CN112292203A - 色谱珠、其生产及用途 - Google Patents
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Abstract
本发明涉及色谱珠、其生产和用途。更接近地,本发明涉及小的、刚性的和不可渗透的琼脂糖珠,其适合于例如在高效液相色谱(HPLC)中作为固定相,用于分析生物分子,例如肽和蛋白质;和生产所述珠的方法。
Description
技术领域
本发明涉及色谱珠、其生产和用途。更接近地,本发明涉及小的、刚性的和不可渗透的琼脂糖珠,其主要适合于例如在高效液相色谱(HPLC)中作为固定相,用于分析生物分子,例如肽和蛋白质;和生产所述珠的方法。
背景技术
HPLC(高效液相色谱)是一种液相色谱方法,其对于化学和生化化合物的研究、制造和诊断至关重要。通常由于获得的速度和分辨率,常常在高压色谱系统中对生物分子(例如肽、蛋白质及其潜在的变体)进行表征和分析。两种类型的柱主要用于常规分析。它们是填充柱或整体柱。填充柱包含最常呈球形珠形状的颗粒固定相,所述球形珠彼此紧密堆积,且柱床高度范围为3-30 cm。由于其刚性,用于HPLC柱的市售固定相通常基于二氧化硅或合成有机树脂例如聚苯乙烯。在较短的柱中使用较小直径的珠是一种非常有效的方式,以便以快速率实现高效能分离,但同时也意味着相当高的背压。因此,仅使用非常刚性的固定相材料。最常用的技术是反相色谱。
填充柱目前可用的HPLC固定相是基于1.7-10µm的珠状二氧化硅或合成聚合物,其中小直径的珠使得色谱效率提高。
分析性蛋白质分离的主要挑战是获得具有高分辨率和低非特异性吸附的快速分离。填充柱中的珠是多孔的或无孔的,具有各自的优点和缺点。二氧化硅和合成的无孔颗粒具有扩散路径短,仅在珠表面上结合的优势,这意味着更快的质量转移。多孔珠提供更大的表面积,这意味着更多的与分析物相互作用的位点和更高的结合能力。
已经描述了用于HPLC的其它类型的固定相。在US 5135650中,Hjertén等人描述了通过收缩珠或填充孔将多孔琼脂糖珠转化成基本上不渗透蛋白质的珠的方法。通过用不同的有机溶剂(例如二噁烷和氯仿)进行顺序处理并结合交联,进行Hjertén方法的收缩。填充是通过添加可聚合物质例如缩水甘油进行的,所述可聚合物质允许部分结合到内部孔以及珠表面。最终的珠被描述为具有足够降低的孔隙率,以防止分子量为3 kDa或更高的蛋白质渗透,并且填充有这些珠的柱已显示最高可承受40巴的压力。收缩方法的主要缺点是使用溶剂,例如二噁烷,其由于环境、健康和安全原因通常避免使用。
发明内容
本发明提供小的、刚性的和不可渗透的琼脂糖珠,其适合于在高效液相色谱(HPLC)中作为固定相,用于分析生物分子,例如肽和蛋白质;并且还用于在更大规模中的制备性用途。
本发明提供固体琼脂糖珠,并具有刚性以排除小至100g/mol的化合物,即基本上排除甚至最简单的二肽。
在第一方面,本发明涉及固体琼脂糖珠,其中所述琼脂糖珠是刚性的,并且对于分子量低至3000g/mol,优选100g/mol的化合物是不可渗透的。
优选地,所述珠具有1-25μm的直径,对于HPLC而言优选1-15μm,对于制备性应用而言优选15-25μm。
根据本发明的固体琼脂糖珠承受超过100巴的压力,优选≥300巴的压力,用于高压下的快速分析性应用和低压下的更大规模的制备性应用。小珠-高压。大珠-低压。
优选地,固体琼脂糖珠提供有触角/接枝聚合物,以增加表面积并实现功能化。
触角或珠表面可提供有配体,例如阳离子配体、阴离子配体、亲和(蛋白A、IMAC)配体、疏水相互作用配体或其组合。
在本发明的一个实施方案中,固体琼脂糖珠的直径为1-15μm,并装填在HPLC柱中。一个或多个HPLC柱可以连接至HPLC系统。当提供多于一个柱时,柱的珠优选具有不同的功能性(连接不同的配体)。
在第二方面,本发明涉及用于生产上述固体琼脂糖珠的方法,其包括以下步骤:在琼脂糖浓度为8-20%(w/w)的溶液中提供直径为5-50μm的琼脂糖珠;加热所述溶液至约45-99℃的恒温并使所述溶液中的所述琼脂糖珠乳化;将所述珠交联至少一次;通过例如烯丙基化使所述珠活化;和任选地将聚合物触角接枝在所述珠上以连接配体。或者,可以将配体连接至珠的表面。活化的其它实例是环氧活化、NHS或CNBr活化。
优选地,在所述聚合物触角上提供配体,例如亲和配体、离子交换配体和疏水相互作用配体。
在一个实施方案中,在乳化步骤之前,将磁性颗粒例如磁铁矿颗粒添加到溶液中。
在第三方面,本发明涉及上述固体琼脂糖珠用于分析生物分子,例如蛋白质和肽的用途,例如就分析物浓度、带电或疏水变体和表征而言。所述珠中排除小至100 g/mol的分子。
在一个实施方案中,所述珠的直径为1-15μm,并用于高效液相色谱(HPLC)应用。
在另一个实施方案中,所述珠的直径为15-25μm,并用于制备性和/或大规模应用,例如精制。
附图说明
图1显示了显示在填充有来自实施例1的无孔琼脂糖珠的连接的4.6mm(内径)×10cm PEEK柱上在不同流速下的平均HPLC系统压力的图表。
图2显示了抑肽酶分离的比较色谱图。图2A显示了来自实施例2的填充在4.6mm(内径)×10cm PEEK柱中的无孔琼脂糖珠上的抑肽酶的分离,所述柱采用10分钟、0-500mM氯化钠的线性梯度,1.66 ml/min。图2B显示了在3.2mm(内径)×3cm的现有技术Mini S柱上的抑肽酶的分离,所述柱采用10分钟、0-500mM氯化钠的线性梯度,0.80 ml/min。mAU - 214 nm处的单位。
图3显示了图3A:来自实施例2的无孔琼脂糖珠在PEEK柱中和图3B:现有技术MonoS柱上mAb分离的峰分辨率的比较色谱图。使用递增的pH梯度在离子交换柱上进行单克隆抗体带电变体的分离。各柱上装载46 µg单克隆抗体。mAU - 280 nm处的单位。
发明详述
本发明人假设琼脂糖珠可以通过塌陷天然孔结构而制成足够坚硬以承受高压,并且足够小以在分辨率和效率方面与目前可用的HPLC树脂竞争。同样,填充有这些无孔珠的柱中分析物的传质也会得到改善,从而导致更尖的峰。为了增加分析物与固定在珠上的配体之间的相互作用,在塌陷后进行表面接枝以增加可用的表面积。该生产方法是基于第一琼脂糖乳化步骤,随后是第二孔塌陷步骤,联合在珠经聚合物-触角接枝之前交联。珠随后可以用与常规的多孔基于琼脂糖的树脂所用的相同类型的配体进行功能化,从而例如实现亲和色谱、离子交换色谱和疏水相互作用色谱。与基于二氧化硅的HPLC珠相比,优势在于提高了pH耐受性,尤其是在碱性pH下。与非极性合成聚合物HPLC珠相比,基于琼脂糖的珠还是天然亲水的。这在希望最小化分析物和凝胶树脂之间不想要的疏水相互作用的技术中是有利的。
本发明人已经表明,可以生产平均直径为3-6μm的琼脂糖珠,并且填充有这些珠的柱是HPLC兼容的,并且可以在至少300巴的压力下操作。出乎意料的是,珠排除小至100 g/mol的化合物,基本上排除甚至最简单的二肽。这对于所有尺寸的相关生物分子都能实现有效的传质和尖而窄的峰。此外,信噪比将受益于更尖的峰。
在本发明的一个实施方案中,本发明的珠已经用弱阳离子交换配体进行功能化,并且功能测试已经使用具有范围从6kDa至160kDa的肽和蛋白质的样品进行,与现有技术相比,具有优良的分辨率和分析速度。
本发明描述了生产接枝有聚合物触角的固体或无孔琼脂糖珠的方法,用于分析性HPLC或高分辨率制备性应用,与先前描述的固定相相比,这提供独特的益处组合:碱性稳定性、亲水性树脂和快速传质,并具有增加的表面积。
下面描述了一些用于生产根据本发明的固体琼脂糖珠的一般和示例性原理:
1.乳化
将直径为5-50μm、水中琼脂糖浓度为8-20%(w/w)的琼脂糖珠用作生产本发明的小琼脂糖珠的起点。可以使用任何合适的乳化方法,例如使用有机连续相,优选甲苯,有或没有具有合适孔径的膜装置或通过搅拌。用于乳化琼脂糖珠的方法在US6602990和US7396467中描述。
2.固化和塌陷
交联前的高温步骤用于固化珠状的琼脂糖孔结构,并使珠无孔。所述预步骤使琼脂糖孔结构塌陷并使之在交联之前变得更致密。在此步骤中,温度通常设置为45-99℃。
3.交联
所述预步骤之后是在氢氧化钠存在下使用交联剂例如表氯醇的交联步骤。用于交联琼脂糖的方法在US6602990和US7396467中描述。
4.孔填充
通过在氢氧化钠存在下使用例如表氯醇的广泛交联的几个附加步骤来进行剩余孔体积的填充。
剩余孔体积还可以通过在氢氧化钠存在下偶联烯丙基缩水甘油醚(AGE),然后失活和水解来填充。用烯丙基缩水甘油醚活化和用溴失活的方法在EP1357988A1中描述。
5.功能化(任选)
功能化通过聚合将聚合物接枝到活化的基质上来进行。活化可以通过烯丙基缩水甘油醚(AGE)的偶联来进行。用于该步骤的方法在EP2841177B1中描述。
烯丙基缩水甘油醚的偶联,其可用于活化或填充剩余孔体积
通过将一种、两种或几种不同的单体与在水中的颗粒悬浮液和自由基引发剂混合,可以将单体接枝到活化的珠上。接枝到活化的珠上通过自由基聚合进行。将单体接枝到活化的珠上的方法亦在EP2841177B1中描述。
实验部分
实施例1:无孔琼脂糖珠的制备
乳化
固体珠通过以下程序由珠状琼脂糖制备。将209 g琼脂糖悬浮在1800 ml水中并加热至95℃以溶解琼脂糖(10%(w/w)),然后将温度降至70℃。在60℃将琼脂糖溶液添加到含甲苯2250ml、表面活性剂(tenside)和纤维素乳化剂的有机相中,以形成粗乳液。将该乳液泵送(加压)通过疏水性SPG膜重复通道,以获得窄的粒度分布。将收集的乳液冷却至22-20℃,并用乙醇洗涤以除去甲苯和乳化剂。乳化的琼脂糖珠最后用水洗涤,以在交联前除去乙醇。
进行两个平行程序以产生不同的乳化琼脂糖珠尺寸,分别为5μm和10μm。
固化和塌陷
称重乳化的琼脂糖凝胶树脂,并将浆液在水中的浓度设定为75% (419ml凝胶体积,559ml浆液)。将该凝胶在搅拌下转移至反应器中,并置于水浴中。将凝胶树脂加热至35℃,并将218g的Na2SO4加入混合物中。将混合物在35℃下放置70分钟。然后将混合物加热至设定的收缩温度,评估温度为87℃。将反应在设定温度下放置60分钟。温度降低至47.5(±1℃)之后,将5.6ml的25M NaOH溶液添加到反应中,然后将0.5g的NaBH4添加到反应中。
交联
用加料装置添加交联剂(表氯醇)和25 M NaOH溶液,持续5小时,添加的25 M NaOH和表氯醇的总量分别为61.5 ml体积。在搅拌下于47.5℃下将反应放置19±2小时(包括添加交联剂的5小时)。凝胶树脂然后在玻璃滤器上用蒸馏水(6x2凝胶体积)洗涤。
称重交联的凝胶树脂,并将浆液在水中的浓度设定为70%。在搅拌下将凝胶转移至圆底烧瓶中。加入4.32g NaAc,使其溶解15分钟。然后向该浆液中添加溴化物0.345ml,并在混合物变黄后将反应放置15分钟。15分钟后,用0.7克甲酸钠破坏残留的溴化物,直至浆液变白。将甲酸钠放置溶解15分钟,然后将温度升至40℃并加入48.1g Na2SO4并将反应放置60分钟。将13.75ml的25M NaOH溶液添加至混合物中,并将反应在搅拌下于40℃放置16-20小时。凝胶树脂然后在玻璃滤器上用蒸馏水(6x2凝胶体积)洗涤。
通过重复交联填充孔
称重水解的凝胶树脂,并将浆液浓度设定为75%凝胶和其余为蒸馏水。将该凝胶树脂转移至具有水浴并搅拌的反应器中。将凝胶加热至33℃,并将42.6g Na2SO4加入混合物中。将混合物在33℃下放置70分钟。之后,将温度升至47.5(±1℃),并在搅拌的同时将混合物放置60分钟。将1.1ml 25M NaOH溶液加入反应中。用dosimats添加交联剂(表氯醇)和NaOH持续5h,加入的NaOH和表氯醇的总量各为12 ml体积。在搅拌下将反应在47.5℃下放置19(±2小时)。凝胶然后在玻璃滤器上用蒸馏水(6x2凝胶体积)洗涤。
通过烯丙基化填充孔
称重凝胶浆液,将其加入玻璃滤器中,并用50%NaOH溶液(2x1凝胶体积)洗涤。排干凝胶,并将其与等量的25M NaOH一起加入反应器中(例如100g排干的凝胶加100ml 25MNaOH)。以200rpm开始搅拌。之后,添加NaBH4(对于100g凝胶为1g)。将凝胶浆液加热至50℃,然后加入烯丙基缩水甘油醚(AGE) (100g凝胶加200ml AGE)。搅拌增加到300rpm。将反应在50℃下放置过夜(16-20小时)。第二天,将凝胶浆液添加到玻璃滤器中,并用蒸馏水(3x2凝胶体积)、乙醇(5x2凝胶体积)和蒸馏水(5x2凝胶体积)洗涤。
结果
尺寸的确定
通过使用激光衍射粒度分析仪进行尺寸测量来分析所得的珠。当分别从最初的5和10µm乳化琼脂糖珠开始时,在此程序后,收缩的琼脂糖珠的平均直径为约3和5 µm。
孔隙率的测定
在100巴的恒定压力下,将4.6mm(内径)×10cm PEEK柱填充有来自实施例1的5μm无孔珠,持续15分钟。为确定KAV值,必须知道空隙,空隙是通过大分子量的化合物(例如BlueDextran 2000)的保留体积来测量的。孔隙率的测定是通过测量注入化合物,Blue Dextran2000 (2x106g/mol)、硝酸钾(101.1 g/mol)和丙酮(58.1 g/mol)的洗脱体积来进行的。Blue Dextran 2000的保留体积为0.78 ml,并且使用几何体积1.66 ml作为Vt得出的硝酸钾(Ve 0.78 ml)和丙酮(0.79 ml)的KAV值分别为0和0.01。通常通过从小分子的洗脱体积中减去空隙体积来测量柱的内部体积,在这种情况下,对于除非常小的化合物以外的所有化合物,这得到0-0.01ml,即无孔色谱固定相。
实施例2:制备具有阳离子交换配体的无孔琼脂糖珠
本实施例说明从5μm无孔琼脂糖珠开始,通过衍生化实施例1的无孔琼脂糖珠来制备阳离子交换剂。
将排干的凝胶树脂倒入三头圆烧瓶中。添加离子单体VSA、中性单体VP和水,参见以下方案。用乙酸和NaOH调节pH至pH 7-8。然后加入引发剂2,2’-偶氮二(2-甲基-丙脒)二盐酸盐(ADBA),并将具有连续氮气流的管降低到分散体中。然后将烧瓶降低到水或甘油浴中,并在搅拌下将反应在48-50℃下放置16-20小时。然后将凝胶树脂在玻璃滤器上用蒸馏水(6x2凝胶体积)洗涤。滴定凝胶树脂以测量反应后的离子容量。
使用乙烯基吡咯烷酮(VP)和乙烯基磺酸盐(VSA)将单体聚合到活化珠上
实施例3:无孔琼脂糖珠的压力与流速性能
本实施例证明了本发明的无孔琼脂糖珠的压力-流速性能。
在100巴的恒定压力下,在4.6mm(内径)×10cm PEEK柱上填充来自实施例1的5μm平均直径的无孔珠,持续15分钟。然后,使用PEEK手拧接头将PEEK柱连接至高压HPLC系统(Agilent 1260 Infinity II)。从流速为0.5 ml/min(181 cm/h线性流速)开始,将水泵送到柱上,并逐渐增加至3.9 ml/min(1408 cm/h)。记录不同流速下的平均系统压力。图1显示流速与系统压力之间的关系在整个范围内是线性的,表明柱可以在最高至少4 ml/min的流速下运行。
实施例4:色谱分离(比较例)
4A
本实施例比较了在Mini S柱(GE Healthcare)和填充有实施例2的无孔珠的PEEK柱上从低盐到高盐的梯度分离的抑肽酶(6.5 kDa)的峰效率。此外,在Mono S柱和实施例PEEK柱之间显示了用pH 6-9的递增pH梯度的单克隆抗体(160 kDa)分离的色谱图比较。
如实施例3中那样制备填充柱,该填充柱包含平均直径为5μm且离子容量为37μmol/ ml的实施例2的珠。该柱用20mM的磷酸钠(pH 6.5)平衡。将抑肽酶(1 mg)溶于1ml平衡缓冲液中,然后将20µl (相当于20 µg)注入柱上。在10分钟内以600 cm/h的线性流速以0-500 mM NaCl的线性梯度洗脱结合的抑肽酶。与相同方法和线性流速但注入5 µg抑肽酶相比,运行Mini S柱,3.2 mm(内径)×3 cm。以半峰高处的宽度表示的峰效率,对于填充无孔琼脂糖珠的柱为5.5秒,相比之下对于Mini S柱为17.5秒(分别为图2A和B)。
4B
用10 mM柠檬酸钠、10 mM磷酸钠、10 mM Tris (pH 5.3)平衡填充有来自实施例2的无孔琼脂糖珠的PEEK柱。在10 mM磷酸钠(pH 6.5)中将单克隆抗体(在CHO细胞中重组产生并在蛋白A亲和柱上纯化)从28 g/L稀释至2 g/L,并将23 µl (相当于46 µg抗体)注入柱上。在10分钟内以600 cm/h从pH 5.3的平衡缓冲液到pH 8.7的洗脱缓冲液以线性梯度洗脱结合的抗体。与相同方法但线性流速降低和梯度时间增加(300 cm/h持续20分钟)相比,运行Mono S柱,5 mm(内径)x 5 cm。主峰(最高峰)与分别在主峰前后的酸性和碱性变体之间的分辨率比较可从图3看出。填充无孔珠的PEEK柱在两倍速度时显示性能提高。
Claims (14)
1.固体琼脂糖珠,其中所述琼脂糖珠是刚性的,并且对于具有100g/mol的分子量的化合物是不可渗透的。
2.根据权利要求1所述的固体琼脂糖珠,其中所述珠具有1-25µm的直径。
3.根据权利要求1或2所述的固体琼脂糖珠,其中所述珠抵抗≥100巴,优选≥300巴的压力。
4.根据上述权利要求中一项或多项所述的固体琼脂糖珠,其中所述珠在表面上提供有触角/接枝聚合物。
5.根据上述权利要求中一项或多项所述的固体琼脂糖珠,其中触角/接枝聚合物或珠表面提供有配体,例如阳离子配体、阴离子配体、亲和配体(例如蛋白A、蛋白G、金属离子螯合剂)、疏水相互作用配体或其组合。
6.根据上述权利要求中一项或多项所述的固体琼脂糖珠,其中所述珠的直径为1-15μm,优选3-6μm并填充在HPLC柱中。
7.根据权利要求6所述的固体琼脂糖珠,其中将一个或多个HPLC柱连接至HPLC系统。
8.根据上述权利要求中一项或多项所述的固体琼脂糖珠的生产方法,包括以下步骤:在琼脂糖浓度为8-20%(w/w)的溶液中提供直径为5-50μm的琼脂糖珠;加热所述溶液并使所述溶液中的所述琼脂糖珠乳化;乳化后将所述珠交联至少一次;通过例如使所述珠烯丙基化来活化,以收缩并填充所述琼脂糖珠中的剩余孔;和任选地将配体添加至所述珠。
9.根据权利要求8所述的方法,其中所述配体被提供在接枝在所述珠上的聚合物触角上,例如亲和配体、离子交换配体和疏水相互作用配体。
10.根据权利要求8或9所述的方法,其中在乳化之前将磁性颗粒加入到溶液中。
11.根据上述权利要求1-7中一项或多项的无孔琼脂糖珠在分析生物分子例如蛋白质和肽中的用途。
12.根据权利要求10所述的用途,其中所述珠中排除小至100g/mol的分子。
13.根据权利要求10或11所述的用途,其中所述珠的直径为1-15μm并用于高效液相色谱(HPLC)应用。
14.根据权利要求10或11所述的用途,其中所述珠的直径为15-25μm并用于制备性应用。
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US5135650A (en) * | 1988-12-22 | 1992-08-04 | Bio-Rad Laboratories, Inc. | Chromatography stationary phase material for high performance liquid chromatography |
CN102989400A (zh) * | 2012-10-25 | 2013-03-27 | 江南大学 | 一种琼脂糖4b微球的交联方法 |
CN105713212A (zh) * | 2016-03-23 | 2016-06-29 | 艾美科健(中国)生物医药有限公司 | 一种琼脂糖交联凝胶微球的制备方法 |
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WO2003007814A1 (en) * | 2001-07-18 | 2003-01-30 | Agilex Biosciences, Inc. | Device and method for collecting, transporting and recovering low molecular weight analytes in saliva |
SE0402322D0 (sv) | 2004-09-22 | 2004-09-22 | Amersham Biosciences Ab | Method of preparing a chromatography matrix |
KR100754409B1 (ko) * | 2006-08-30 | 2007-08-31 | 삼성전자주식회사 | 원심력을 이용한 자성비드 팩킹 유닛, 이를 구비한미세유동 장치 및 상기 미세유동 장치를 이용한 면역학적검정 방법 |
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CN105713212A (zh) * | 2016-03-23 | 2016-06-29 | 艾美科健(中国)生物医药有限公司 | 一种琼脂糖交联凝胶微球的制备方法 |
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