【作者】 马泓冰；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2005， 硕士

【摘要】 单克隆抗体（mAb）的纯化是其理论研究和临床应用的基础,文献报道的纯化方法较多,包括各种沉淀法、离子交换层析法（IEX）、固定化金属螯合亲和层析法（IMAC）、凝胶层析法（GF）、羟基磷灰石层析法（CHT）、疏水作用层析法（HIC）和亲和层析（AC）法等,但各种方法的适用性及其规律至今尚需进一步研究。本文选择gp130、CD28和CD80三种鼠抗人mAb作为研究对象,采用IEX、IMAC、CHT、GF、AC、（NH₄）₂SO₄沉淀、辛酸沉淀等方法对上述三种小鼠腹水来源的mAb的纯化进行了比较性研究,在此基础上进一步从纯化后的抗体纯度、回收率和生物学活性等方面对多种不同纯化方法进行了分析,并优化了实验条件,从而建立了可获得高纯度、生物学活性好和纯化过程易于放大的mAb纯化方法,为其理论研究和临床应用提供了必要的实验基础。具体如下: 1.离子交换一步法快速纯化gP130激发型单克隆抗体B-S12的研究 采用中高压层析仪（Biologic Duo-Flow）和阴离子交换层析柱（Uno Q1或Bio-Scale Q5）,建立了从小鼠腹水中纯化抗gp130 mAb B-S12的一步层析方法。 本论文分别研究了上样pH值和离子强度梯度洗脱等条件对mAb B-S12纯度的影响,结果表明:经预处理后的样品以Tris-HCl缓冲溶液（pH7.0,20mmol/l）稀释10倍后上Uno Q1柱,0-0.5mol/l NaCl 10柱体积（CV）梯度洗脱,可获得纯度大于95%的mAb B-S12,抗体回收率达66%,纯化后的抗体在体外对XG-2细胞有明显的促增殖作用。同时,采用Bio-Scale Q5层析柱对本方法的放大可行性进行了研究,结果令人满意。 在方法筛选、优化过程中,比较了阴离子交换一步层析法（AIEX）、阳离子交换一步层析法（CIEX）以及ProteinG AC法在纯度、回收率和生物学活性等方面的差异。结果表明:虽然CIEX一步层析法也能获得纯度较高的mAb B-S12（纯度90%）,但纯度和回收率（52%）均不如AIEX一步法。而ProteinG AC法,虽然其纯化后的抗体纯度较高（>90%）,但其在体外对XG-2细胞的促增殖作用均低于更多还原

【Abstract】 The purification of monoclonal antibodies is the basis of their theoretical and clinical study. Many purification methods have been reported, such as precipitation, ion exchange (IEX), immobilized metal affinity chromatography (IMAC), gel filtration (GF), ceramic hydroxyapatite (CHT), hydrophobic interaction (HIC), and affinity chromatography (AC). In this work, a series of methods including IEX, IMAC, GF, CHT, AC, ammonium sulphate precipitation and caprylic acid precipitation were studied for the purification of three antibodies in mouse ascites, i.e., anti-gp130, anti-CD28, and anti-CD80. Advantages and disadvantages of these methods were compared in terms of purity of antibodies, recovery, bioactivity and amplificability. Experimental parameters were optimized in order to establish the purification methods characterized by high-purity, excellent bioactivity and easy amplification. These purification methods may build an important basis for their further applications.1. One-step method for purification of monoclonal antibody B-S12 against gpl30 from mouse ascites using ion exchange chromatographyA one-step method for purification of monoclonal antibody (mAb) B-S12 against gpl30 from mouse ascites was developed by using an anion cloumn (Uno Q1, or Bio-Scale Q5).In this work, the effects of sample loading pH and concentraion gradient of eluction salt on the purity of mAb B-S12 obtained were studied. It showed that antibody purity and recovery of mAbB-S12 were up to 95% and 66% respectively.when the ascites sample was diluted ten folds with buffer (pH7.0, 20mmol/l Tris-HCl) prior to loading on an anion exchange cloumn and then eluted with 0-0.5mol/l NaCl gradient. The purified mAb could stimulate the proliferation of XG-2 cells significantly in vitro. Meanwhile,the ampliation of this method was studied by using a 5-ml anion exchange column and the result was satisfactory.During the method screening and optimization, anion ion-exchange (AIEX), cation ion-exchange (CIEX) and Protein G AC techniques were evaluated in terms of purity, recovery and bioactivity. The results showed that mAb B-S12 with a relative high purity of >90% could be obtained; however the purity and recovery (52%) were worse than those of AIEX. Although the Protein G AC method could get a high purity (>90%) of mAb B-S12, the ability to stimulate the proliferation of XG-2 cells in vitro were lower than that of AIEX and CIEX.In conclusion, the establised method in this work was characterized with simple, rapid, high purity, excellent bioactivity and easy amplication. It built an important basis for its further application.2. Three-step method for purification of monoclonal antibody 2F5 against CD28 from mouse ascitesIn this work, the purification of mAb 2F5 against CD28 from mouse ascties was initially studied by using a neotype IMAC. Yet, the results showed that mAb 2F5 with a satisfying purity could not be achieved simply through the IMAC method. Therefore, a three-step method employing IMAC, CIEX and GF in series was used in order to obtain the mAb with a high purity.The ascites was purified by IMAC, CIEX and GF in sequence after centrifugation and filteration. The experimental parameters of each step were optimized respectively. The optimized procedure is shown as follows: The sample was first loaded on the IMAC cloumn using a phosphate buffer (pH 8.0, 50mmol/l Na2HPO4-NaH2PO4) containing 0.5mol/l NaCl, followed by elution with a pH gradient from 8.0 to 4.0. The fraction containing the targeting protein was desalted and buffer-exchanged on a desalting column. The desalted fraction was directly loaded on a CIEX column (Uno S1) at pH4.0 (50mmol/l acetate buffer) and subsequently step-eluted by sodium chloride. The fractionfrom CIEX was finally loaded on a GF column at pH7.2 (20mmol/l PB) and the fraction containing the mAb of interest was collected. With this three-step method, purity up to 95% was achieved with a total recovery of 58%. The purified mAb could distinctly stimulate the proliferation of PBTC in vitro.In addition, the present method was compared with a classic Protein G AC method. The results showed that the purity of the mAb obtained was higher than that of Protein G method and so is the stimulating effect on the proliferation of PBTC in vitro.3. Purification of monoclonal antibody 4E5 against CD80 from mouse ascites using a tandem method of AIEX and GFIn order to purify mAb 4E5 against CD80 from mouse ascites, ammonium sulphate precipitation (AS), caprylic acid precipitation (CA), IMAC, AIEX, CHT, and Protein G AC were studied. The results showed that high purity protein of interest could not be achieved with a single AS, CA, IMAC, CHT, AIE1X and AS+CA separation. Although Protein G AC could get a satisfing result in purity, it suffered from poor recovery, economy and difficulties of ampliation. While AIEX could effiently capture the mAb 4E5, further improvement in purity ought to make. Thus, in this work, a two-step method for the purification of mAb 4E5 against CD80 from mouse ascites was developed using AIEX and GF in combination.The ascites was first purified by AIEX after centrifugation and filteration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0, 50mmol/l Tris-HCl, and satisfactory results were obtained using a NaCl step elution. The fraction containing the protein of interest was directly loaded on a GF column and eluted using a 20mmol/l PB at pH7.2. The purity of mAb 4E5 obtained was more than 95% with a total recovery of 61%. The purified mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was sastifing.In addition, the present method was compared with the Protein G AC method. Itshowed that although a high purity of 90% could be attained with Protein G AC, the recovery (60%) was relatively low and the inhibition effect on the growth of Daudi cells in viro was lower than that of the present method.Therefore, it can be concluded that the establised AIEX+GF two-step purification method was characterized with high purity, excellent bioactivity and easy amplication.The above studies have showed that for the purification of mAb from mouse ascites, methods such as precipitation, IEX and IMAC could first be used to capture and purify the protein of interest in order to remove the major impurities and increase the concentration of the target antibody. Then according to the practical needs, high-resolution separation methods, such as HIC, CHT and IEX could be used for further purification in order to obtain high purity products. When tandem steps were used, different separation mechanisms should be selected. GF was usually used in the final polishing step. Protein G AC suffers from loss of bioactivity and difficulties of amplification although relatively high purity antibodies could be obtained quickly.更多还原