CN108452775A - 一种功能化的高密度层析基质、其制备方法及应用 - Google Patents

一种功能化的高密度层析基质、其制备方法及应用 Download PDF

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CN108452775A
CN108452775A CN201810283350.1A CN201810283350A CN108452775A CN 108452775 A CN108452775 A CN 108452775A CN 201810283350 A CN201810283350 A CN 201810283350A CN 108452775 A CN108452775 A CN 108452775A
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CN108452775B (zh
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程昉
李明洋
何炜
孙冰冰
马晓春
曲景平
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Dalian University of Technology
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Abstract

本发明公开了一种功能化的高密度层析基质、其制备方法及应用。所述的层析基质功能化方法，以表面多羟基结构的亲水性微球或表面包被有多羟基聚合物的亲水性微球作为层析基质，在无水条件下利用催化剂催化层析基质表面羟基与二乙烯基砜反应，实现对基质表面羟基进行功能化。本发明的层析基质表面功能化方法步骤少，过程简单，功能化密度高，所用的试剂和溶剂均为常规试剂并且可以回收重复利用，该反应在反应过程中没有副产物生成，原子经济性高，成本低廉；通过调控反应时间和催化剂种类能够实现密度控制。利用本发明所述方法制备的层析基质，其在亲和层析填料制备以及抗体药物纯化中有广泛的应用前景。

Description

一种功能化的高密度层析基质、其制备方法及应用
技术领域
本发明属于生物化工领域中层析材料制备技术领域，具体涉及一种功能化的高密度层析基质、其制备方法及应用。
技术背景
抗体及其相关产品在临床治疗和免疫学诊断方面具有重要的作用。随着近年来上游细胞培养技术突飞猛进的发展，对于下游抗体纯化工艺的要求不断升高。具有高反应活性且反应可控的层析基质活化方法对于提高基质的吸附容量和优化吸附特异性具有重要的意义。现在广泛应用的层析基质的活化试剂为环氧氯(溴)丙烷和烯丙基溴，Harding等(J.Chromatogr.A,1997,775:29)详细研究了环氧氯(溴)丙烷和烯丙基溴在不同条件下对不同多羟基层析基质功能化效果。基于该功能化方法并以巯乙基吡啶为配基的层析基质已由Pall Biosepara公司商业化生产。但是该方法步骤复杂，成本高；反应条件苛刻，需要强碱条件；并且反应过程中会生成卤化氢副产物，原子经济性差。
1975年Porath等提出利用二乙烯基砜(DVS)作为层析基质功能化试剂(Porath etal.J.Chromatogr.A,1975,73:1767)，该功能化方法的优势在于没有副产物生成；反应后的乙烯砜基(VS)可以与含有巯基、氨基和羟基的配基高效连接，具有广泛的适用性；砜基在进行抗体结合过程中可以提供亲硫作用，提高抗体结合的选择性。专利(CN 101284224A)中利用该功能化方法制备了用于分离抗体的扩张床吸附基质。但是现在利用二乙烯基砜进行层析基质功能化是在碱性水溶液中进行的，其反应效率低，最高功能化密度仅能达到60μmol/g树脂，且反应不可控，限制了层析基质的吸附容量和吸附特异性的提高。此外，在碱性水溶液的反应条件下，乙烯砜基容易发生水解，进一步降低了配基偶联量。因此，提高二乙烯基砜功能化反应效率，实现对层析基质密度可控的高密度乙烯砜基功能化具有重要意义。
发明内容
本发明的目的在于提供一种反应效率高、密度可控的高密度多羟基层析基质的功能化方法。为实现上述目的，本发明采用了以下技术方案：
一种密度可控的高密度层析基质功能化方法，其步骤如下：
以吡啶衍生物或三取代有机磷作为催化剂，将此催化剂和二乙烯基砜溶于有机溶剂，混合均匀，再加入彻底除水后的层析基质获得反应液，15-60℃下反应0-48h；获得功能化的层析基质。
对于上文所述的技术方案中，优选的情况下，所述的催化剂选自：三苯基膦、三环己基膦、三异丙基膦、三甲苯基膦、三对甲苯基膦、三苯基膦三间磺酸盐、吡啶、吡啶二甲酸和4-二甲氨基吡啶。
对于上文所述的技术方案中，优选的情况下，所述的有机溶剂为非质子性溶剂。更为优选的情况下，所述的有机溶剂为二氯甲烷、丙酮、乙腈、二甲基亚砜、二甲基甲酰胺。
对于上文所述的技术方案中，优选的情况下，所述的层析基质为表面多羟基结构的亲水性微球或表面包被有多羟基聚合物的亲水性微球。更为优选的情况下，所述的层析基质为琼脂糖凝胶，或者为由PVA、葡聚糖或纤维素包被的微球。
对于上文所述的技术方案中，优选的情况下，所述的催化剂与二乙烯基砜的摩尔比为1:10-1000。更为优选的情况下，所述的催化剂与二乙烯基砜的摩尔比为1:10-1:100。
对于上文所述的技术方案中，优选的情况下，所述的反应温度为15-45℃。更为优选的情况下，所述的反应温度为20-35℃。
对于上文所述的技术方案中，优选的情况下，在15-60℃下反应0-48h后，还包括对产物进行抽滤，冲洗的过程，最后获得功能化的层析基质。
对于上文所述的技术方案中，优选的情况下，所述的15-60℃下反应0-48h，是在恒温摇床中300-1000rmp条件下反应，且反应的时间在0-24h内也能取得很好的效果。
通常可以利用有机溶剂对层析基质进行彻底除水；本领域技术人员还可以按照下述标准选择合适的除水方式，即：凡是符合1、能够彻底去除层析基质中水分；2、不会对层析基质造成损伤的除水方式均可。
对于上文所述的技术方案中，优选的情况下，所述的二乙烯基砜在有机溶剂中的浓度为1-20％(v/v)。
对于上文所述的技术方案中，优选的情况下，所述的层析基质在反应液中的终浓度为0.1-0.3g/mL。
此外，本发明还保护一种利用本发明上文所述的方法制备的层析基质。以及所述的层析基质在亲和层析填料制备以及抗体药物纯化中的应用。具体的，所述的亲和层析填料的制备，是指用本发明所述方法活化后的基质可以偶联多种亲和配基，理论上只要含有巯基，氨基，羟基的配基均可以实现偶联，例如常用的氨三乙酸(NTA),多肽(谷胱甘肽等)，多糖等。
有益效果：
(1)高密度：利用本发明上文所述的方法制备的层析基质，优选的情况下，采用三苯基膦作催化剂，三苯基膦与二乙烯基砜摩尔比1:10，二乙烯基砜在有机溶剂中浓度10％(v/v)，层析基质0.1g/mL；恒温摇床中25℃1000rpm条件下反应12h。其表面的乙烯砜基密度最高可达200μmol/g，较传统方法提高了三倍。
(2)密度可控：利用本发明上文所述的方法制备的层析基质，能够通过调控催化反应时间和催化剂种类实现表面乙烯砜基密度的调控，密度调控范围为60-200μmol/g。即：不同种类的催化剂催化效率不同，相同反应条件下利用不同催化剂催化，表面乙烯砜基密度不同。该催化反应符合一级反应动力学，相同催化剂催化不同时间其表面乙烯砜基密度不同。
(3)高反应效率：利用本发明上文所述的方法制备的层析基质，具有较高的反应效率，其反应速率常数可达0.011min-1。
(4)操作简单，成本低，原子经济性高：本发明上文所述的一种密度可控的高密度层析基质功能化方法，步骤少，操作简单；所用的试剂和溶剂均为常规试剂并且可以回收重复利用，该反应在反应过程中没有副产物生成，原子经济性高，成本低廉。
附图说明
本发明附图幅，用以对本发明的进一步说明，并且构成说明书的一部分，与以下面的具体实施例方式一起用于来解释本发明，并不构成对本发明的限制。
图1为实施例5中对每一修饰步骤的树脂进行的XPS表征结果。其中(a)图为全谱；
(b)图为S 2p谱图；(c)图为C 1s谱图。(1)为未修饰的琼脂糖凝胶；(2)为利用催化剂催化VS功能化反应后的琼脂糖凝胶。
图2为实施例6中采用三种方法对树脂进行重复功能化修饰后的表面VS密度变化。
图3为实施例7中树脂对人IgG的静态吸附曲线。
图4为实施例9中树脂对单克隆抗体料液纯化的色谱图。其中(1)为实施例7修饰得到的琼脂糖树脂；(2)为MEP HyperCel；(3)为碱性条件下修饰的琼脂糖凝胶。(a)为上样步骤；(b)为pH 4醋酸缓冲液洗脱步骤；(c)为0.1M NaOH溶液在线清洗步骤。
图5为实施例9中树脂对单克隆抗体料液纯化后的GPC谱图。其中(1)为实施例7修饰得到的琼脂糖树脂纯化得到的单抗；(2)为MEP HyperCel纯化得到的单抗；(3)为碱性条件下修饰的琼脂糖凝胶纯化得到的单抗；(B)为pH 4醋酸缓冲液；(S)为商业ProteinA树脂纯化得到的单抗；(F)为未纯化的单克隆抗体料液。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1
取0.3g琼脂糖树脂(Bastarose 6FF：高度交联的6％琼脂糖，平均粒径90μm；博格隆生物技术有限公司)，抽滤并用乙腈充分洗涤以去除其中水分，加入1mL含有4-二甲氨基吡啶的10％(v/v)二乙烯基砜的乙腈溶液，其中4-二甲氨基吡啶与二乙烯基砜摩尔比为1:10，于25℃条件下反应12h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜和催化剂残留，得到VS功能化的琼脂糖树脂。
对照组将0.3g琼脂糖树脂加入1mL含有10％(v/v)二乙烯基砜和10％丙酮的碳酸盐缓冲液(0.5M,pH 11)中，于25℃条件下反应12h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜残留。取定量的功能化后的琼脂糖树脂，加入过量的半胱氨酸溶液充分与表面VS进行反应，利用Ellman法测定反应前后溶液中半胱氨酸的变化值从而可以得到树脂表面VS密度，经计算催化法得到的功能化密度可达120μmol/g。密度为传统碱性条件反应的2倍。
实施例2
取0.3g琼脂糖树脂(Bastarose 6FF：高度交联的6％琼脂糖，平均粒径90μm；博格隆生物技术有限公司)，抽滤并用乙腈充分洗涤以去除其中水分，加入1mL含有三苯基膦的10％(v/v)二乙烯基砜的乙腈溶液，其中三苯基膦与二乙烯基砜摩尔比为1:10，于25℃条件下反应1h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜和催化剂残留，得到VS功能化的琼脂糖树脂。
对照组将0.3g琼脂糖树脂加入1mL含有10％(v/v)二乙烯基砜和10％丙酮的碳酸盐缓冲液(0.5M,pH 11)中，于25℃条件下反应12h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜残留。取定量的功能化后的琼脂糖树脂，加入过量的半胱氨酸溶液充分与表面VS进行反应，利用Ellman法测定反应前后溶液中半胱氨酸的变化值从而可以得到树脂表面VS密度，经计算功能化密度可达80μmol/g。密度为传统碱性条件反应的1.3倍。
实施例3
取0.3g琼脂糖树脂(Bastarose 6FF：高度交联的6％琼脂糖，平均粒径90μm；博格隆生物技术有限公司)，抽滤并用乙腈充分洗涤以去除其中水分，加入1mL含有三苯基膦的10％(v/v)二乙烯基砜的乙腈溶液，其中三苯基膦与二乙烯基砜摩尔比为1:10，于25℃条件下反应2h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜和催化剂残留，得到VS功能化的琼脂糖树脂。
对照组将0.3g琼脂糖树脂加入1mL含有10％(v/v)二乙烯基砜和10％丙酮的碳酸盐缓冲液(0.5M,pH 11)中，于25℃条件下反应12h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜残留。取定量的功能化后的琼脂糖树脂，加入过量的半胱氨酸溶液充分与表面VS进行反应，利用Ellman法测定反应前后溶液中半胱氨酸的变化值从而可以得到树脂表面VS密度，经计算功能化密度可达120μmol/g。密度为传统碱性条件反应的2倍。
实施例4
取0.3g琼脂糖树脂(Bastarose 6FF：高度交联的6％琼脂糖，平均粒径90μm；博格隆生物技术有限公司)，抽滤并用乙腈充分洗涤以去除其中水分，加入1mL含有三苯基膦的10％(v/v)二乙烯基砜的乙腈溶液，其中三苯基膦与二乙烯基砜摩尔比为1:10，于25℃条件下反应12h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜和催化剂残留，得到VS功能化的琼脂糖树脂。
对照组将0.3g琼脂糖树脂加入1mL含有10％(v/v)二乙烯基砜的碳酸盐缓冲液(0.5M,pH 11)中，于25℃条件下反应12h，反应结束后抽滤并用乙腈洗涤彻底去除二乙烯基砜残留。取定量的功能化后的琼脂糖树脂，加入过量的半胱氨酸溶液充分与表面VS进行反应，利用Ellman法测定反应前后溶液中半胱氨酸的变化值从而可以得到树脂表面VS密度，经计算功能化密度可达160μmol/g。密度为传统碱性条件反应的2.7倍。
实施例5
根据实施例4所述的步骤，对琼脂糖树脂进行功能化，取功能化前后的树脂进行冻干，并采用XPS(X射线光电子能谱)进行表征。在未进行功能化的琼脂糖树脂表面未检测到S元素；在进行了VS功能化后在169eV处检测到S 2p峰，归属为砜基峰。说明催化剂条件下成功实现了VS功能化。
实施例6
分别选择三苯基膦和4-二甲氨基吡啶作为催化剂，并以pH11功能化条件作为对照。根据实施例1和实施例4所述方法分别对琼脂糖树脂进行三次重复功能化修饰，并对每次反应后的VS密度进行检测。结果显示采用催化剂法进行功能化的树脂在第一次功能化修饰时密度即达到了最高，之后的重复修饰步骤其VS密度没有发生显著增长，而采用pH11功能化条件的树脂在每次重复修饰后VS密度均发生显著升高。这表明催化剂法具有较传统方法更高的反应效率。
实施例7
取0.2g实施例4所述方法得到的功能化琼脂糖树脂加入1mL含有10mg/mL MEP的HEPEs缓冲液(20mM,pH 8.0)，于25℃条件下反应6h，得到MEP修饰的琼脂糖树脂。利用该树脂对IgG的静态吸附性能进行测试。
首先用去离子水清洗树脂，并用缓冲液进行平衡。准确称取0.04g树脂置于2mL离心管中，分别加入1mL不同浓度的人IgG浓度的缓冲溶液，恒温25℃下吸附3h,达到吸附平衡后，离心分离，取出上清液测定人IgG的浓度，根据为物料平衡计算树脂的吸附容量，绘制吸附等温线，并根据Langmuir方程拟合得到饱和吸附容量和解离常数。本发明实施例4制备的树脂在进行MEP修饰后对人IgG的饱和吸附容量为141.4mg/g树脂，解离常数为1.61×10-5M-1。
实施例8
取实施例7所得到的MEP修饰的树脂对人IgG(日本和光纯药工业株式会社)的动态载量进行测试。取1mL树脂装填于1mL柱中，分别计算不同流速下的10％穿透时的动态载量。本发明实施例1制备的树脂在0.25mL/min流速下对人IgG的10％穿透时的动态载量为84.4mg/g树脂，在0.5mL/min流速下对人IgG的10％穿透时的动态载量为29.6mg/g树脂，在1.0mL/min流速下对人IgG的10％穿透时的动态载量为15.9mg/g树脂。
实施例9
取实施例7所得到的MEP修饰的树脂对无血清细胞培养上清液中的单克隆抗体(奥马珠单抗)进行纯化，并以商业产品MEP HyperCel(PALL)和传统碱性条件反应得到的树脂作为对照。取1mL树脂装填于1mL柱中，在流速0.5mL/min条件下上样，pH 4的20mM的醋酸钠缓冲溶液洗脱，最后用0.1M NaOH进行原位清洗。洗脱得到的样品用GPC分析。发明实施例7制备的树脂纯化得到的单克隆抗体纯度高于95％，纯化量为MEP HyperCel的1.43倍。
对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应仍属于本发明技术方案保护的范围内。

Claims (12)

1.一种功能化的高密度层析基质，其特征在于，所述方法包括以下步骤：以吡啶衍生物或三取代有机磷作为催化剂，将此催化剂与二乙烯基砜溶于有机溶剂中，再加入彻底除水后的层析基质获得反应液，15-60℃下反应0-48h；获得功能化的层析基质。
2.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的催化剂选自：三苯基膦、三环己基膦、三异丙基膦、三甲苯基膦、三对甲苯基膦、三苯基膦三间磺酸盐、吡啶、吡啶二甲酸和4-二甲氨基吡啶。
3.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的有机溶剂为非质子性溶剂。
4.根据权利要求3所述的一种功能化的高密度层析基质，其特征在于，所述的有机溶剂为二氯甲烷、丙酮、乙腈、二甲基亚砜、二甲基甲酰胺。
5.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的层析基质为表面多羟基结构的亲水性微球。
6.根据权利要求5所述的一种功能化的高密度层析基质，其特征在于，所述的层析基质为琼脂糖凝胶，或者为由PVA、葡聚糖或纤维素包被的微球。
7.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的催化剂与二乙烯基砜的摩尔比为1:10-1000。
8.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的反应温度为15-45℃。
9.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的二乙烯基砜在有机溶剂中的浓度为1-20％(v/v)。
10.根据权利要求1所述的一种功能化的高密度层析基质，其特征在于，所述的层析基质在反应液中的终浓度为0.1-0.3g/mL。
11.利用权利要求1所述的方法制备的层析基质。
12.如权利要求1所述的层析基质在亲和层析填料制备以及抗体药物纯化中的应用。
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