Large-Scale Displacement High Performance Liquid Chromatography for Separation of Peptides and Proteins

本提案旨在确定在色谱置换模式下扩大高效液相色谱（HPLC）分离用于生产制药级合成肽和重组蛋白的可行性。在某些情况下，高效液相色谱法是唯一的大规模工艺步骤，可产生人类治疗用途所需的纯度水平。传统的洗脱色谱法可以方便地扩大到制造所需的水平，但价格昂贵，利用柱容量差，难以重复操作。置换模式是在类似于传统HPLC操作的条件下进行的，但不是洗脱液强度的梯度，而是通过精心选择的溶液将进料成分从色谱柱中置换出来，该溶液中含有比任何进料成分吸附更强的成分。纯化后的产物在置换剂前端的相邻各组分带中退出色谱柱。在这种色谱模式中，浓度和柱负载通常比洗脱色谱高10到100倍，因此置换更充分地利用了柱和设备的容量。这些因素加上控制方面的简化，使置换色谱法成为一个很有前途的制造步骤。

This proposal seeks to determine the feasibility of scaling up High Performance Liquid Chromatography (HPLC) separations in the displacement mode of chromatography for the production of pharmaceutical grade synthetic peptides and recombinant proteins. In certain cases, HPLC is the only large scale process step that yields the purity levels required for human therapeutic use. Conventional elution chromatography can be conveniently scaled up to the level required in manufacturing, but is expensive, poorly utilizes column capacity and is difficult to reproducible operate. The displacement mode is performed under conditions similar to conventional HPLC operations, but instead of a gradient in eluent strength, the feed components are displaced from the column by a carefully chosen solution containing a component that is more strongly adsorbed than any of the feed components. The purified product exits the column in adjacent bands of individual components just ahead of the displacer front. Concentrations and column loadings in this mode of chromatography are typically ten- to one hundred-fold higher than in elution chromatography, and so displacement more fully exploits the column and equipment capacity. These factors together with simplified control aspects make displacement chromatography a promising manufacturing step.