CN112969533A - 色谱质量控制系统 - Google Patents
色谱质量控制系统 Download PDFInfo
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Abstract
本发明涉及通过使用可变路径长度分光光度计进行质量测试来实时控制色谱过程的方法。
Description
相关申请
本申请要求于2018年10月9日提交的美国专利申请系列号62/766,253的优先权,其整体并入本申请。
技术领域
本发明涉及通过使用可变路径长度分光光度计进行质量测试来实时控制色谱过程的方法。
背景技术
纯化生物分子的色谱过程是麻烦且耗时的过程。它需要能够监测UV吸收度、电导率、pH、流速和其他参数的设备。亲和色谱通常是纯化过程中的第一色谱步骤,并且是将感兴趣的蛋白主要从收获的细胞培养液或发酵收获物的复杂混合物中分离出来的地方。装载在色谱柱上的物料量、物料在色谱柱上的流速以及色谱柱尺寸或床高决定了物料在色谱柱中的停留时间。停留时间与动态结合容量(GE纸)有直接关系。色谱介质的动态结合容量是在未结合的蛋白发生显著穿透之前,在实际流动条件下该介质将结合的目标蛋白的量。对于任何给定的停留时间,存在与动态结合容量相关的穿透曲线。动态结合容量反映随着流速增加可能发生的传质限制的影响,并且与饱和或静态容量的测定相比动态结合容量在预测实际过程性能时更有用。亲和色谱过程中的穿透曲线描述离开色谱柱且未结合的物料的百分比。为了设计有效且有用的过程,应当确定取决于必须处理的质量的量的给定批次的适当的停留时间、装载和循环次数。通常,动态容量将随着停留时间的减少而减少,但是动态容量减少的速率会因介质不同而显著变化。理想的介质将在流速范围内具有有效的传质性能,但实际上流速有一个由介质的机械强度确定的上限。最大动态结合容量的过程标准的优化导致较少的过度工艺放大需求,以及减少了工艺时间、成本和蛋白质损失。即使单色谱柱色谱步骤也是如此,并且当纯化过程中使用几个色谱柱进行连续色谱时,这是复杂的。在进料浓度和/或流速随时间变化或色谱柱材料不同的情况下,动态结合容量将不同或将随时间变化。此外,色谱柱中材料的使用会随着时间而变化,并且当色谱柱是新的时使用的过程条件将与色谱柱较旧时不同。因此,需要提供关于在给定穿透水平下动态结合容量以及蛋白质滴度和质量信息的实时信息。
使用可变路径长度紫外分光光度计,而不是使用具有有限的线性范围的单路径长度紫外吸收传感器。由于可变路径长度分光光度计可以提供以吸光度/mm为单位的斜率值,所述斜率值可以使用消光系数(mL/cm*mg)容易且准确地转换为蛋白质的浓度,因此可以计算出准确的质量。
发明内容
在过去,使用线性范围有限的单路径长度紫外吸收传感器来确定色谱参数。在本发明中,使用可变路径长度紫外分光光度计,因为于可变路径长度分光光度计可以提供以吸光度/mm为单位的斜率值,所述斜率值可以使用消光系数(mL/cm*mg)容易且准确地转换为蛋白质的浓度,因此可以计算出准确的质量。
本发明涉及用于确定色谱柱的穿透百分比的方法,所述方法通过以下步骤进行:通过将收获的细胞培养液流经色谱柱足够长的时间以建立不变的信号来用斜率光谱确定给定蛋白质的初始斜率(m0),以及通过将传感器放置在色谱柱的入口并用斜率光谱测量斜率来确定第一斜率(m1),以及通过将传感器放置在色谱柱的出口并用斜率光谱测量斜率来确定第二斜率(m2),以及通过计算%BT=(m2-m0)/(m1-m0)*100来计算穿透百分比。
本发明还涉及方法用于确定色谱柱的蛋白质滴度的方法,所述方法通过以下步骤进行:通过将收获的培养细胞液流经色谱柱足够长的时间以建立不变的信号来确定初始斜率(m0),其中初始斜率由斜率光谱确定,然后通过将传感器放置在色谱柱的入口并用斜率光谱测量斜率来确定第一斜率(m1),然后通过计算滴度=(m1-m0)/EC来计算色谱柱的滴度,其中EC是蛋白质的消光系数,其单位为mL/mg*cm。
本发明涉及用于确定装载在色谱柱上的蛋白质的实时质量的方法,所述方法包括如上所述确定色谱柱的蛋白质滴度,并通过计算质量柱1(mg)=滴度*流速*时间,来计算装载在色谱柱上的蛋白质的实时质量。
本发明涉及用于在具有两个色谱柱的色谱过程中确定装载在第二色谱柱上的蛋白质的实时质量的方法,所述方法包括如上所述确定第一色谱柱的穿透百分比,并通过计算质量柱2(mg)=%BT*滴定度*流速*时间,来计算装载在第二色谱柱上的蛋白质的实时质量。
相似类型的控制方案可用于后续的修正步骤,例如阴离子交换色谱、阳离子交换色谱或混合模式色谱。
具体实施方式
将已知波长λ(即紫外、红外、可见光等)和强度(I)的电磁辐射(光)入射到比色皿的一侧。将测量出射光强度I的检测器放置在比色皿的另一侧。光通过样品传播的长度是距离d。大多数标准紫外/可见分光光度计使用具有1cm路径长度、通常容纳50至2000μL样品的标准比色皿。对于由浓度为c的单一均匀物质组成的样品,透射过该样品的光将遵循称为比尔定律的关系:A=εcl,其中A是吸光度(也称为样品在波长λ下的光密度(OD),其中OD=透射光与入射光之比的负对数),ε是吸光系数或消光系数(通常在给定的波长下恒定),c是样品的浓度,l是光穿过样品的路径长度。
通常溶液中的感兴趣的化合物是高度浓缩的。例如,当测量吸光度时,某些生物样品(如蛋白质、DNA或RNA)通常被分离出来的浓度落在分光光度计的线性范围之外。因此,通常需要稀释样品以测量落在仪器线性范围之内的吸光度值。需要对样品进行多次稀释,这导致稀释误差以及用于任何下游应用的稀释样品的移出。因此,在不知道可能浓度的情况下获取现有样品,并在不稀释的情况下测量样品的吸光度是理想的。
在例如蛋白质纯化的连续过程中,本发明的一个或多个流量传感器可用于过程的每个步骤或过程中的特定位置。在过程的第1步骤中,收获的材料是目标蛋白、宿主细胞、介质、DNA和其他杂质的组合。斜率信号将给出所有这些成分的吸光度贡献。通过特征分析,可以使用光谱信号来定量成分。光谱可用作柱前指示剂,其与柱后斜率信号比较以确定分批或连续过程中的色谱柱装载。或者,在柱前和柱后使用斜率信号可以确定产物滴度。一旦将产物滴度与浓度信号进行比较,就可以确定装载过程中的实时质量。这允许在色谱柱之前的物料含有装载物料的全部补体。一旦色谱柱被装载,目标蛋白吸附或结合到色谱柱上,并且流过色谱柱的物料是来自收获物料的杂质。相反地,在排阻色谱柱中会捕获杂质并允许目标物料通过色谱柱。在亲和柱之后,过程的第二步骤可能是监测该过程的最佳位置。这个步骤是大部分物质纯化发生的地方。斜率信号可用于查看色谱柱何时满载。这可以通过比较流经传感器时的背景信号(仅由于收获物料)与在收获物料和装载物料一起的稍后时间的信号来实现。这发生在树脂装载到容量时。或者,通过获得产物滴度和实时浓度,可以通过装载的总蛋白质的质量来控制色谱柱上的装载。像pH、流速、电导率、树脂的尺寸和构型、树脂的类型或温度的参数可能影响装载容量。仅用该斜率信号,可快速确定并通过实验改变装载容量以获得理想的过程参数。在连续过程中,可能会有许多亲和色谱柱被单独装载至容量然后洗脱。色谱柱之间的洗脱峰的长期比较可以表明树脂容量是否随时间下降,这表明需要更换色谱柱或过程中的其他变化。在洗脱过程中添加光谱测量可以允许溶液中存在的单个成分的量化。步骤3和4是修正步骤,并且在每个修正步骤中斜率传感器为该过程提供浓度的连续量化和总产率值。由于流量传感器的大动态范围,在离子交换色谱分离中可以对多种物质进行量化,否则会进行离线分析。在步骤5中,在UF/DF阶段之后的传感器给出浓度值,该浓度值是已被处理/纯化的药物物质的最终浓度。浓度可以在整个过程中容易地监测,无需大量表征,这与例如折射率监测的其他方法形成对比。斜率值在多数情况下与缓冲无关。渗透液也可以在正常处理或结合中监测。在最后步骤,填充站处的流量传感器将给出最终的药瓶浓度(vial concentration)。它可以用于捕获所有剩余的物料并用于确定最终过程收率。虽然在本发明的方法的许多实施方案中可以监测单个波长,但是在某些情况下监测两个或多个波长可能是有利的。例如随着时间的推移,产品线中的污染物可能积聚,使得污染物沉积,使得最终到达检测器的光部分或完全被阻挡。在连续过程中监测非峰值波长可以在成为难题之前检测到该问题。
一种可变路径长度分光光度计,其通过软件控制响应实时测量动态调整参数以扩展传统分光光度计的动态范围,使得可以在不稀释或浓缩原始样品的情况下测量几乎任何浓度的样品。此外,本发明的方法不需要已知路径长度来确定样品的浓度。
本发明的方法提供了一种新的技术,该技术通过在加载曲线期间建立单位为Abs/mm的初始斜率(m0)并将其从色谱柱前后的斜率中减去来确定装载质量。然后应用流速(mL/min)和消光系数并实时积分,以确定装载在色谱柱和/或后续色谱柱上的质量。在本发明中,使用1个或2个传感器的组合。在有2个传感器的方案中,一个放置在产生第一斜率值(m1,Abs/mm)的色谱柱入口处,一个放置在产生第二斜率值(m2,Abs/mm)的色谱柱出口处。可以使用入口的离线斜率测量的组合来代替m1。初始斜率(m0)是通过将收获的细胞培养液(HCCF)流经色谱柱足够长的时间以建立在一个在一段时间内保持相对不变的信号来确定。该体积通常在至少1-2个柱体积流过色谱柱后确定。在信号稳定之前,可能需要多达4个柱体积(CV)通过色谱柱。在建立了m0斜率(Abs/mm)之后,可以将该值输入控制系统以开始绘制%穿透率相对时间的曲线。
%BT=(m2-m0)/(m1-m0)*100
蛋白质滴度也可如下确定:
滴度=(m1-m0)/EC
滴度以mg/ml为单位,m1和m0以Abs/mm为单位,且EC以mL/mg*cm为单位。
装载在色谱柱上的实时质量为
质量柱1(mg)=滴度*流速*时间
装载在后面色谱柱上的实时质量为
质量柱2(mg)=%BT*滴度*流速*时间。
该控制方案可用于单柱或多柱亲和色谱中。在单柱色谱中,质量控制允许色谱柱上的最大装载。该方法的使用将增加分批过程的灵活性和控制。不再需要考虑树脂降解,因为控制系统适应任何结合能力。
在多柱过程中,质量控制实时提供第一色谱柱和第二色谱柱的装载。然后该控制系统可以适应滴度可能是动态的灌注生物反应器。有了质量控制系统,可以快速且准确地确定时间。在连接的分批多柱过程中,它提供与单柱相似的优点。
流通装置可以充当用于本发明的方法形成的测量的容器。流通装置包含流动池体,该流动池体允许样品溶液流入和流出流动池装置。流动池体具有至少一个对电磁源(通常为200至1100nm)范围内的电磁辐射透明的窗口。窗口可以由各种材料制成,但是对于紫外线应用,可能需要石英、环烯烃聚合物(COP)、环烯烃共聚物(COC)、聚苯乙烯(PS)或聚甲基丙烯酸甲酯(PMMA)。窗口可以是不同的尺寸和形状,只要电磁辐射可以穿过窗口并撞击检测器。在流动池系统中,检测器和探针尖端可以处于基本水平的方向,并且样品在检测器和探针之间流动。在一个替代实施方案中,可以使用镜子将电磁辐射反射到窗口并穿过窗口。镜子和窗口的放置不受限制,只要镜子可以反射电磁辐射通过窗口使得辐射被检测器检测即可。在某些实施方案中,镜子和窗户可以彼此相对或彼此成直角。不管探针和检测器的绝对空间取向如何,探针尖端和检测器表面应当基本相互垂直。流动池主体还包括探针尖端可穿过的端口。该端口用动态密封件密封,使得探针尖端可以通过端口而样品溶液不从流通装置泄露。这样的密封件包括可从犹他州西盐湖城的Parker HannifinCorporation EPS Division获得的FlexiSeal Rod和Piston Seals。在示意图中,样品溶液具有一条流入入口和流出出口的路径。替代实施方案可以包括多个路径以及多个入口和出口。在流动池装置中,探针尖端基本垂直于样品溶液流移动并且基本垂直于检测器。流动池可以具有各种内径。各种流动池直径是给定过程中所需的体积和流速的函数。
流动池可以通过各种配件结合到流动流中。3mm ID流动池使用倒钩配件或鲁尔配件。10mm ID流动池使用三夹钳配件。在流动池的一个优选实施方案中,流动池由不锈钢316制成,具有石英窗口和包在不锈钢中的光纤。在该优选实施方案中,在光纤(fibrette)的任一侧上有2个特氟龙密封件,所述光纤在流动池中上下活塞运动以进行读数。或者,固定在光纤上的并固定在流动池中的垫圈可以在确保准确的路径长度变化的同时提供适当的密封。在流动池的一个优选实施方案中,光纤的外径与静态系统相比增加。在优选的实施方案中光纤的外径可以小于1mm或大于25mm。光纤的尺寸将取决于应用,所述应用将影响流动池的尺寸和流过流动池的流体速率。在优选的实施方案中,光纤有足够的直径,以使其不会振动、弯曲或断裂。光纤外径的增加减少了影响测量精度的设备振动。在流动池的一个优选实施方案中,在特氟隆密封件之间放置有一个不锈钢塞子。塞子填充了流动池中可能存在清洁挑战的空隙。随着空隙被填充,流动池更容易清洁。流动池中的其他密封件可由铂固化的有机硅制成。随着时间的流逝,标准的EPDM密封件可能释放出一些可能会污染流动池的物质,而铂固化的有机硅的使用避免了这个潜在问题。本发明的流动池能够被消毒或清洁,使得它们可以用于需要消毒或无菌环境的过程中。
检测器包括能够将检测到的光的能量转换成可由装置处理的信号的任何机制。合适的检测器包括光电倍增管、光电二极管、雪崩光电二极管、电荷耦合器件(CCD)和增强型CCD等等。根据检测器、光源和分析模式,这样的检测器可以用于各种检测模式,包括但不限于离散、模拟、点或成像模式。检测器可用于测量吸光度、光致发光和散射。尽管在优选实施方案中使用单个检测器,本发明的装置可以使用一个或多个检测器。在优选实施方案中,光电倍增管用作检测器。本发明仪器的检测器可以集成到仪器中,或者可以通过可操作地将检测器连接到光传送装置而位于远处,所述光传送装置可以将穿过样品的电磁辐射带到检测器。光传送装置可以是熔融石英、玻璃、塑料或适合电磁源和检测器波长范围的任何可透射材料。光传输装置可以由单根纤维或多根纤维组成,并且这些纤维可以根据仪器的使用而有不同的直径。纤维可以是几乎任何直径,但是在大多数实施方案中,纤维直径在约0.005mm至约20.0mm的范围内。
控制软件将基于各种标准调整设备行为,所述标准例如但不限于波长、路径长度、数据采集模式(对于波长/路径长度)、动力学、触发器/目标、离散路径长度/波长带,以提供光谱不同区域的不同的动态范围/分辨率、横截面图以创建吸光度/路径长度曲线、回归算法和斜率确定、根据斜率值的浓度确定、消光系数确定、基线校正和散射校正。该软件被配置为提供扫描或离散波长读取选项、信号平均时间、波长间隔、扫描或离散路径长度读取选项、数据处理选项例如基线校正、散射校正、实时波长横截面、阈值选项(例如波长、路径长度、吸光度、斜率、截距、测定系数等)动态/连续测量选项。
Claims (4)
1.一种用于确定具有入口和出口的色谱柱的穿透百分比的方法,其包括:
通过将收获的细胞培养液流经色谱柱足够长的时间以建立不变的信号来确定初始斜率(m0),其中所述初始斜率由斜率光谱确定;
通过将传感器放置在色谱柱的入口并用斜率光谱测量斜率来确定第一斜率(m1);
通过将传感器放置在色谱柱的出口并用斜率光谱测量斜率来确定第二斜率(m2);和
通过计算%BT=(m2-m0)/(m1-m0)*100来确定穿透百分比。
2.一种用于确定具有入口和出口的色谱柱的蛋白质滴度的方法,其包括:
通过将收获的细胞培养液流经色谱柱足够长的时间以建立不变的信号来确定初始斜率(m0),其中所述初始斜率由斜率光谱确定;
通过将传感器放置在色谱柱的入口并用斜率光谱测量斜率来确定第一斜率(m1);
通过计算滴度=(m1-m0)/EC来计算色谱柱的滴度,其中EC是蛋白质的消光系数,其单位为mL/mg*cm。
3.一种用于确定装载在色谱柱上的蛋白质的实时质量的方法,其包括根据权利要求2所述确定色谱柱的蛋白质滴度,并通过计算质量柱1(mg)=滴度*流速*时间,来计算装载在所述色谱柱上的蛋白质的实时质量。
4.一种用于在含有第一色谱柱和第二色谱柱的色谱过程中确定装载在第二色谱柱上的蛋白质的实时质量的方法,其包括根据权利要求1所述确定第一色谱柱的穿透百分比,并通过计算质量柱2(mg)=%BT*滴定度*流速*时间,来计算装载在所述第二色谱柱上的蛋白质的实时质量。
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WO2018153743A2 (en) * | 2017-02-21 | 2018-08-30 | Ge Healthcare Bio-Sciences Ab | Method for adapting uv cell pathlength in a chromatography system |
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KR102489233B1 (ko) | 2023-01-17 |
SG11202103572VA (en) | 2021-05-28 |
JP2022512659A (ja) | 2022-02-07 |
EP3863763A1 (en) | 2021-08-18 |
JP7173671B2 (ja) | 2022-11-16 |
WO2020076818A1 (en) | 2020-04-16 |
CA3123030C (en) | 2023-04-04 |
US20220042969A1 (en) | 2022-02-10 |
CA3123030A1 (en) | 2020-04-16 |
KR20210066912A (ko) | 2021-06-07 |
AU2019359263B2 (en) | 2022-09-15 |
EP3863763A4 (en) | 2022-10-19 |
AU2019359263A1 (en) | 2021-05-20 |
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