The capacity to locate efficiently a subset of experimental conditions necessary for the identification of an operating envelope is a key objective in many studies. We have shown previously how this can be performed by using the simplex algorithm and this paper now extends the approach by augmenting the established simplex method to form a novel hybrid experimental simplex algorithm (HESA) for identifying 'sweet spots' during scouting development studies. The paper describes the new algorithm and illustrates its use in two bioprocessing case studies conducted in a 96-well filter plate format. The first investigates the effect of pH and salt concentration on the binding of green fluorescent protein, isolated from Escherichia coli homogenate, to a weak anion exchange resin and the second examines the impact of salt concentration, pH and initial feed concentration upon the binding capacities of a FAb', isolated from E. coli lysate, to a strong cation exchange resin. Compared with the established algorithm, HESA was better at delivering valuable information regarding the size, shape and location of operating 'sweet spots' that could then be further investigated and optimized with follow up studies. To test how favorably these features of HESA compared with conventional DoE (design of experiments) methods, HESA results were also compared with approaches including response surface modeling experimental designs. The results show that HESA can return 'sweet spots' that are equivalently or better defined than those obtained from DoE approaches. At the same time the deployment of HESA to identify bioprocess-relevant operating boundaries was accompanied by comparable experimental costs to those of DoE methods. HESA is therefore a viable and valuable alternative route for identifying 'sweet spots' during scouting studies in bioprocess development.
Key WordsCapillary liquid chromatography Porous graphitic carbon capillary columns Coupled columns bDOPAand metabolites in plasma
SummaryPorous graphitic carbon has been successfully used for the separation of L-DOPA and four metabolites (dopamine, 3,4-dihydroxy-phenyl-acetic acid, homovanillic acid and 3-O-methyl-b DOPA) and with a volatile mobile phase of relatively high percentage organic modifier (methanol-ammonium formate pH 2. 9, 60:40, v/v). The substances were analysed in plasma. Ultrafihration was used to remove the proteins from the sample and a coupled column system to enrich and perform on-line sample clean up. Both precolumn and separation column of i. d. 0.2 mm were packed with porous graphitic carbon.
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