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.
Logan
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KA
Natural Sciences Department, College of Notre Dame, Belmont, California, USA.
Lagerlund
I
I
Chamow
S M
SM
D001709:Biotechnology; D002021:Buffers; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D019382:Human Growth Hormone / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use
D002021:Buffers; D011994:Recombinant Proteins; D019382:Human Growth Hormone