Size-exclusion chromatographic protein refolding: Fundamentals, modeling and operation

Monolithic capillary columns based on pentaerythritol acrylates for molecular‐size‐based separations of synthetic polymers

“…Eighteen polystyrene standards with molar masses from 258 Da to 2.11×10 7 Da with narrow molar mass distribution M w /M n = 1.05 -1.33 were purchased from Polymer Labs, part of Agilent (Church Stretton, UK), MZ-Analysentechnik (Mainz, Germany), and PSS (Mainz, Germany). Benzene, azobisisobutyronitrile, methyl ethyl ketone, alcohols, Silane A-174, PE3A and PE4A and methylene chloride (HPLC grade) were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA) and were used without additional purification.…”

“…Among the latter, the most popular method is SEC (also known as gelpermeation chromatography) due to its simplicity and applicability to separation of synthetic [1][2][3][4] and natural [5][6][7] polymers. Conventional packings for SEC are macroporous polymeric beads having narrow particle size distribution, defined mean pore size and high pore volume [8, www.agilent.com, www.shodex.com, www.gelifesciences.…”

Oxidative protein refolding on size exclusion chromatography at high loading concentrations: Fundamental studies and mathematical modeling

“…Furthermore, in case of aggregate(s) formation, the simplified mechanism is not able to capture the interactions of all intermediates that are prone to aggregation [5,14,15]. In this work, (1) a mathematical model for refolding of denatured/reduced lysozyme in SEC at high loading concentrations was developed to investigate a detailed reaction mechanism previously proposed for this protein which was selected as a model system due to importance of disulfide bond formation in considerable number of proteins [13]; (2) non-reactive native, quenched and equilibrium experiments were executed to find the model parameters for the final product and short-lived kinetic intermediates formed during unimolecular refolding reaction; (3) the model was tested by varying operating conditions, namely: protein loading concentration, injection volume, flow rate and composition of refolding buffer (with and without l-arginine additive); and (4) the effect of low concentration (0.2 M) of l-arginine on mass transfer and kinetic parameters in SEC was studied.…”

“…For these reasons SEC has been widely used at lab scale for protein refolding in either batch or continuous mode using single or multiple column configurations (i.e. simulated moving bed) [1][2][3][4][5][6][7][8][9]. Existing work has also developed mathematical models for separation in SEC using various model proteins in their native forms [10,11].…”

“…Existing work has also developed mathematical models for separation in SEC using various model proteins in their native forms [10,11]. For protein refolding in SEC (reaction-separation SEC), the refolding reaction is incorporated into the mathematical model as well as interactions of aggregating species in case they are formed under the operating conditions being studied and the model parameters must be obtained for kinetic species formed during these reactions [5,12]. Development of an experimentally verified and robust model helps minimizing the number of screening for a broader range of operation condition and is essential for systematic process optimization.…”