Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli.

Apolipoprotein A 1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train.

Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A-I(Milano) in an industrial HIC unit operation.

We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A-I(Milano) (apo A-I(M)) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale-up of biopharmaceutical manufacturing processes.

Separation of recombinant apolipoprotein A-I(Milano) modified forms and aggregates in an industrial ion-exchange chromatography unit operation.

We have shown how protein self-association impacts the ion-exchange separation of modified forms and aggregates for apolipoprotein A-I(Milano). It is well known that reversible self-association of a protein can lead to chromatographic band broadening, peak splitting, merging, fronting, and tailing. To mitigate these effects, urea or an organic modifier can be added to the chromatography buffers to shift the equilibrium distribution of the target molecule to the dissociated form.

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

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.

Impact of denaturation with urea on recombinant apolipoprotein A-IMilano ion-exchange adsorption: equilibrium uptake behavior and protein mass transfer kinetics.

We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I(Milano) (apo A-I(M)) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic alpha helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution.