Application Of Agarose Based Hydrophilic Interaction Chromatography In Separation And Purification Of Peptides

Great attention has been paid to peptides in the field of new drugs development in the past few years due to the special properties of peptides, including lower molecular weight than that of proteins, simple structure, easy to be synthesized and uptaken by the organism and the diversity of administration route. Peptides have a lot of biological functions, such as antibiosis, antivirus, antioxidation, antitumor, neuroregulation and immunoregulation. Some structural analogues are formed during the process of chemical synthesis of peptides, and natural derived peptide crudes also contain a variety of impurities. At present, the items of available separation and purification methods of peptide are very limited and mainly based on reversed phase chromatography. Those methods can not meet the demands of quick developing market of peptide drugs.In the present thesis, agarose gels were introduced into the field of separation and purification of peptides as hydrophilic interaction chromatographic media. The purpose is to expand the application field of agarose gel and the method of peptide separation. First, the highly cross-linked 12% agarose gel Superose 12 was used to separate synthetic glycine dipeptide and tripeptide. The general rules of the formation of adsorption between Superose 12 and the framework of peptides (peptide bond and terminal charged groups) were investigated. Second, Superose 12 was introduced into separation and purification of peptides from protein hydrolysis. A preliminary prediction model for peptide retention time of Superose 12-hydrophilic interaction chromatography was established. Finally, four types of agarose gels with different partical sizes and ligands were introduced to separate synthetic oxytocin crude, including Superose 12. The separation results with different media were compared. The main conclusions were as follows:1. Synthetic glycine dipeptide and tripeptide were separated by Superose 12, a agarose gel medium. Glycine oligopeptides can be retardated efficiently on Superose 12 when mobile phase of acetonitrile-water was used. The dipeptide and tripeptide were completely separated from each other. The retention volumes of glycine oligopeptide increased with the fraction of acetonitrile in the mobile phase and decreased with pH value of the mobile phase. The retention volume was reduced when urea or sodium chloride was added into the mobile phase. These results indicated that the adsorption mechanism between Superose 12 and glycine oligopeptides could be a mixed-mode of hydrogen bond adsorption and electrostatic interaction. Dynamic adsorption isotherms were determined by the method of elution by characteristic points. Together with the calculation results of adsorption energy distribution, it was suggested that the interactions between the Superose 12 medium and glycine oligopeptides were slightly heterogonous and the isotherm adsorption data could be fitted by the Toth model excellently.2. Peptides from protein hydrolysis were separated by Superose 12 medium. Lysozyme hydrolysate, ribonuclease A hydrolysate and peptides from trypsin autodigestion were separated with decreased acetonitrile gradients, respectively. Ten to twenty peaks were appeared per run. According to the results of amino acid sequence analysis by mass spectra, forty seven peptides were recognized and collected from three hydrolysates. A prediction model of peptide retention time on Superose 12 column was established based on the retention coefficients corresponding to 20 amino acids and peptide bond. According to the retention datas of 47 peptides, the coefficients as unknown variables were solved. It can be demonstrated that amino acid residues with ionogen, phenolic hydroxyl group or amide group could have a positive contribution to the retention of peptides.3. Synthetic oxytocin crude was separated by four types of agarose gel media including Superose 12, Superose Prototype, Sepharose HP-β-CD and Novarose SE-100/40-Tris. Using an optimized gradient elution method with acetonitrile-water-acetic acid sequentially, the purity of oxytocin crude increased from 56.7% to 92.8% after separation on Superose 12 column (10μm particle size, hydrogen bond acceptor type). The purification folds were 1.64 and the recovery rate of oxytocin was 78.7%. The sample capacity of Superose 12 was very high and exceeded 0.34 mg oxytocin per ml gel. The elution profiles and separation results of Superose Prototype (hydrogen bond acceptor type) and Sepharose HPβ-CD (mixed type) media were similar to that of Superose 12. These media with 30μm particle size could be used to scale up. However, oxytocin could not be retardated effectively by Novarose SE-100/40 Tris (hydrogen bond donor type) medium under the acetonitrile-water mobile phase condition.