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Use of the design‐of‐experiments approach for the development of a refolding technology for progenipoietin‐1, a recombinant human cytokine fusion protein from Escherichia coli inclusion bodies
Author(s) -
Boyle Denis M.,
Buckley John J.,
Johnson Gary V.,
Rathore Anurag,
Gustafson Mark E.
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20080268
Subject(s) - dithiothreitol , escherichia coli , chemistry , urea , tris , cystine , inclusion bodies , dissolution , fusion protein , dimer , dilution , chromatography , recombinant dna , biochemistry , cysteine , organic chemistry , enzyme , physics , gene , thermodynamics
Optimization of refolding conditions for progenipoietin was performed. The molecule has five disulfide bonds and, hence, is a challenge to refold. Variables studied included pH, DTT (dithiothreitol) concentration, cystine concentration, urea concentration, protein concentration, dissolution hold time and oxygen availability. In view of the complexity of the reaction with respect to the number of parameters that can impact the refold efficiency, some variables were examined via single‐parameter studies, whereas others were looked at via a DOE (design of experiments) approach. The DOE approach allowed us to look at the effect of these variables over wide ranges, as well as their interactions, in a very efficient manner. We were able to obtain a maximal refolding efficiency of 57%, defined as a percentage of correctly folded, bioactive dimer protein from inclusion‐body slurries produced from Escherichia coli . The final method involved dissolution of IBs for 30 min at 2 mg/ml protein, 6 M urea, 2 mM DTT and 50 mM Tris (pH 10.2) for approx. 30 min, followed by the addition of 4 mM cystine just prior to a 10‐fold dilution with 50 mM Tris (pH 10.2) buffer and reaction for 72 h at 2–10 °C. The use of the DOE approach allowed us to understand the interactions between the various parameters, in particular those between cystine and urea concentrations. The results were used to create a process model that demonstrated satisfactory accuracy and that could be used during commercialization of the product.