Ameskamp N, Priesner C, Lehmann J, Lütkemeyer D
Institute of Cell Culture Technology, Faculty of Technology, University of Bielefeld, Germany.
Bioseparation. 1999;8(1-5):169-88.
The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG1 from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell-containing broth resulted in an additional decrease of the value down to 0.5 mg ml-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline 25 to a Streamline 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix.
这些研究的目的是评估在扩张床模式下使用离子交换基质从未稀释、未澄清的杂交瘤细胞培养液中回收鼠源IgG1的高效色谱首次捕获步骤的放大特性。所测试的新型基于磺丙基的离子交换基质（Streamline SP XL，Amersham Pharmacia Biotech）因其与前身（Streamline SP）相比容量增强而脱颖而出。在初步电泳分析（滴定曲线、SDS-PAGE）和小规模色谱结合研究中确定工作pH值表明，IgG纯化的最佳值为pH 4.6，此时培养基补充白蛋白（500 mg l-1，pI = 4.8）的共色谱现象无法避免。进一步的探索性实验研究了基质的动态容量，通过前沿吸附分析对其进行评估。在填充床模式下，即使每毫升基质施加6.5 mg IgG后，目标蛋白也未出现穿透。在无细胞上清液的扩张床模式下无法重现这些结果，在该模式下动态容量仅为1.5 mg IgG/ml SP XL。处理含细胞的培养液导致该值进一步降至0.5 mg ml-1，这可能是由于大量生物质吸附到基质上所致。对原因的探究导致对流体动力学条件进行研究。用示踪物质（丙酮）进行的缓冲液实验指出，扩张床中的流动比填充床中受返混效应和通道形成的影响显著更大。这些效应可以通过增加液相粘度来补偿，这可通过添加葡萄糖来实现。由于扩张床中流体动力学条件的改善，对于用400 mM葡萄糖处理细胞培养液，动态容量可从0.5 mg IgG/ml基质增加到超过4.5 mg IgG/ml基质。最后，在已证明为最佳的条件下进行了从Streamline 25柱到Streamline 200柱的放大：100 L未澄清的杂交瘤培养液在不到3.5小时内以95%的结合率进行浓缩。上样到柱中时目标蛋白未出现穿透。然而，洗脱液中仍含有碎片和细胞，这指出了该方法的主要缺点：生物质附着到基质上。
