Optimization studies in preparative chromatography of biomolecules.

摘要

Optimization of preparative nonlinear chromatography was carried out for the first time for a biomolecule mixture. Conventional wisdom on optimization, which roots from analytical chromatography, dictates optimizing resolution in an analytical column and obtaining similar separation in a large column for isolation. Such a method of optimization significantly under uses the capacity of the column and consumes large quantities of mobile phase. Hence, in preparative chromatography, the objective function is productivity, a measure of compromise between the amount of feed that can be loaded on to the column and time. Here, we report results from optimization studies carried out on a closely related binary peptide mixture on an analytical reversed-phase column. The goal is to optimize productivity under various chromatographic modes—nonlinear isocratic elution, gradient elution, stepwise elution and displacement chromatography. In each mode, feed mixtures at highest possible concentration (limited by solubility), for increasing feed volumes was used. Productivity was monitored for increasing feed volumes, and loading was stopped as it went through a maximum. However, in some cases, solubility limitations from one of the feed components prevented further increase in loading. Even with this constraint, high productivities (5–10 g product/L stationary phase-h) were achieved. Separate experiments were carried out to measure the adsorption isotherms of these peptides over the range permitted by solubility.; Separations under nonlinear chromatographic conditions were applied to isolate commercially significant two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C ₁₈ stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column.; We also report simple and economical process to purify phycocyanins and allophycocyanins from a cyanobacterial process waste stream for two kinds of applications: food colorant and biomedical marker. A detailed design for the large-scale production of biliproteins for both applications is also presented. Economic evaluation of the process resulted in comparable costs with the current market price for food-grade product and substantially lower cost for the biomedical grade product.