NaCl could lead to a shift on the binodal curve of the phase diagram of PEG/HPS ATPS, which caused the increase on the differences of hydrophobicity of two phases. The effect of NaCl addition on the surface hydrophobicity of IgG molecule was determined using fluorescence method. The results indicated that the addition of NaCl could increase the hydrophobicity of IgG, which would be the main reason for the selective partition of IgG in top phase. In addition, the effects of NaCl addition on the solubility of IgG were investigated. It was found that the addition of NaCl could increase the precipitation of IgG in the presence of (NH4)2SO4while improve the solubility of IgG in the presence of PEG, which could explain the experimental phenomenon of the difference of IgG recovery in PEG/(NH4)2SO4and PEG/HPS ATPSs. Furthermore, the effect of NaCl on the interaction between IgG and PEG was inveatigated using isothermal titration calorimetry (ITC). It was found that the addition of NaCl might make a shift on the IgG-PEG interaction mode and the hydrophobic interaction of IgG-PEG would increase with high NaCl concentration. This would be the reason that the NaCl addition could improve the solubility of IgG in the presence of PEG, and the possible mechanism was proposed.
(3) A two-step extration process with ATPSs were studied to purify monoclonal antibody MAB from chinese hamster ovary (CHO) cell culture supernatant. The effects of NaCl addition, pH and feedstock loading on the extraction of MAB with PEG/HPS ATPS were investigated. The optimal conditions were12%PEG,18%HPS,15%NaCl, pH6.0and6%feedstock loading, and the yield of MAB in top phase was96.7%with the purity of96.0%. PEG/phosphate ATPS was used for the back extractions of MAB, and the optimal conditions were8%phosphate (pH7.0) and the mass ratio of top phase to phosphate stock solution (40%w/w) of2.5:1. After two-step extraction, the purity of MAB could reach97.6±0.5%with the yield of86.8±1.0%. It was found that the purity of MAB obtained with ATPE was comparable to that of Protein A chromatography. The results indicated that the ATPE process could be used as the alternative technique for the purification of monoclonal antibody from cells culture broth.
(4) The separation of antibody using ATPS with mixed-mode ligand was studied. Firstly, the ligand-PEG was prapared by coupling mixed-mode ligand onto PEG efficiently. PEG was activated by epichlorohydrin, and the epoxide content of activated PEG could reach460μmol·g-1PEG The PEG-expoxide was then coupled with mixed-mode ligands with above90%coupling rate. The effects of the ligand-PEG on the partition of IgG in ATPS were investigated, including the hydrophobic ligand (MMI-PEG) and the charged ligands (MBA-PEG, AHNSA-PEG, MBIA-PEG and MBIS-PEG). It was found that2-mercapto-5-Benzimidazole-carboxylic acid (MBIA) was a proper ligand for IgG separation. At pH5.0, the addition of MBIA-PEG could make IgG riched in top phase while most of HSA still partitioned in bottom phase. The results indicated that ATPE with MBIA-PEG could be used for the separation of IgG. However, more follow-up research should be done to improve the separation efficency.
In this thesis, the separation of IgG and monoclonal antibody with ATPE was focused. In order to improve the separation efficiency, two strategies have been developed:(1) NaCl was added into PEG/HPS ATPS to tranfer antibody from bottom phase into top phase by influencing the phase equilibrium and the hydrophobic property of antibody;(2) The mixed-mode ligands were coupled with PEG to induce antibody into top phase to purify antibody from the impurity proteins by the specific interactions between ligand and antibody. The feasibility of two strategies was verified and the mechanism was analyzed, which provided some new ideas for the development of new techniques for antibody separation.
(1)从含有HSA的混合蛋白中双水相萃取分离IgG。比较分析了不同类型双水相系统中IgG的分配情况,选择聚乙二醇(PEG)/羟丙基淀粉(HPS)为合适的双水相系统。考察了PEG浓度、HPS浓度、NaCl浓度和pH对IgG和HSA在PEG/HPS系统中分配的影响,以IgG上相收率(Ytop)和纯化因子(PF)为响应值,利用响应面法评价了四个因素的显著性,并优化了萃取条件,确定最佳分离条件是12%PEG、18%HPS、10%NaCl和pH8.0, Ytop为99.2%,PF为5.28。进一步考察了PEG/磷酸盐双水相系统反萃取IgG,确定反萃取最适条件为磷酸盐(pH7)浓度10%,第一步萃取的上相量与磷酸盐母液(40%,w/w)的质量比为1.6：1。经萃取和反萃取两步分离,IgG收率为84.0%,纯化因子为5.73,实现了从含HSA料液中高效分离IgG。
(2)探讨了NaCl对IgG在双水相系统中分配和溶解性影响的作用机制。首先考察了NaCl对kG分配的影响机制,发现添加NaCl造成PEG/HPS双水相系统相图的双节点曲线向偏离坐标原点方向移动,两相间的疏水差异性增大；利用荧光法测定了NaCl对IgG疏水性的影响,发现随着NaCl浓度增大,IgG分子疏水性增大,这正是IgG选择性分配至上相的主要原因。其次考察了添加NaCl对IgG溶解性的影响,发现添加NaCl加剧了IgG在(NH4)2SO4溶液中沉淀析出,却增大了IgG在PEG溶液中的溶解性,解释了PEG/(NH4)2SO4和PEG/HPS双水相系统中IgG收率的差异。进一步采用等温滴定量热法分析了NaCl对IgG-PEG间相互作用的影响,发现随NaCl浓度增大,IgG-PEG间的相互作用方式发生了改变,高NaCl浓度下IgG-PEG间疏水相互作用增强,明确了添加NaCl增大IgG在PEG溶液中溶解性的内在原因,并提出了NaCl影响的可能机制。
(3)采用两步双水相萃取法从中国仓鼠卵巢(CHO)细胞培养液中分离单克隆抗体MAB。考察了PEG/HPS双水相系统萃取分离MAB,优化了NaCl添加量、pH和料液添加量,确定了最佳条件为12%PEG、18%HPS、15%NaCl、pH6.0和6%CHO细胞培养液,MAB收率和纯度达96.7%和96.0%。采用PEG/磷酸盐双水相系统实现反萃取,得到合适的分离条件为系统中磷酸盐浓度为8%,第一步萃取的上相量与磷酸盐母液的质量比为2.5：1。经两步萃取,MAB收率和纯度分别为86.8±1.0%和97.6±0.5%。比较了双水相萃取和Protein A亲和层析,发现二者得到的MAB纯度相近,表明双水相萃取可作为Protein A亲和层析的潜在替代方法,用于从细胞培养液中高效分离单克隆抗体。
(4)探讨了混合模式配基双水相系统分离抗体的可行性。首先实现了PEG分子与配基的高效偶联,制备了系列配基-PEG。采用环氧氯丙烷法活化PEG,环氧基接入量可达到460μmol·g-1PEG;再将活化PEG与配基偶联,配基接入率达到90%以上。考察了混合模式配基对IgG在双水相系统中分配的影响,包括疏水型配基(MMI-PEG)和荷电配基(MBA-PEG、AHNSA-PEG、MBIA-PEG和MBIS-PEG)。比较分析后发现巯基咪唑苯甲酸(MBIA)较合适,当pH5.0时,添加MBIA-PEG可使IgG富集于上相而大部分HSA分配在下相中。结果表明,混合模式配基双水相系统分离IgG具有一定可行性,相关系统研究还需进一步开展。
综上所述,本文探讨了双水相萃取分离IgG和单克隆抗体,为了提高分离效率,采取了两个策略：(1)添加中性盐,在聚合物／聚合物双水相系统中添加NaCl,通过影响相平衡和抗体疏水特性,促进抗体从下相转移分配入上相；(2)引入混合模式配基,PEG分子与功能配基偶联,通过配基与抗体发生相互作用,吸引抗体进入上相,实现与杂蛋白分离。本文证实了两种方法的有效性,并分析了相关机制,为抗体分离新方法的开发提供了新思路。
Antibodies have been widely used for the therapeutic drug, diagnostic reagent and immunoaffinity separation, which has a broad market demand and prospects. Antibodies are normally separated from animal blood, ascites and mammalian cell culture. The manufacturing of monoclonal antibody from mammalian cell culture has been applied in large-scale production. As we know, the high-cost downstream process for antibody production has become one bottleneck of antibody industry. It is of great importance to develop new techniques with low cost and high efficiency for antibody separation. As new bioseparation technology, aqueous two-phase extraction (ATPE) has some advantages such as good biocompatibility, mild condition, relative low cost, easy to scale-up and so on. In this thesis, the feasibility of antibody separation with ATPE was investigated, which aimed at two typical processes of the separation of immunoglobulin G (IgG) from human serum albumin (HSA) containing feedstock and the purification of monoclonal antibody from mammalian cell culture broth.
Main contents are summerized as follows:
(1) Aqueous two-phase system (ATPS) was studied for the extraction of IgG from HSA containing feedstock. The partition behaviors of IgG in different types of ATPSs were compared firstly, and polyethylene glycol (PEG)/hydroxypropyl starch (HPS) ATPS was chosen as the suitable system. The effects of the concentrations of PEG, HPS and NaCl and pH on the partition of IgG and HSA in PEG/HPS ATPS were investigated. With the yield of IgG in top phase (Ytop) and the purification factor (PF) as the objective parameters, the response surface methodology (RSM) was used to evaluate the effects of some key factors and the separation conditions were optimized. The optimal conditions were obtained as12%(w/w) PEG4000,18%(w/w) HPS and10%(w/w) NaCl at pH8.0, and Ytop was99.2%with PF of5.28. The back extraction of IgG with PEG/phosphate ATPS were studied, the optimal conditions were10%phosphate (pH7) and the mass ratio of top phase of the first extraction to phosphate stock solution (40%w/w) of1.6:1. After two steps of ATPE, the total yield of IgG was84.3%with PF of5.73. The results demonstrated that IgG could be efficiently separated from HSA containing feedstock with suitable ATPE process.
(2) The mechanisms of NaCl effects on the partition and solubility of IgG in ATPS were investigated. It was found that the addition of NaCl could lead to a shift on the binodal curve of the phase diagram of PEG/HPS ATPS, which caused the increase on the differences of hydrophobicity of two phases. The effect of NaCl addition on the surface hydrophobicity of IgG molecule was determined using fluorescence method. The results indicated that the addition of NaCl could increase the hydrophobicity of IgG, which would be the main reason for the selective partition of IgG in top phase. In addition, the effects of NaCl addition on the solubility of IgG were investigated. It was found that the addition of NaCl could increase the precipitation of IgG in the presence of (NH4)2SO4while improve the solubility of IgG in the presence of PEG, which could explain the experimental phenomenon of the difference of IgG recovery in PEG/(NH4)2SO4and PEG/HPS ATPSs. Furthermore, the effect of NaCl on the interaction between IgG and PEG was inveatigated using isothermal titration calorimetry (ITC). It was found that the addition of NaCl might make a shift on the IgG-PEG interaction mode and the hydrophobic interaction of IgG-PEG would increase with high NaCl concentration. This would be the reason that the NaCl addition could improve the solubility of IgG in the presence of PEG, and the possible mechanism was proposed.
(3) A two-step extration process with ATPSs were studied to purify monoclonal antibody MAB from chinese hamster ovary (CHO) cell culture supernatant. The effects of NaCl addition, pH and feedstock loading on the extraction of MAB with PEG/HPS ATPS were investigated. The optimal conditions were12%PEG,18%HPS,15%NaCl, pH6.0and6%feedstock loading, and the yield of MAB in top phase was96.7%with the purity of96.0%. PEG/phosphate ATPS was used for the back extractions of MAB, and the optimal conditions were8%phosphate (pH7.0) and the mass ratio of top phase to phosphate stock solution (40%w/w) of2.5:1. After two-step extraction, the purity of MAB could reach97.6±0.5%with the yield of86.8±1.0%. It was found that the purity of MAB obtained with ATPE was comparable to that of Protein A chromatography. The results indicated that the ATPE process could be used as the alternative technique for the purification of monoclonal antibody from cells culture broth.
(4) The separation of antibody using ATPS with mixed-mode ligand was studied. Firstly, the ligand-PEG was prapared by coupling mixed-mode ligand onto PEG efficiently. PEG was activated by epichlorohydrin, and the epoxide content of activated PEG could reach460μmol·g-1PEG The PEG-expoxide was then coupled with mixed-mode ligands with above90%coupling rate. The effects of the ligand-PEG on the partition of IgG in ATPS were investigated, including the hydrophobic ligand (MMI-PEG) and the charged ligands (MBA-PEG, AHNSA-PEG, MBIA-PEG and MBIS-PEG). It was found that2-mercapto-5-Benzimidazole-carboxylic acid (MBIA) was a proper ligand for IgG separation. At pH5.0, the addition of MBIA-PEG could make IgG riched in top phase while most of HSA still partitioned in bottom phase. The results indicated that ATPE with MBIA-PEG could be used for the separation of IgG. However, more follow-up research should be done to improve the separation efficency.
In this thesis, the separation of IgG and monoclonal antibody with ATPE was focused. In order to improve the separation efficiency, two strategies have been developed:(1) NaCl was added into PEG/HPS ATPS to tranfer antibody from bottom phase into top phase by influencing the phase equilibrium and the hydrophobic property of antibody;(2) The mixed-mode ligands were coupled with PEG to induce antibody into top phase to purify antibody from the impurity proteins by the specific interactions between ligand and antibody. The feasibility of two strategies was verified and the mechanism was analyzed, which provided some new ideas for the development of new techniques for antibody separation.
引文
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