Bill E, Lutz U, Karlsson B M, Sparrman M, Allgaier H
Dr Karl Thomae GmbH, Germany.
J Mol Recognit. 1995 Jan-Apr;8(1-2):90-4. doi: 10.1002/jmr.300080116.
Compared to more stable chromatographic media the use of affinity media with biological ligands such as Protein G Sepharose 4 Fast Flow poses special challenges regarding regeneration and sanitization. This is especially critical for the purification of pharmaceutical proteins, where complete regeneration of the column between runs is of paramount importance. Here, the problems encountered during process development and upscaling of regeneration methods for a Protein G Sepharose Fast Flow column intended for the large-scale purification of pharmaceutical monoclonal antibodies are reported. The initially chosen alkaline regeneration buffer led to an increase in the affinity of Protein G towards antibodies which made elution increasingly difficult. A combination of urea and acetic acid was selected to ensure efficient cleaning of the matrix without affecting ligand properties. Validation experiments were done to demonstrate the functional integrity of the matrix after repeated cycles of use and regeneration, as well as the efficiency of the cleaning process.
与更稳定的色谱介质相比，使用具有生物配体的亲和介质（如蛋白G琼脂糖4快速流动介质）在再生和消毒方面带来了特殊挑战。这对于药物蛋白的纯化尤为关键，因为每次运行之间柱子的完全再生至关重要。本文报道了在用于大规模纯化药物单克隆抗体的蛋白G琼脂糖快速流动柱的工艺开发和再生方法放大过程中遇到的问题。最初选择的碱性再生缓冲液导致蛋白G与抗体的亲和力增加，使得洗脱变得越来越困难。选择尿素和乙酸的组合以确保在不影响配体性质的情况下有效清洁基质。进行了验证实验，以证明基质在重复使用和再生循环后的功能完整性以及清洁过程的效率。
