| Size Exclusion Chromatography (SEC) |
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SEC, also known as gel filtration when applied in an aqueous mobile phase separates molecules based on differences in size. As a non-binding chromatographic mode SEC does not provide binding capacities known from adsorptive modes such as IEC. At manufacturing scale, the main applications of SEC are desalting of the feedstock and virus purification.
TOYOPEARL polymeric SEC materials are available in a number of pore sizes and particle diameters. It is important to understand the pore sizes, particle sizes, and physical properties of these beads, as they are the base beads for TOYOPEARL IEX, HIC, mixed-mode and AFC products.
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| Ion Exchange Chromatography (IEC) |
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Toyopearl and TSKgel polymeric ion exchange resins utilize an aqueous buffer solution whose pH is between the pI of the target protein and the pKa of the resin. Protein elution occurs by either increasing the salt strength of the buffer or a change in the buffer pH itself during the run. The strong ionic interactions give IEC resins very high dynamic binding capacities (DBC). This makes them ideal for any stage in the purification process but more often used for the Capture step.
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| Hydrophobic Interaction Chromatography (HIC) |
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Toyopearl and TSKgel polymeric HIC resins utilize a decreasing salt gradient buffer system for elution. A high initial salt concentration promotes binding between a protein's surface hydrophobic patches and the resin's hydrophobic ligand. Resin ligand density is less than a reversed phase resin. This allows an organic solvent free elution mechanism with high mass and biological activity recoveries. Tosoh's advanced resin design has created HIC resins with DBCs approaching those of reversed phase materials, with developers frequently using them for Intermediate and Polishing purification steps.
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| Mixed Mode Chromatography (MMC) |
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Toyopearl polymeric MMC resins have both ionic and hydrophobic groups in their composition. These unique ligands allow the separation of acidic, basic, and neutral proteins by one resin. Unlike with traditional ion exchange resins, elevated conductivity feedstocks and buffers can be used without diminishing binding capacity.
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| Affinity Chromatography (AFC) |
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Toyopearl polymeric AFC resins are available in a variety of group specific ligands. In addition, activated and reactive forms are available for the immobilization of target specific ligands. In AFC the mechanism of binding and elution varies depending on the immobilized ligand used. In some cases such as with Toyopearl AF-Chelate-650M (for Immobilized Metal Affinity Chromatography or IMAC) the binding and elution mechanism may change through the use of different metals or chemical modifiers. DBC values for AFC materials vary with ligand and are typically on the low side. However, because AFC has high selectivity and most of the capacity is used for the target, with feedstock impurities remaining unbound, AFC resins are typically used for the Capture and Intermediate purification steps of a manufacturing process.
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