Evaluation of refolding conditions for a human recombinant fusion cytokine protein, promegapoietin‐1a

Abstract

Conditions to obtain correctly folded PMP-1a (promegapoietin-1a), an engineered fusion IL-3 (interleukin-3) and thrombopoietin receptor agonist from recombinant Escherichia coli IBs (inclusion bodies), were defined to generate sufficient amounts of protein for evaluation as a potential therapeutic compound. Several ionic and non-ionic detergents, as well as the chaotrope urea, in combination with selected additives, were screened for their ability to dissolve IB protein and promote formation of monomeric, oxidized protein. Upon dissolution, soluble aggregates constituted 50-60% of total protein in detergent-solubilized IBs depending on the level of detergent used, whereas use of urea increased aggregation to approx. 70%. Subsequent addition of 5 mM cysteine or DTT (dithiothreitol) reduced the levels of aggregation, but never lower than approx. 20%. Refolds from detergent-solubilized IBs with or without organic modifiers characteristically produced multiple persistent misfolded species. However, the addition of a 12:1 molar excess of cystine (cystine/DTT) to urea-dissolved IBs containing DTT, followed by dilution, promoted the formation of correctly oxidized, disulfide-paired PMP-1a monomer with minimal misfolds present. Thus treatment of urea-dissolved proteins with thiol-group-containing additives and control of dilution, pH, protein concentration and order of addition were able to produce a maximum refold efficiency of 40-50% of correctly paired protein monomer.

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

“…In summary, the results presented here are similar to those obtained in previous work from our laboratory in developing scaleable refolding protocols for recombinant proteins from insoluble IBs [17,18]. Although empirically derived refold conditions and procedures must generally be tailored to the requirements of any given protein, the results presented here show that certain properties common to different proteins can be identified and exploited for refold development.…”

“…The results presented here represent an effort to develop commercially viable refolding technology for potential biotherapeutic candidates [16,17]. The goals of the refold development experiments described here were to increase refolding efficiency, increase refold protein concentration and understand, if possible, the chemical interactions for better control of the refolding mechanism and adaptation to large scale.…”

A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies

“…The recent work by Boyle et al (2008) complements our study and gives more insight into the optimization of refolding for PMP and the addition of reducing agents during the solubilization step.…”

“…PMP contains 306 amino acids with a molecular mass of 33,263 Da. More recently, Boyle et al (2008) proposed a refolding procedure as part of a production process for this protein. They were able to produce 40-50% refolded protein by optimizing the addition of urea and thiol-group-containing additives and control of dilution, pH, protein concentration and their order of addition.…”

Role of growth factors and thrombopoietic agents in the treatment of chronic hepatitis C

“…Data looked promising in vitro and in vivo in nonprimates, but the program has not been developed. However, there seems to be some recent interest [37].…”