Abstract
Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety.
Highlights
® etanercept biosimilar GP2015 (Erelzi ) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain
® relationship was found to be the molecular basis for reduced potency in recent Enbrel batches, which exhibit higher levels of incorrect disulfide bridging
Incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should be carefully monitored and controlled to guarantee patient safety
Summary
Samples were subsequently transferred into 50 mM Tris buffer pH 8.0 containing 0.5% RapiGest SF (Waters) and 50 mM iodoacetamide and denatured for 30 min at 50 °C. Samples were buffer exchanged into 50 mM Tris buffer pH 8.0 containing 0.1% Rapigest SF solution and digested with 0.1 μg/μL AspN (Roche), 1 μg/μL Trypsin (Promega Gold) and 1 μg/μL chymotrypsin (Roche) for 16 hours at 37 °C. A Q Exactive mass spectrometer (Thermo scientific) was used for identification of the disulfide bridge peptides in the sample (see Supplementary Table S1 for details). This non reducing peptide map allows quantitation of a tryptic peptide T7 containing the incorrectly disulfide bridge C78-C88 relative to an internal peptide standard T27 in etanercept samples. The data were processed using the programs XDS and XSCALE
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