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 (35×7 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°=1 mg/ml) in 100 m M 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.