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Optimization of a refolding step for a therapeutic fusion protein in the quality by design (QbD) paradigm
Author(s) -
Bade Pratap D.,
Kotu Susmitha P.,
Rathore Anurag S.
Publication year - 2012
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201200476
Subject(s) - quality by design , fractional factorial design , factorial experiment , yield (engineering) , recombinant dna , escherichia coli , design of experiments , inclusion bodies , central composite design , biochemical engineering , fusion protein , chemistry , chromatography , microbiology and biotechnology , computational biology , process engineering , mathematics , biochemistry , biology , response surface methodology , materials science , engineering , statistics , particle size , metallurgy , gene
Production of biotech therapeutics in Escherichia coli involves protein expression as insoluble inclusion bodies that need to be denatured and the resulting protein refolded into the native structure. In this paper, we apply a Quality by Design approach using Design of Experiments for optimization of the refolding process for a recombinant biotech therapeutic, granulocyte colony stimulating factor. First, risk analysis was performed to identify process parameters that require experimental examination. Next, the chosen parameters were examined using a fractional factorial screening design. Based on the results of this study, parameters that have significant effect on refold yield and product quality were identified and examined using a full factorial Design of Experiments for their interactions. The final model was statistically significant and delivered a refolding yield of 77%. Further, kinetics of refolding was evaluated under optimal conditions and was found to be of first order with a rate constant of 0.132/min. Design space was established for the three parameters for a given permissible range of yield, protein concentration, and purity. The primary objective of this paper is to provide a roadmap for implementing Quality by Design for development of a protein refolding step.