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Capture |
feedstock
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A chromatographic "capture" or "concentration" step is a column just downstream of the harvested feedstock produced either by a bacterial fermentation or cell culture expression . Typically the harvested feedstock is clarified The chromatographic step's primary function is to "capture" the target from solution using the smallest amount of resin possible. It is then eluted into a significantly smaller volume of buffer for further downstream processing. A resin is selected which has the best combination of dynamic binding capacity, mass recovery, and retention of the target's biological activity. Resin particle sizes tend to be larger, typically 100 micron or more, to allow for the feedstock's viscosity.
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Intermediate
Purification |
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The partially purified feedstock from the capture step will have a higher target concentration and relatively less complex impurity profile (also at higher concentrations). The intermediate purification step uses chromatography's resolving power to separate the target away from other impurities of the feedstock. Because of this, and the improved fluid flow properties, resin particle sizes of 65 micron and smaller are used. In many cases, more than one intermediate purification column may be necessary to achieve the desired degree of product purity.
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Polishing |
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The final column in a production train is often referred to as the polishing step. At this point the target protein is very pure. The residual impurities however may be very closely related and may be dimer, trimer, or other target isoforms. In the case of synthetic peptides or oligonucleotides, "n-1", "n-2", etc. forms may remain. Resins with high selectivity or efficiency are selected for the polishing step. Dynamic binding capacity remains important but buffer volume has been already significantly reduced. Particle sizes of 35 micron and smaller are typically used.
NOTE: As raw treated feedstocks have become more clarified and less viscous, there is a trend to use smaller particle materials earlier in the production process.
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QC |
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Many Toyopearl resins have prepacked analogous TSK-GEL analytical columns available which have the same resin chemistry.
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