Abstract: Based on the fact that the resolution of intact protein separation is almost independent of column length, the effect on the resolution for intact protein separation causing from dynamic factors in hydrophobic interaction chromatography (HIC) was investigated. A concept of “conditional plate height” (H) for protein separation is firstly suggested for characterizing this effect for protein separation under linear gradient elution. Standard proteins were separated with conventional chromatographic column and chromatographic cake, and the plot of the H vs the linear velocity of mobile phase (u) was made, respectively. It was found that the obtained plot is similar to the conventional van Deemter Plot but has some differences. The optimized u corresponding to the minimum H was determined to be approximate 0.2 mm/s for the chromatographic cake and 1~3 mm/s for the conventional column. Furthermore, in comparison with the latter, optimized u value for the former has much broader range. Based on this fact, the resolutions and speeds for standard protein separation between the chromatographic cake packed with silica-base HIC material and the conventional column packed with soft HIC media were compared. The chromatographic cake (10 mm×20 mm i.d.) was found to perform a complete separation of seven standard proteins in 10 min, while with the latter (55 mm×12 mm i. d.) only five standard proteins can be completely separated in 140 min, even though the sample load for the former having bed volume of 3.14 mL, five times of that of the latter. The HIC chromatographic cake was also employed for the renaturation with simultaneous purification of the recombinant human granulocyte colony stimulating factor. The obtained purity was ≥97%, mass recovery was 39%, specific bioactivity was 1×108 IU/mg with only one step HIC in 50 min. It would be expected that when a kind of packings having very small particle size is packed into a chromatographic cake with diameter to be greater than its thickness and is employed to separate, and/or renature proteins, a result of high speed and high resolution with simultaneous renaturation under high protein loading (“three H” target) could be obtained.

Key words: biotechnology, chromatographic cake, dynamic factor, granulocyte colony stimulating factor , protein separation, resolution, hydrophobic interaction chromatography