Preparative Liquid Chromatography As A Whole Protein Is Rapid And Efficient Separation Of The New Strategy,

With the coming of great interesting in proteomics research, the separation and preparation of active intact proteins have became difficult problems that retard the development of proteomic research. To obtain more information on the structure and function of proteins, active proteins with high purity and enough amounts are required in the investigation. In the other aspect, in order to apply the achievements obtained from proteomic research for developing and producing protein drugs, there is an urgent need to improve the efficiency of separation method and to lower production cost. Therefore, to develop new strategies for rapid and efficient separation of intact proteins on preparative scale is the key for solving the above problems. A new approach termed the "five-S" strategy is firstly presented for rapid, high-resolution separation of proteins on a preparative scale. The five-S strategy means that a Short column of very large diameter termed the chromatographic cake (or cake) is packed with very Small particles for protein separation under Small overloading per unit cross-Sectional area of the packed bed at a Suitably low linear velocity.Introduction. The significance of intact protein separation and preparation of intact protein, the development of separation technologies and media for liquid chromatography (LC) were introduced. The principle and technology of chromatographic cake were reviewed. In addition, the development of preparative liquid chromatography was introduced and the similarities and differences between analytical and preparative LC were compared. This thesis cited 150 references.Experiments. All of instruments, materials and methods are included in this part.The five-S strategy for intact protein separation in LC. The resolution and peak capacity of protein separation with both cakes and columns were comprehensively compared. The cake was found to have a comparable resolution with a thin and long column on analytical scale, but the former to be much better than the latter with the same bed volume on preparative scale. Additionally, under an optimized condition the resolution of intact proteins separation by the cake was found to be much better than the column under sample overloading at a high flow rate. It would be expected that the cake packed with very fine silica materials can possibly replace conventional soft media and/or polymer-based packings. A conclusion is that the five-S strategy is firstly reported for rapid and high-resolution protein separation on a preparative scale using a chromatographic cake.The interaction of five-S strategy and the thermodynamics. The investigation of retention behavior of biopolymers shows that each protein has its own concentration window during separation process. The stoichiometric displacement theory (SDT) is employed to explain this characteristic, the interactions between the solute and the stationary phase and mobile phase are used to elucidate the complicated adsorption and desorption process and the intrinsic role. Three new concepts, conditional resolution, ratio of available column length and density per unit cross-sectional area are suggested for elucidating the obtained excellent resolution under sample overloading condition. The only approach to achieve this goal is to enlarge the diameter of the employed chromatographic column and decrease the width and density of the adsorbed sample layer on the top of the packed bed. For enlarging output of protein purification at very fast flow rate of mobile phase, the column length must be decreased. The only approach to the five-S strategy is to employ chromatographic cake.The effect of the five-S strategy on the dynamics. The effect of particle size, density and characteristics of the column and cake beds were studied. The results showed that a better result can be obtained with decreasing of the packed particle size. The geometric structure of cake provides a possibility to pack a high packing density than the same bed volume of a conventional column, resulting in a less band broadening of proteins. The Plots of the conditional plate height (H) and conditional resolution (R_S) versus the linear velocity of the mobile phase (u) indicates a relative lower minimum linear velocity u_min and broader optimum values for the cake than that from the column. The R_S～u plot for intact proteins is presented and can be used for the selection of the better condition in chromatography. The conditions for the fast separation of proteins on cake were also optimized and a conclusion is thus obtained that with compared to the data reported from literatures, cake has a larger sample loading, better resolution and higher mass recovery and higher sample concentration.The application of the five-S strategy. Human serum was separated within 6 min on hydrophobic chromatographic cake. The cake was used in the two-dimensional liquid chromatography (2D-LC) for a rapid buffer exchange, and a fast 2D-LC separation of the human serum was achieved on the cake with a diameter of 2 cm in 70 min by means of an on-line manner, the total protein mass is 40 mg by one step.