Clinical-scale production of granulocyte progenitor and post-progenitor cells using daniplestim, leridistim, Progenipoietin, Promegapoietin and autologous plasma

Patel, Sanjay; Guo, Ran; Miller, William M.; Papoutsakis, Eleftherios T.; Minster, N.I.; Baum, Charles M.; Winter, Jane N.

doi:10.1080/146532400539080

Cited by 1 publication

(1 citation statement)

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“…It is a recombinant fusion homodimer protein consisting of two identical subunits, and each subunit contains a G‐CSF and a flt‐3 receptor agonist domain [13]. It has been investigated clinically to stimulate differentiation of dendritic cells and production of white blood cells for chemotherapy‐generated thrombocytopenia [14].…”

Section: Introductionmentioning

confidence: 99%

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

Boyle¹,

Buckley²,

Johnson³

et al. 2009

Biotech and App Biochem

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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.

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Section: Introductionmentioning

confidence: 99%