Department of Chemical Engineering, University of California, Davis, California 95616, USA.
Biotechnol Bioeng. 1992 May;39(11):1086-96. doi: 10.1002/bit.260391104.
One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity column performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.
亲和层析放大过程中的一个关键问题是支撑基质对压力的机械强度。由于亲和层析用凝胶基质和配体的成本都非常高，因此凝胶基质的可重复使用性直接关系到总生产成本。在某些情况下，原料粘性较大（例如血浆），凝胶基质可能会发生不可逆变形，这就需要对流速施加严格的限制。因此，生产效率较低。我们对系统参数进行了表征，并对各种市售基质的性能进行了研究。本研究采用的实验系统是从人血浆中免疫亲和纯化蛋白 C（一种抗凝蛋白）。所研究的支撑基质为交联琼脂糖、聚甲基丙烯酸、纤维素和聚乙烯醇聚合物。研究的主要系统参数为耐压能力、偶联效率、吸附效率以及单克隆抗体（MAb）-基质复合物上蛋白 C 的批吸附/解吸动力学。此外，通过脉冲测试，还对洗脱液中产物浓度分布的表观平衡常数和带宽进行了表征。建立了一种评估免疫亲和柱用于蛋白 C 分离性能的方法。通过利用实验测量的参数，计算了每个纯化步骤的流速限制。然后，根据单位时间的产率来评估基质的纯化性能。在所测试的基质中，纤维素在使用 10 cm x 10 cm 柱对蛋白 C 进行免疫亲和纯化时具有整体性能优势。
