Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL.

Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells. Although significant improvements have been made, in terms of expression, more improvements are needed to compete with the yields achievable in stable gene expression. Much progress has come from optimization of transfection media and parameters, as well as altering culturing conditions to enhance productivity. Recent studies have included cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL. In this study, we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of Bcl-xL and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing Bcl-xL using polyethylenimine. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta, in CHO cells, or through the co-transfection with Bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a stable cell line and subsequent screening, which are both time- and resource-consuming. See accompanying commentary by Matthias Hackl and Nicole Borth DOI: 10.1002/biot.201400104.