CN110785219A - 配体结合的mbp膜、用途和制造方法 - Google Patents

配体结合的mbp膜、用途和制造方法 Download PDF

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CN110785219A
CN110785219A CN201880020331.8A CN201880020331A CN110785219A CN 110785219 A CN110785219 A CN 110785219A CN 201880020331 A CN201880020331 A CN 201880020331A CN 110785219 A CN110785219 A CN 110785219A
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affinity ligand
mbp
linker
membrane
ligand
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CN110785219B (zh
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拉谢尔·M·多林
斯宾塞·罗宾斯
马克·胡尔维茨
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Trapolay Technology Co Ltd
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Abstract

描述了具有大孔、介孔或微孔中至少一种的自组装聚合物材料的组合物和方法。

Description

配体结合的MBP膜、用途和制造方法
技术领域
本发明涉及配体结合的多嵌段聚合物(multi-block polymer，“MBP”)均孔材料、其制备以及在实验室环境、放大环境或商业环境的分析、检测和分离方案中的用途，例如但不限于蛋白质纯化和生产过程所必需的分离技术，并且特别是作为基于柱的亲和分离的替代方案，以及用于监测和检测目的分析物的传感器。
背景技术
从R&D到大规模制造，蛋白质纯化在生物分子分离市场中是关键且具有挑战性的一个方面。普遍认为整个蛋白质纯化过程的关键瓶颈涉及许多分离方案。目前蛋白质纯化的一个方面涉及使用带有具有多于一种尺寸方案之连通孔(interconnected pore)的支持多孔材料(supported porous material)、或多孔膜的亲和分离。通常来说，可用于亲和分离的多孔膜通过将反应性基团(公开了羧酸基团等)与生物活性剂(例如蛋白A(Protein A)等)连接而形成。该膜由聚合物材料制成。反应性基团可以直接地是聚合物的一部分或者由通过后续处理形成反应性基团的前体形成。
该膜还通过使亲和配体与反应性双官能单体连接(直接地或通过连接部分)并单独地或在存在另外的单体和“致孔剂”(例如醇)的情况下使亲和配体/单体材料聚合来形成。为了在聚合期间保护亲和配体，可通过可切割部分对其进行保护，所述可切割部分在聚合之后通过处理(例如用酸)来去除。
亲和分离可包括具有聚(芳醚酮)和“致孔剂”(例如聚酰亚胺)的膜，其已经进行浇铸并且随后通过化学反应选择性地去除聚酰亚胺来在铸件中形成孔。然后，通过使膜中的酮基团与携带另外的连接基团的胺反应来使所得多孔膜官能化，所述另外的连接基团可用于进一步连接多种物质，例如蛋白质。
用于亲和分离的多孔膜也由共聚物(包括包含(一般来说)苯乙烯衍生物和丙烯酸烷基酯衍生物的共聚物)制成，并且通过酰胺键与“亲和配体”材料(包括蛋白质)连接。共聚物不是自组装的嵌段共聚物或者通过与多种“活化剂”反应而被/用羟基或氨基官能化，然后使其进一步与亲和配体(公开了蛋白A)反应。
另一些亲和膜通过在有机溶剂中浇铸嵌段共聚物(PEO/PPO聚醚)溶液和聚砜，随后进行水淬灭步骤来形成。所得聚合物膜在表面上具有羟基，其随后进一步衍生化以使生物材料与膜共价结合。另外，在浇铸之前将共聚物衍生化，并且在膜形成之后释放羟基。
来自微相分离结构的膜由来源于具有“极性基团”的丙烯酸烷基酯和苯乙烯的自组装的嵌段聚合物制备。含有携带羧基的单体单元(糖、脂质、蛋白质、肽及其复合材料中的一种或更多种)的共聚物膜也是已知的。
在过去的十年中，对膜色谱的关注逐渐提高，但是膜色谱作为柱色谱的替代方案的广泛商业应用由于在可用材料平台种类以及膜结构和性能缺乏显著技术进步两方面中的限制而受到阻碍。特别地，市售膜色谱材料已建立在具有宽孔径分布的常规膜结构上。这种孔径变化导致跨膜的流动模式不均匀，使穿透曲线变宽并且降低介质容量。在目前的实践中，通过将膜层堆叠在一起如此使沿流线(streamline)的平均渗透率更加均一来使穿透曲线锐化。已经提出了其中膜层的堆叠替代更典型的填充珠柱的色谱装置(参见例如WO2000050888A1)，但是柱的深度由于驱动流过具有亚微孔的膜的堆叠所需的压力而受到严重限制。
然而，尽管在了解蛋白质表达、结构和功能方面取得了重大进展，但是从复杂混合物纯化蛋白质在所有方法规模上对蛋白质开发者而言仍然是重大挑战，并且它需要一系列分离技术。
附图简述
图1是描绘了MBP材料的一般分级孔隙率(hierarchical porosity)的图示。描绘了孔隙率的多个长度尺度。
图2是描绘了在多孔嵌段共聚物膜的表面上的亲和配体的图示。
图3是描绘了用蛋白A配体与EDC进行膜表面修饰的图示。
发明详述
公开了嵌段共聚物、三元共聚物、四元共聚物、共同地多嵌段聚合物(MBP)材料/结构，其中所述材料/结构中的至少一部分通过自组装形成使得所述材料或结构是分级多孔的(携带具有多个长度尺度的孔)。自组装聚合物材料包含大孔、介孔或微孔中的至少一种，所述孔中的至少一些是均孔的。微孔限定为具有0.1至1nm的直径。介孔限定为具有1至200nm的直径。大孔限定为具有200至1000μm的直径。
在一个实施方案中，自组装聚合物材料包含具有窄孔径分布的介孔或微孔，并且MBP材料或结构部分地或完全地被亲和配体(或用于连接亲和配体的接头)修饰。聚合物材料形成为膜、有支持或无支持的、有褶或无褶的、三维有褶构造，其形状是平的、圆周的或螺旋的，作为用于多种容器、管或吸收池(cuvette)的衬里或插入物。该材料可形成为多种形状，例如珠、球体或多维固体介质。
在一些实施方案中，MBP包含介孔。在一个实施方案中，介孔为约1nm至约200nm。在一个实施方案中，介孔为约3nm至约200nm。在一个实施方案中，介孔为约5nm至约200nm。在一个实施方案中，介孔为约5nm至约100nm。在一个实施方案中，介孔为约5nm至约50nm。在一些实施方案中，MBP包含微孔。在一个实施方案中，微孔为约0.1nm至约1nm。
在一些实施方案中，MBP包含大孔。在一个实施方案中，大孔为约200nm至约1000μm。在一个实施方案中，大孔为约200nm至约100μm。在一个实施方案中，大孔为200nm至约10μm。在一个实施方案中，大孔为1μm至约10μm。在一个实施方案中，大孔为约500nm至约10μm。
通过使用高容量MBP膜结构/膜，本发明的MBP结构/材料提供了对现有柱亲和色谱的困难的解决方案，所述高容量MBP膜结构/膜被修饰以部分地或完全地用作膜吸附剂，其相对于现有的柱亲和色谱介质，在一部分处理时间中具有相同或更好的分离质量。目前的下游纯化过程使用多个步骤并且分批进行。纯化线(purification line)中的第一步骤是基于柱的亲和分离，其被广泛认为是整个过程的关键瓶颈。亲和柱占总处理时间的近25％，使下游设备空闲并且导致低制造效率。该步骤也代表了整个纯化线中最大的劳动力支出。此外，柱色谱所需的高消耗品量极大地导致蛋白质开发和制造的高成本。当使用本发明的MBP结构/材料膜/膜吸附剂时，通过显著缩短处理时间并相应地降低制造成本且同时提高生产效率，由现有柱亲和色谱引起的现有瓶颈得以缓解。
除了消除现有纯化过程的瓶颈之外，本发明还通过使高度均匀地流过单层膜成为可能而提供了过程改进。在其中均孔的介孔或微孔在膜的下游表面上形成连续层的构造中，这些孔提供均一的流阻，其尽管小于在具有类似孔径的填充柱(paced column)中发生的流动的阻力但是比由较大的不均匀孔所提供的大得多。当使用小膜层数(优选不超过3，更优选1)将膜包装为过滤装置时，均孔层的阻力使得在每个地方流速都是均一的，因而高效地利用所有结合位点。此外，在这样的装置中，所述装置例如可以是包含有褶包装物的筒(cartridge)、包含以平膜片(flat sheet)为边界的流动通道的错流盒或组件(crossflowcassette or module)、或者螺旋卷式筒(spiral wound cartridge)，例如水过滤工业中所常见的，颗粒或大分子过滤可与色谱分离组合在一个步骤中，因而降低所需单元操作的总数。
本发明的多嵌段聚合物依赖于自组装技术来形成膜样膜(membrane film)，例如美国专利公布No.9,527,041中公开的那些，或国际公布No.WO2015048244的混杂材料(hybrid material)，其各自通过引用整体并入。
或者，如Hierarchically Porous Materials from Block Copolymers，Dorin etal.中所述的，其全部内容通过引用并入。膜被修饰以包含结合的配体，并且提供高选择性，同时具有高通量。该过程为膜提供具有非常高表面积的分级多孔结构，并且将大孔连续域(continuous domain)与介孔壁结构组合在单个可扩缩(scalable)材料中。大孔结构特征提供对流溶液流(convective solution flow)，从而提供迅速处理，同时介孔壁产生高表面积，从而提供进行高密度亲和官能化的独特潜力。通过介孔基质结构添加高容量潜力使其适于生物分子分离市场中的工业应用。
本发明的多嵌段聚合物中均一介孔性与大孔性的组合提供了具有高通量和高表面积的膜。大孔区域允许高通量，而均一的介孔提供高表面积和均匀的流分布。在膜表面上的亲和配体提供了用于目的物质/目的分析物与膜进行基于亲和力的相互作用的平台。
无论是以膜还是三维构造，本发明的MBP材料单独地由纯有机模板、混杂材料、其组合中的一种形成或者与嵌入或位于表面的纳米颗粒组合，并且任选地被官能化。
本发明利用自组装技术来制备本发明的多嵌段聚合物，其具有至少一个包含官能团的嵌段以形成具有非常高表面积的分级多孔膜。该膜的MBP中的至少一个嵌段被修饰成具有与亲和配体的共价或非共价连接。这样的结构不需要基底来形成，并且将大孔连续域与介孔壁结构组合在单个可扩缩结构中。大孔结构特征提供改善的对流溶液流，从而提供迅速处理，同时介孔壁产生高表面积，从而提供用于高密度表面官能化的潜力。与已知膜相比，通过介孔基质结构添加高容量潜力对于蛋白质分离市场中的工业应用具有高度地有前景的意义。
在一些应用中，均孔层占据膜的整个下游表面，尤其是在其中材料是有褶的膜的一些实施方案中。
本发明的分级多孔多嵌段聚合物(“MBP”)材料/结构是经亲和配体官能化的。MBP包含两个或更多个化学上不同的嵌段(A-B)；也可以是A-B-C或B-A-C三元共聚物；或者是A-B-C-B、或A-B-C-D、或A-B-C-B-A、或A-B-C-D-E形式的更高级多嵌段共聚物体系；或者这些更高级体系的其他可变排列。多嵌段共聚物可通过本领域中已知的方法来合成。多嵌段共聚物的合成方法的一些实例包括：阴离子聚合、阳离子聚合、可逆加成-断裂链转移聚合、原子转移自由基聚合，以及所列合成方法的任意组合。每个嵌段可包含但并非必须包含化学成分的混合物，只要相邻嵌段在化学上足够不同因而使得能够自组装即可。在一个实施方案中，包含MBP的至少一种嵌段共聚物中的至少一种嵌段包含亲水性或氢键键合嵌段化学成分。例如，合适的亲水性或氢键键合嵌段化学成分包括：聚((4-乙烯基)吡啶)、聚((2-乙烯基)吡啶)、聚(环氧乙烷)、聚(甲基丙烯酸酯)、聚(甲基丙烯酸甲酯)、聚(二甲基乙基氨基乙基甲基丙烯酸酯)、聚(丙烯酸)、聚(二甲基丙烯酰胺)、聚(苯乙烯磺酸盐)、聚(甲基丙烯酸2-羟乙酯)、聚(丙烯酰胺)和聚(羟基苯乙烯)。在一些实施方案中，包含MBP的至少一种嵌段共聚物还包含至少一种疏水性嵌段化学成分。合适的疏水性嵌段化学成分的一些实例包括：聚(苯乙烯)、聚(异戊二烯)、聚(丁二烯)、聚(乙烯)、聚(丙烯)。合适的嵌段共聚物的一些实例包括例如聚(异戊二烯-b-苯乙烯-b-4-乙烯基-吡啶)、聚(异戊二烯-b-苯乙烯-b-2-乙烯基-吡啶)、聚(异戊二烯-b-苯乙烯-b-环氧乙烷)、聚(异戊二烯-b-苯乙烯-b-甲基丙烯酸酯)、聚(异戊二烯-b-苯乙烯-b-甲基丙烯酸甲酯)、聚(异戊二烯-b-苯乙烯-b-二甲基乙基氨基乙基甲基丙烯酸酯)、聚(异戊二烯-b-苯乙烯-b-丙烯酸)、聚(异戊二烯-b-苯乙烯-b-二甲基乙基氨基乙基甲基丙烯酸酯)、聚(异戊二烯-b-苯乙烯-b-二甲基丙烯酰胺)、聚(异戊二烯-b-苯乙烯-b-苯乙烯磺酸盐)、聚(异戊二烯-b-苯乙烯-b-2-羟乙基甲基丙烯酸酯)、聚(异戊二烯-b-苯乙烯-b-丙烯酰胺)、聚(异戊二烯-b-苯乙烯-b-羟基苯乙烯)、聚(苯乙烯-b-4-乙烯基吡啶)、聚(苯乙烯-b-2-乙烯基吡啶)、聚(苯乙烯-b-环氧乙烷)、聚(苯乙烯-b-甲基丙烯酸酯)、聚(苯乙烯-b-甲基丙烯酸甲酯)、聚(苯乙烯-b-二甲基乙基氨基乙基甲基丙烯酸酯)、聚(苯乙烯-b-丙烯酸)、聚(苯乙烯-b-二甲基丙烯酰胺)、聚(苯乙烯-b-苯乙烯磺酸盐)、聚(苯乙烯-b-2-羟乙基甲基丙烯酸酯)、聚(苯乙烯-b-丙烯酰胺)、聚(苯乙烯-b-羟基苯乙烯)、聚(丙烯-b-4-乙烯基吡啶)、聚(苯乙烯-b-2-乙烯基吡啶-b-异戊二烯-羟基苯乙烯)、聚(苯乙烯-b-丁二烯-b-二甲基丙烯酰胺-b-异戊二烯-苯乙烯-4-乙烯基吡啶)。列举以上聚合物作为举例说明性实例，并且其他化学成分、组合以及嵌段数目和取向也是可能的，只要材料满足本发明的结构特征即可。
嵌段不必由单个单元或数个具有不同化学成分的单元分开，所述单元可能不被认为是不同的“嵌段”。嵌段可以但并非必须连接成在嵌段之间具有梯度化学成分(即，共聚物中的单个单元在化学成分方面没有急剧转变)。分级多孔材料通常携带具有多于一种尺寸方案的连通孔。膜包含介孔以及大孔。由于在材料或结构形成(例如膜)期间嵌段共聚物的自组装，介孔表现出窄孔径分布。材料具有整体各向同性(isotropic)或不对称的结构。膜表面部分地或完全地被亲和配体进行表面修饰，如下所示。
亲和配体是能够以非常高的亲和力与对其具有特异性的部分或针对其产生的抗体结合的分子。在蛋白质-配体结合中，配体通常是与靶蛋白质上的位点结合的信号触发分子。在DNA-配体结合中，配体通常是与DNA双螺旋结合的任何小分子或离子或者甚至是蛋白质。结合通过分子间力(例如离子键、氢键和范德瓦耳斯力(van der Waals force))发生。对接(缔合)通常是可逆的(解离)。将这样的配体并入到分级多孔膜形式的嵌段共聚物中允许使用合适的亲和配体通过亲和色谱来捕获和纯化特定的生物分子部分。在一个实施方案中，蛋白A是通过化学反应直接地或通过连接分子与嵌段共聚物连接的亲和配体。得到的具有共价结合的蛋白A的膜可用于捕获或纯化人免疫球蛋白(IgG)。亲和配体的另一些实施方案是蛋白G、蛋白A/G和蛋白L。亲和配体/靶部分的另一些实例还包括生物素(配体)-链霉亲和素(部分)、地高辛配基(digoxigenin)(配体)-抗DIG抗体和二硝基酚(配体)-抗DNP抗体，以及核酸。对于亲和配体的共价连接，在材料表面上的合适官能团的一些实例是：羧酸、羟基、氨基、巯基和包含可电离或可去除氢的其他基团。亲和配体的共价连接的一个实施方案是蛋白A与膜的羧基表面连接。这通过首先用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺氯化物(EDC)活化MBP材料表面来实现。随后，将活化的材料表面暴露于亲和配体(蛋白A)，从而形成配体与材料表面的共价连接。
合适的连接剂包括但不限于亚氨酸酯(imidoester)，例如辛二亚氨酸二甲酯(dimethyl subenmidate)；N-羟基琥珀酰亚胺酯，例如BS3；碳二亚胺，例如EDC、SMCC或其水溶性类似物磺基-SMCC、DCC或DIC；苯并三唑衍生物，例如BOP、HATU、PyBOP等；通过酰卤(acid halide)、酰基叠氮化物或磺酰卤(sulfonyl halide)的酐或混合酐形成；或者中间体硝基苯酯。尽管羧酸是优选的，但是当在嵌段共聚物或嵌段共聚物膜上存在其他种类的官能团时，可使用其他偶联剂。
在另一个实施方案中，MBP材料携带选自以下的部分：适于与可接枝化合物(graftable compound)的反应性基团反应以将该化合物共价接枝至材料的化学反应性基团；pH敏感性基团，适于直接固定分析物的基团；染料、荧光团、生色团或猝灭剂；固定化蛋白质；以及固定化天然或人工核酸分子。
在另一个实施方案中，MBP材料包含以下侧链或基团中的至少一种：羟基、氨基、羧基、聚乙二醇、烷基、马来酰亚胺、琥珀酰亚胺、酰卤、巯基或叠氮化物。
在另一个实施方案中，至少一种官能化单体是氨基甲基丙烯酸酯、氨基丙烯酸酯、丙烯酸、二甲基丙烯酰胺或甲基丙烯酸。
在另一个实施方案中，MBP材料表面通过非共价附接(例如吸附或浸渍)用亲和配体进行修饰。
在本发明的另一个实施方案中，MBP材料是用于基于亲和力的分离(例如蛋白质纯化)的分离介质，其用作结合-洗脱操作，其中靶物质与亲和配体结合以将其分离。随后，靶物质可从材料解连接(unbound)以记录该靶物质。在本发明的另一个实施方案中，结合特定杂质用于在靶化合物流过材料的同时从混合物中除去该杂质。
本发明的另一个应用是作为传感器的一部分，例如化学或生物化学检测和/或量化。在靶物质与亲和配体结合之后，膜上的特定响应、阻力、容量、颜色被激活。在该实施方案中，靶物质与亲和配体的结合引起材料的可检测变化或响应(例如膜的分光光度谱的变化)，从而允许检测和/或量化靶物质。
本发明的MBP膜的一种或更多种嵌段被接头修饰，其为随后连接亲和配体提供官能团；本发明的MBP具有包括分级孔隙率并且用接头或亲和配体修饰的三维嵌段共聚物结构，并且不限于“膜(film)”或“膜(membrane)”所典型的纵横比(aspect ratio)。MBP材料可以是整体材料(monolithic material)。该材料可模制或以其他方式形成为多种三维形状。形状可包含具有不同孔隙率或配体的区域。在制造成膜之前或之后提供用亲和配体或接头对嵌段共聚物的修饰。通过改变时间、温度、修饰剂浓度等，相对于嵌段共聚物的化学计量来用接头或亲和配体对嵌段共聚物进行修饰。对于大多数应用，相关的修饰范围或程度是10％至100％的可用位点。本发明的MBP膜包含多于一种亲和配体或接头、含有多于一个官能团的亲和配体或接头、或者部分地或完全地在多于一种聚合物嵌段上的亲和配体或接头。
本发明的带有MBP配体的材料/膜包括通过共价或非共价方式的连接材料的共形涂层(conformal coating)，产生接头或亲和配体的物理层；将材料形成或固定在支持材料上，以提供机械稳定性。
本发明的带有MBP配体的材料/膜包括整合到纺织品或传感器装置中。
除了其介孔结构之外，本发明的带有MBP配体的材料/膜还在材料/结构中包含微孔，其通过对MBP进行加工以将微孔材料(例如沸石、微孔碳)并入到材料/结构之中/之上来提供。微孔是介孔的补充，或者部分地或全部地替代由介孔提供的微孔。
本发明的MBP膜有助于控制用亲和配体或接头修饰的材料/结构的几何结构和面积。几何控制是二维或三维的，或者其一些组合。涂层的这种几何控制的一个实施方案可以是图案化，例如通过刻蚀(lithographically)。这种几何控制的另一个实施方案是将经修饰材料/结构的一个部分物理地附接于未经修饰的膜或其他基底。
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Claims (20)

1.自组装聚合物材料，其包含大孔、介孔或微孔中的至少一种，所述孔中的至少一些是均孔的，所述自组装聚合物材料包含至少一种多嵌段聚合物(MBP)，该聚合物嵌段中的至少两种是化学上不同的，所述化学上不同的嵌段中的至少一种被修饰以包含共价或非共价部分连接，以及与所述部分连接结合的亲和配体。
2.权利要求1所述的材料，其是不对称的或对称的中的一种。
3.权利要求1所述的材料，其还包含单个、整体可扩缩结构中的大孔域和介孔壁结构。
4.权利要求2所述的材料，其还包含连续的大孔域。
5.权利要求4所述的材料，其中所述材料是不对称的并且具有介孔和大孔。
6.权利要求1所述的材料，其还包含，
a.多于一种亲和配体或接头，或者
b.含有多于一个官能团的亲和配体或接头，或者
c.部分地或完全地位于多于一种聚合物嵌段上的亲和配体或接头。
7.制备权利要求1所述材料的方法，其包括，
a.通过非共价附接例如吸附用亲和配体对所述MBP的表面进行修饰；或，
b.用接头对所述MBP进行修饰，所述接头提供用于随后连接亲和配体的官能团；或，
c.所述MPB是具有接头或亲和配体的包含分级孔隙率的三维嵌段三元共聚物结构，其中在制成膜之前用亲和配体或接头对所述MBP的嵌段进行修饰；或，
d.相对于所述MBP的化学计量，改变用接头或亲和配体修饰的MBP的量，包括改变时间、温度和修饰剂浓度，以修饰10％至100％的可用位点；或，
e.通过共价或非共价部分将连接材料共形地涂覆至所述材料的表面以提供所述接头或亲和配体的完整但离散的物理层；或，
f.除了MBP介孔-大孔结构的介孔之外，还通过将微孔材料例如沸石、微孔碳并入到膜中/膜上、并入到所述材料中来向所述材料提供微孔；或，
g.控制经亲和配体或接头修饰的材料的几何结构和面积以使其
i.以二维排列，或，
ii.以三维排列，或，
iii.通过刻蚀以图案排列，或
iv.通过将经修饰材料的一部分附接于未经修饰的膜或其他基底来进行排列。
8.包含权利要求1所述材料的制品，其包括将所述材料固定在支持材料上以提供机械稳定性。
9.包含权利要求1所述材料的制品，其包括将所述材料与纺织品整合。
10.传感器，其包含权利要求1所述材料。
11.分离和/或检测目的分析物的方法，其使包含所述目的分析物的介质与权利要求1所述材料接触。
12.权利要求11所述的方法，其中所述目的分析物是蛋白质。
13.权利要求11所述的方法，其中分离涉及结合-洗脱操作，其中所述分析物是靶物质并且与所述亲和配体结合以使它分离，其中所述靶物质可从所述材料解连接以记录所述靶物质。
14.检测目的分析物或目的物质的方法，其包括接触包含权利要求1所述材料的传感器，并测量作为所述目的分析物或目的物质与材料配体之间相互作用之结果的光学/电学变化。
15.权利要求1所述的材料，其还包含单个、整体可扩缩结构中的大孔域和介孔壁结构。
16.权利要求1所述的材料，其中所述大孔结构特征提供改善的对流溶液流、迅速处理，并且所述介孔壁产生用于高密度表面官能化的高表面积。
17.权利要求11所述的方法，其中所述分析物包含核酸，并且是分离的。
18.使用权利要求1所述材料的装置，其中所述材料形成为有褶的包装物、形成为在错流盒或组件中的平片、或形成在螺旋卷组件中。
19.使用权利要求18所述装置的方法，其通过结合和洗脱来分离分子。
20.使用权利要求18所述装置的方法，其用于通过过滤来分离分子或悬浮的颗粒。
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