Continuous-bed chromatography for the analysis and purification of recombinant human basic fibroblast growth factor.
The chromatographic properties of the commercial cation exchanger UNO-S1 (35x7 mm) was investigated using lysozyme from hen egg white as model protein and recombinant human basic fibroblast growth factor (rh-bFGF) from a high cell density cultivation of E. coli. The dynamic capacity for lysozyme (c(o) = 1 mg/ml) in 100 mM acetate buffer, pH 5 was 27 mg per ml sorbent. It was found independent of the flow-rate from 78 to 935 cm/h owing to the absence of mass transfer restrictions with this column concept. Regarding the selectivity for rh-bFGF and the capacity for lysozyme, no changes were apparent after cleaning-in-place (CIP) procedures with 0.5 M NaOH. Clogging of the column by a clarified crude cell homogenate of E. coli was not critical as precipitates were removed by reversal of the flow during CIP. Rh-bFGF elutes in three consequent peaks from the UNO-S1 column, which could be attributed to soluble rh-bFGF aggregates of different size. The dynamics of rh-bFGF aggregation and reaggregation in the crude feedstock was monitored by fast gradient elution chromatography.
Garke
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G
Biochemical Engineering Division, GBF-Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, Braunschweig, Germany.
Radtschenko
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I
Anspach
F B
FB
D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000032:analysis* / Q000302:isolation & purification*; D006801:Humans; D008855:Microscopy, Electron, Scanning
D002411:Cation Exchange Resins; D016222:Fibroblast Growth Factor 2