A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein.

Binding characteristics of tumor necrosis factor receptor-Fc fusion proteins vs anti-tumor necrosis factor mAbs.

Tumor necrosis factor (TNF) antagonists are efficacious in the treatment of various autoimmune diseases. Two classes of TNF antagonists are currently commercially available: soluble TNF receptor-Fc fusion proteins (etanercept) and anti-TNF mAbs (adalimumab and infliximab). The classes differ in molecular structures and mechanisms of action. The interactions between TNF antagonists with TNF molecules were characterized. The anti-TNF mAbs, but not the soluble TNF receptor, formed visible lines of precipitation in Ouchterlony assays. The molecular weights of complexes formed by TNF (52 kDa) with either etanercept (130 kDa), adalimumab (150 kDa), or infliximab (average 165 kDa) were determined by size exclusion chromatography-light-scattering assays. Etanercept and TNF formed complexes of 180 and 300 kDa, representing one and two etanercept monomers bound to a TNF trimer, respectively. Adalimumab and infliximab formed a variety of complexes with TNF with molecular weights as high as 4,000 and 14,000 kDa, respectively, suggesting the presence of complexes with a wide range of sizes and stoichiometries. The absence of large complex formation with the binding of soluble receptor-fusion proteins to TNF may account for the different clinical efficacy and safety profiles of the two classes of TNF antagonists.