A simple, two-component buffer enhances use of chromatofocusing for processing of therapeutic proteins

To extend the feasibility of chromatofocusing to industrial use, we have developed a simple chromatofocusing buffer system capable of generating a smooth pH gradient without the use of an external gradient maker. Using two cationic buffering components, an internal pH gradient is produced on appropriate chromatography media over a broad pH range (9.5 to 5.0). The utility of this buffer system is demonstrated with PBE94 and DEAE Sepharose fast flow ion‐exchangers, as well as with experimental fast flow chromatofocusing gels. Using a rapid flow rate, we evaluated this buffer system for recovery of a therapeutic protein from a bacterial cell extract. The simplicity of the buffer system requiring no external gradient maker, coupled with the use of fast flow chromatographic media to produce broad‐range pH gradients, improves the scalability of chromatofocusing for processing of therapeutic proteins. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 208–215, 1999.