Eng. Life Sci. 2015, 15, 140–151 www.els-journal.com Sylwia Ry´ s Wojciech Pi ˛ atkowski Dorota Antos Department of Chemical and Process Engineering, Rzesz´ ow University of Technology, Rzesz´ ow, Poland Research Article Predictions of matrix-assisted refolding of α -lactalbumin: Process efficiency versus batch dilution method Protein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial-and- error method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to op- timize the operating conditions. In this article, we suggest a design procedure for matrix-assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion-exchange chro- matography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α-lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix-assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used. Keywords: Batch dilution / Matrix-assisted refolding / Process dynamics / Protein chromatog- raphy / Protein refolding  Additional supporting information may be found in the online version of this article at the publisher’s web-site Received: May 18, 2014; revised: July 3, 2014; accepted: July 18, 2014 DOI: 10.1002/elsc.201400105 1 Introduction The overproduction of the recombinant proteins in Escherichia coli often leads to inclusion body formation. This is due to aggre- gation of partially folded proteins that accumulate in the cyto- plasm of E. coli. Processing of proteins extracted from inclusion bodies is very common technique due to high concentration of the target protein and its relative high purity in inclusion body [1–5]. Correspondence: Dr. Dorota Antos (dorota.antos@prz.edu.pl) De- partment of Chemical and Process Engineering, Rzesz ´ ow Univer- sity of Technology, Powsta´ nc´ ow Warszawy Ave. 6, 35-959 Rzesz´ ow, Poland. Abbreviations: HIC, hydrophobic interaction chromatography; IEC, ion-exchange chromatography; MAR, matrix-assisted refolding; MTG, monothioglycerol Inclusion body proteins are devoid of biological activity and the recovery of their biologically active form requires solubiliza- tion, refolding, and purification. Solubilization is accomplished by means of strong denatu- rants and reducing agents, which are used to break intermolec- ular bonds of the protein. The solubilized proteins have to be refolded to recover their native activities. The refolding process is realized by reducing the concentration of denaturants in the presence of oxidizing agents inducing formation of the correct disulfide bonds [6, 7]. In most systems refolding is accompa- nied with competitive reaction of intermolecular aggregation, which reduces yield of the operation. Therefore, the composi- tion of the refolding buffer is critical operating parameter for the improvement of the refolding efficiency, that is accelerating the rate of refolding, reducing the rate of aggregation, and sta- bilization of the refolded form of the protein. To increase yield of the operation various additives are used acting as (i) so-called “disulfide-shuffling” reagents, such as reduced glutathione, cys- tine, and cysteine or foldases, which increase the rate of oxidation 140 C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim