摘要
层析是目前应用最广泛的蛋白分离方法,介质性能决定了层析分离过程的效率,关注点常集中于介质偶联的功能配基,而孔径和配基密度往往被忽视。本文以抗体分离为目标,探讨两种不同作用机制的层析方法,离子交换层析和疏水电荷诱导层析,制备不同配基密度与孔径的系列介质,比较吸附性能,分析配基密度与孔径的影响。主要结果如下：
以3种不同孔径的琼脂糖凝胶(Bestarose3.5HF、4FF和6FF)为基质,优化配基偶联条件,制备了不同配基密度的阴离子交换层析介质(以diethylamuboethyl, DEAE为配基)和疏水电荷诱导层析介质(以2-mercapto-1-methylimidazole, MMI为配基)。对于DEAE介质,最高配基密度分别达到110、280和410μmol/g;对于MMI介质,分别达到50、110和130μmol/g。
以牛免疫球蛋白(bIgG)和牛血清白蛋白(BSA)为模型蛋白,考察了系列DEAE介质和MMI介质的静态吸附性能,并采用Langmuir吸附等温式进行拟合。结果表明,对于DEAE介质,配基密度较低时,增加配基密度有利于蛋白质吸附；密度较高时,吸附量趋于稳定,甚至略有降低；较小孔径介质的比表面积较大,有利于蛋白质吸附；综合考虑孔径和配基密度影响,提出综合参数N,发现两种蛋白的饱和吸附容量均随log(N)呈线性变化。对于MMI介质,配基密度较低时,MMI介质无法有效吸附BSA和bIgG;当单位表面的配基数目较高(>15x10-12μmol/μm2)时,介质才能较好吸附BSA和blgG。
考察了系列DEAE介质和MMI介质对bIgG和BSA的动态吸附性能,包括吸附动力学和穿透曲线。采用孔扩散模型对吸附动力学曲线进行拟合,对于DEAE介质,配基密度对有效扩散系数的影响不大,而孔径增大可促进孔内扩散；对于MMI介质,配基密度和孔径增大都可促进蛋白质的孔内扩散,主要原因在于提高了介质与蛋白之间的疏水作用力。根据穿透曲线获得蛋白的动态载量Q10%,系列DEAE介质对BSA动态载量都比较大,总体上随平衡吸附量而线性增大；而blgG动态载量达到最大值时的配基密度有所偏移,分析可能原因在于动态吸附过程降低了bIgG在介质内部孔道的可及区域,减小了介质内部能够与蛋白相互作用的有效配基。MMI介质对BSA的动态载量很小,对bIgG的动态载量则依赖于介质的配基密度和孔径,控制较低操作流速,配基密度较高、中等孔径的MMI-B-4FF-110介质可实现bIgG的高效吸附。
考察了三种MMI介质MMI-B-3.5HF-50、MMI-B-4FF-110、MMI-B-6FF-110对bIgG和BSA混合蛋白的吸附等温线,发现吸附过程存在竞争性,具有优先吸附bIgG的能力,其中孔径适中、配基密度较大的MMI-B-4FF-110介质最佳,对bIgG的饱和吸附容量为93.51mg/g,而对BSA仅为43.69mg/g。进一步考察了MMI-B-4FF-110对混合蛋白的吸附动力学,发现BSA对bIgG的动态吸附影响不大,而当bIgG存在时改变了MMI介质对BSA的吸附,MMI对BSA的吸附量在吸附初始阶段大于其平衡吸附容量,随后逐渐减小到其平衡吸附量,可能是介质对两种蛋白吸附作用的强弱差异造成。
以bIgG为分离目标,考察了MMI介质的分离性能,包括bIgG/BSA混合液中分离bIgG,以及牛血中分离bIgG。对于bIgG/BSA混合液,优化了层析分离的上样和洗脱条件,确定pH7.0上样和pH4.0洗脱,可以获得高纯度的bIgG;比较了不同配基密度和孔径的MMI介质,发现孔径适中、配基密度较大的MMI-B-4FF-110介质效果最好。进一步采用优化的分离条件,利用MMI-B-4FF-110从牛血中分离bIgG,结果表明,牛血经离心和硫酸铵沉淀预处理后,利用MMI介质一步层析分离,bIgG纯度约95%,收率大于85%。
本文以DEAE离子交换层析为常规层析的典型代表,MMI疏水性电荷诱导层析为新型分离方法代表,系统考察了介质孔径和配基密度对bIgG和BSA吸附性能的影响,得到一些规律性认识,并且发现MMI介质对bIgG有优先吸附能力,优化了分离条件,用于抗体的分离纯化。相关结果有助于层析介质的进一步优化设计,提高抗体分离的效率。
Chromatography is a widely used technique in protein separation, and its separation efficiency is largely affected by chromatographic resins. Research usually focuses on structures of functional ligands coupled onto resins, while the pore size of resin matrices and ligand density are less discussed. This thesis investigated ion-exchange chromatography (IEC) resin and hydrophobic charge induction chromatography (HCIC) re' sins on their application in antibody separation. Series of diethylaminoethyl (DEAE) IEC resins and2-mercapto-l-methylimidazole (MMI) HCIC resins with different ligand densities and pore sizes were prepared. The adsorption performance of these resins was characterized with the influences of ligand density and pore size. The findings of this thesis can be summarized as:
(1) Three cross-linked agarose gels with different pore sizes (Bestarose3.5HF,4FF and6FF) were used as the matrices. DEAE IEC resins and MMI HCIC resins with different ligand densities and pore sizes were prepared by the control of ligand coupling reactions. The results showed that the highest ligand density of DEAE resins could reach to110,280and410μmol/g gel, respectively, and that of MMI were50,110and130μmol/g gel, respectively.
(2) The static adsorption behaviors of the DEAE and MMI resins were investigated using bovine immunoglobulin (bIgG) and bovine serum albumin (BSA) as model proteins. The adsorption isotherms obtained were fitted by Langmuir equation. The results showed that the adsorption capacity of DEAE increased with the increase of ligand density when the ligand density was relatively low, and it reached saturation then deceased slightly when the ligand density further increased. Meanwhile, the adsorption capacity increased with the decrease of pore size, as the specific surface area of the resins was higher with smaller pore size. A parameter N defined as the ratio of ligand density to the square of pore size was introduced to describe the integrative effects of pore size and ligand density, and it was found that the saturated adsorption capacity increased linearly with log (N). For MMI resins, the results showed that BSA and bIgG could not be adsorbed with low ligand densities, and resins with ligand density over15x10-12μmol/μm2were need for effective adsorption of BSA and bIgG
(3) The dynamic adsorption performance for bIgG and BSA were investigated with the DEAE and MMI resins prepared. The adsorption kinetics curves were fitted with the pore diffusion model (PDM). For DEAE resins, the effective diffusion coefficient was found to be influenced by the pore size of DEAE resins, but independent on ligand density. The pore diffusion of proteins in the MMI resins was increased with the increase of ligand density and pore size, which is mainly due to stronger hydrophobic interaction between the resins and the proteins. The dynamic adsorption capacity Q10%could be calculated from the breakthrough curves. For DEAE resins, Q10%of BSA was high and generally increased linearly with the equilibrium adsorption capacity. However the maximum Q10%of bIgG was obtained at the ligand density around100μmol/g gel, which was lower than that in static adsorption process. This behavior could be explained as a result of decreasing accessible surface area for bIgG during the dynamic adsorption process. However, Q10%of BSA with MMI resin was low, but that of bIgG was depended significantly on ligand density and pore size of the resins. Under low operational flow rates, the resin with high ligand density and medium pore size (MMI-B-4FF-110) could efficiently adsorb bIgG
(4) The adsorption isotherms of bIgG/BSA protein mixtures with the three MMI resins, MMI-B-3.5HF-50, MMI-B-4FF-110and MMI-B-6FF-110, were investigated. Competitive adsorption was found during the adsorption processes. The results showed that bIgG could be preferentially adsorbed and the performance of MMI-B-4FF-110was the best among the three resins. The qm of bIgG with MMI-B-4FF-110was93.51mg/g, while that of BSA was only43.69mg/g. It was found that the adsorption of BSA was influenced by the addition of bIgG. Due to the preferential adsorption of bIgG, the adsorption capacity of BSA with MMI resins exceeded the equilibrium adsorption capacity at the beginning of the adsorption, and then decreased to the equilibrium adsorption capacity. This effect may due to the strength difference of the hydrophobic interaction between the MMI resin and the two proteins.
(5) The chromatographic separation processes were developed using MMI resins for IgG separation. The protein loading and elution conditions were optimized for bIgG/BSA mixtures. High purity of bIgG could be obtained when the protein was loaded at pH7.0and eluted at pH4.0. MMI resins with different ligand densities and pore sizes were investigated and MMI-B-4FF-110was found to have the best performance. Furthermore, the optimized conditions were used to purify bIgG from crude bovine serum with MMI-B-4FF-110. The result showed that after the pretreatment with ammonium sulfate precipitation, the purity of bIgG was close to95%and the yield was more than85%with only one-step HCIC separation using MMI-B-4FF-110.
In summary, DEAE ion exchange chromatography and MMI hydrophobic charge induction chromatography were studied and compared as the representative for conventional chromatographic method and the novel separation technique, respectively. The influence of pore size and ligand density was explored. MMI resins were found to have preferential adsorption of IgG. By optimizing the separation conditions, these resins could be used for effective purification of antibodies. The results would be useful to design and optimize the resins and improve the the separation efficiency for antibody purification.
引文
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