PMID- 19435462
OWN - NLM
STAT- MEDLINE
DCOM- 20100105
LR  - 20131121
IS  - 1470-8744 (Electronic)
IS  - 0885-4513 (Linking)
VI  - 54
IP  - 2
DP  - 2009 Jul 14
TI  - 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.
PG  - 85-92
LID - 10.1042/BA20080268 [doi]
AB  - 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 degrees 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.
FAU - Boyle, Denis M
AU  - Boyle DM
AD  - Department of Bioprocess Research and Development, Pfizer Global Biologics, 
      Chesterfield, MO 63017, USA. denis.m.boyle@pfizer.com
FAU - Buckley, John J
AU  - Buckley JJ
FAU - Johnson, Gary V
AU  - Johnson GV
FAU - Rathore, Anurag
AU  - Rathore A
FAU - Gustafson, Mark E
AU  - Gustafson ME
LA  - eng
PT  - Journal Article
DEP - 20090714
PL  - United States
TA  - Biotechnol Appl Biochem
JT  - Biotechnology and applied biochemistry
JID - 8609465
RN  - 0 (Colony-Stimulating Factors)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Recombinant Proteins)
RN  - 0 (progenipoietin-1)
RN  - 48TCX9A1VT (Cystine)
RN  - N762921K75 (Nitrogen)
RN  - S88TT14065 (Oxygen)
RN  - T8ID5YZU6Y (Dithiothreitol)
SB  - IM
MH  - Chromatography, High Pressure Liquid
MH  - Colony-Stimulating Factors/chemistry/genetics/*metabolism
MH  - Cystine/chemistry
MH  - Dithiothreitol/chemistry
MH  - Electrophoresis, Capillary
MH  - Escherichia coli/*genetics
MH  - Humans
MH  - Hydrogen-Ion Concentration
MH  - Inclusion Bodies/*chemistry
MH  - Nitrogen/chemistry
MH  - Oxygen/chemistry
MH  - *Protein Folding
MH  - Recombinant Fusion Proteins/chemistry/genetics/*metabolism
MH  - Recombinant Proteins/chemistry/genetics/*metabolism
MH  - Research Design
EDAT- 2009/05/14 09:00
MHDA- 2010/01/06 06:00
CRDT- 2009/05/14 09:00
PHST- 2009/05/14 09:00 [entrez]
PHST- 2009/05/14 09:00 [pubmed]
PHST- 2010/01/06 06:00 [medline]
AID - BA20080268 [pii]
AID - 10.1042/BA20080268 [doi]
PST - epublish
SO  - Biotechnol Appl Biochem. 2009 Jul 14;54(2):85-92. doi: 10.1042/BA20080268.