| Based on stoichiometric displacement model for retention (SDM-R) in liquid chromatography, a short column theory was firstly proposed. Two equations for evaluating the effective migration column length (Z.err) of a solute and the minimum column length (Z,mjn) for separating a pair-solute were theoretically derived. According to the equations, different sizes of chromatographic pies were designed, manufactured, and applied to the renaturation with simultaneous purification of recombinant human therapeutic interferon-y in a productive scale. A new packing method-radial packing method specially to the chromatographic pies was also explored. The separation efficiency, renaturation ability, the curves of backpressure versus flow rate and the minimum volumes of mobile phase employed were investigated. The extract of rhlFN-y in guanidine hydrochloride solution was directly injected to these designed pies, resulting the purity to be over 95% satisfying the purity request of the domestically biological products, the highest specific activity to be 9.7><107IU/mg being six folds of the domestic criteria. Compared to the total bioactivity of rhIFN-y in the extract before the renaturation by the chromatographic pies, that increased 62 folds after renaturation and purification. This work includes six parts as following:1. Short column theory and the minimum length and its testingWith the SDM-R, a short column theory was proposed. Based on the fact that the resolution of biopolymers by high performance liquid chromatography (HPLC) was almost independent of column length and five assumptions, two equations for calculating the Z,er, and Z,min for a pair of difficultly separated biopolymers were theoretically derived. The Z,mm and Z,etr. were found to relate to the parameters in these equations as follows: the flow rate of mobile phase, gradient steepness, initial concentrations of displacer in the mobile phase, at this moment that the pair ofbiopolymers begin to migrate on a stationary phase, aou and ao2i, respectively, and the parameters characterizing the retention behavior, such as Z (the total moles of solvent released from the contact surface rejoin of 1 mole of the solvated solute absorbed onto solvated stationary phase) and log/ (a constant relating to the affinity of 1 mole of solute to stationary phase). In addition, other parameters, such as the final concentrations of the displacer making the pair of biopolymer be almost completely separated, aoi2 and ap22 were also found to effect on the Lm\n and Le(f. The Z,err and Lmm of six proteins were calculated by the presented equations. The Lmm was also found to vary with flow rate of mobile phase. The obtained experimental results from various sizes packed with the packing of high performance hydrophobic interaction chromatography (HPHIC) fit the expected ones well, resulting to lay a theoretical basis for selecting the column length.2. Design and manufacture of chromatographic piesConsisdering the designed principles, materials, seal methods, the distribution of mobile phase and overall structure of chromatographic pies, seven different sizes of chromatographic pies with diameters from 10 mm to 300mm but only 10 mm in thickness were designed and made. The experimental results indicated that the resolution and renaturation efficiency of proteins, column packing, seal performance, and so on satisfied with the experimentally requests. The chromatographic pies could be used in low backpressure and high flow rate duo to the packing of small particles in it, resulting to have the characteristics of perfusion chromatography and to be favorable for the stability of active proteins because of the systemically low pressure. Chromatographic pies with unique structure and features have been applied for a domestic invention patent (No. 01115263.x) and passed the first review by National Patent Office.3. Packing method for chromatographic pies in radial directionFrom the comparisons of the packing method of the chromatographic pies between radial and the axial on...
|
PDF Full Text Request