| With the rapid development of the biopharmaceutical industry,efficient downstream protein purification technology is of great significance for the large-scale biopharmaceuticals production.Chromatography is an indispensable separation and purification technology in the current production of biopharmaceuticals.As a kind of chromatography technology,mixed-mode chromatography has attracted more and more attention.In this study,three different types of mixed-mode chromatographic media were obtained using polyacrylate microspheres as the matrix.The mixed-mode chromatographic media were optimized including the ligand density and pore size parameters.We investigated the performance of media including the ligand densities,salt tolerance and protein recovery.Furthermore,the effects of different pore sizes on the separation performance of media were studied,and the chromatographic retention mechanism of mixed-mode media was explored.The main research contents are as follows:1)Strong anion exchange/hydrophobic mixed-mode chromatography media(BMEA-MMC)was prepared with polyacrylate microspheres as matrix and N-benzyl-N-methylethanolamine(BMEA)as ligand.The effects of reaction time,reaction temperature and BMEA concentration on the ligand density of BMEA-MMC were investigated.The ligand density of BMEA-MMC reached 0.217 mmol/m L.It was found that with the increase of ligand density,the protein adsorption properties of BMEA-MMC showed an upward trend using bovine serum albumin(BSA)as a model protein.Compared with NM90 Agarose-HAM,BMEA-MMC showed good salt tolerance under medium and high salt concentration.At the same time,in terms of protein recovery,BMEA-MMC with high ligand density had similar performance with Capto Adhere.The BMEA-MMC with different pore sizes was employed to the separation and purification of porcine blue ear virus.The result showed that no target protein was detected in the flow-through fraction.At the same time,the larger pore size BMEA-MMC showed excellent mass transfer performance and separation efficiency.This indicates that BMEA-MMC with the large pore size of 275 nm has a large potential in the separation and purification of biological macromolecules such as viruses.2)The weak anion exchange/hydrophobic mixed-mode chromatography media(AMP-MMC)was prepared by optimizing the reaction time,reaction temperature and AMP ligand concentration with polyacrylate microspheres as matrix and 2-amino-3-methylpyridine(AMP)as ligand.The ligand density of AMP-MMC can reach 0.209mmol/m L.The preparation method of AMP-MMC was established.The effects of p H and Na Cl concentration on protein recovery were investigated.It was found that the recovery of BSA increased significantly with the decrease of p H and the increase of Na Cl concentration.AMP-MMC eventually showed a recovery of more than90 %.The salt tolerance of the media was investigated.It was found that the salt tolerance of AMP-MMC decreased with the increase of the ligand density within a certain range of ligand density.When the ligand density was about 0.18 mmol/m L,the salt tolerance increased instead.The ion exchange and hydrophobic interaction modes was verified through measuring retention factor of BSA on AMP-MMC.For the separation of human immunoglobulin,p H 8.0 and 0.25 mol/L Na Cl were used as the loading conditions.p H 5.0 acetate buffer was used as the elution condition.In the separation process,the chromatographic performance of AMP-MMC media with large pore size of 275 nm was significantly better than that of AMP-MMC with pore size of 49 nm.The separation of bovine serum albumin and human immunoglobulin was realized,which was suitable for the separation and purification of biological macromolecules such as antibodies.3)The cationic exchange/hydrophobic chromatography media(MPS-MMC)with sodium 3-mercaptopropane sulfonate(MPS)as ligand was prepared by thiol-epoxy method using polyacrylate microspheres as matrix under alkaline conditions.The media contains three interaction modes including cation exchange,hydrophobic and sulfur affinity.Through the investigation of the main reaction conditions,the optimal reaction conditions for ligand coupling were established,and the static and dynamic binding capacities of lysozyme on MPS-MMC were tested.The static binding capacity reached 105 mg/m L,and the dynamic binding capacity reached 34 mg/m L.By testing the retention factor of lysozyme on MPS-MMC,it was confirmed that the media had ion exchange and hydrophobic chromatographic mode.When the MPS-MMC with different pore sizes was applied to the separation and purification of lysozyme,the acidic protein in egg white showed flow-through phenomenon.The final results showed that the MPS-MMC with different pore sizes had high separation performance for lysozyme in egg white.To sum up,this paper established a method for preparation of mixed-mode chromatography media,which can be adjusted and designed according to different ligand density requirements.The influence of ligand density of mixed-mode chromatography media on chromatographic performance was explored in detail.It was found that three kinds of polymer mixed-mode chromatography media can be applied to the separation and purification of biological macromolecules.But it is necessary to select a media with the reasonable pore size according to the size of the target protein.At the same time,it is necessary to further optimize the structure of mixed-mode chromatography media to improve the protein binding capacity of the media to obtain better chromatographic performance. |
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