Establishment And Application Of PH-regulated Salt-independent Hydrophobic Chromatography

Chromatographic separation plays an important role in the downstream technology of bioengineering.How to improve the effectiveness and economy of chromatographic separation is very important in this field.Theoretically speaking,orthogonal combinations of chromatographic methods can provide a common technology platform for protein separation,depending on the surface charge,molecular size,and hydrophobicity of proteins.However,hydrophobic chromatography has not played an important role in the process of protein separation in recent years.This is mainly due to the problems caused by the mode of classical hydrophobic chromatography which closely depends on high concentrations of salts to achieve desired separation.In this paper,a pH-dependent hydrophobic chromatography method is developed for the problem of hydrophobic chromatography.The method can achieve salt-independent protein adsorption and elution by increasing the hydrophobicity and coupling density of the ligand.The research contents of this paper include the following aspects:pH-dependent hydrophobic chromatography: To improve the adsorption selectivity,we use 1-naphthaleneacetic acid,more hydrophobic than ibuprofen,as a new hydrophobic ligand to synthesize the adsorbent.The effect of solution conditions and material properties on lysozyme adsorption was investigated.After density optimization,pH-dependent adsorption under low salt conditions can be achieved.In medium density adsorption medium,the adsorption capacity of lysozyme can reach 28.5 mg/mL under the condit ion of pH 10,which is only 0.8 mg/mL at pH 5.0.By SDS-PAGE analysis,1-naphthaleneacetic acid adsorption medium has a obvious separation effect on lysozyme in complex samples.The adsorbent can effectively separate lysozyme from egg white by usage of phosphate buffer at pH 5.0,after which the purity of lysozyme is over 90% and the recovery rate is about 80% by gray scal e scanning.The development of the application of pH-dependent hydrophobic chromatography method: The effects of solution conditions and material properties on the adsorption of antibodies by 1-naphthaleneacetic acid adsorbent were further explored.Similarly,pH-dependent adsorption under low salt conditions can also be achieved.Adsorption capacity of hIg G on medium density adsorption medium can reach 34.3 mg/mL under neutral conditions.And 1-naphthaleneacetic acid adsorption medium also has a obvious separation effect on hIgG in complex systems.The pH optimization result shows that the optimal loading pH for bIgG was 7.0 and the elution pH is 3.5.The optimization result of sample loading shows that the best loading volume for bIgG is 1:1.After optimization of the conditions,the purity of the antibody after one-step purification can reach more than 90%,and the recovery rate can reach over 70%.Moreover,the adsorbent can still maintain stability after soaking in acid and alkali,and the amount of antibody adsorbed still reached 30 mg/mL after five round of uses.The results of this paper indicate that the pH-dependent hydrophobic chromatographic mode based on 1-naphthylacetic acid can achieve efficient separation of lysozyme and bovine serum antibody.Meanwhile,it provides a new direction for designing and screening of hydrophobic chromatography ligands.