Enbrel TNF-Alpha Neutralisation Assays

The Enbrel (etanercept) TNF-Alpha neutralization bioassay measures activity of the Fab binding affinity against TNF-Alpha, reporting the inhibition of TNF-Alpha mediated signal, commensurate to the concentration of Enbrel (etanercept). BioOutsource are industry experts in the development, optimization and qualification/validation of bioassays and have applied this expertise to Enbrel (etanercept) biosimilars and biobetters to offer a wide range of TNF-Alpha neutralization assays to our customers. We have provided services to support clone selection, characterization and similarity activities with assays performed using our existing methodology, adapted to incorporate the customer’s reference standard of choice.

The primary mechanism of action of Enbrel is the neutralization of circulating TNF-Alpha. TNF-Alpha itself is involved in an array of different signalling pathways and through binding to, and neutralization of TNF-Alpha, Enbrel has the capacity to neutralize this wide range of different biological activities that are mediated by TNF-Alpha. Historically, anti-TNF-Alpha therapeutic antibody products have used, as the primary potency bioassay, a murine cell line (L929 or WeHi) with a cell death endpoint. Increasingly, however Enbrel biosimilar manufacturers are looking to perform more extensive characterization that is aligned with originator filings to study multiple different mechanisms of action of TNF-Alpha neutralization. BioOutsource currently offers six different versions of the Enbrel TNF-Alpha neutralization assay:

  • Enbrel TNF-Alpha Neutralisation Assay using L929 cells (Measurement of cell death/viability)
  • Enbrel TNF-Alpha Neutralisation Assay using U937 cells (Measurement of cell death/viability)
  • Enbrel TNF-Alpha Neutralisation Assay using U937 cells (Measurement of apoptosis)
  • Enbrel TNF-Alpha Neutralisation Assay using HUVEC cells (Measurement of ELAM-1 Expression)
  • Enbrel TNF-Alpha Neutralisation Assay using HUVEC cells (Measurement of ICAM-1 Expression)
  • Enbrel TNF-Alpha Neutralisation Assay using HUVEC cells (Measurement of VCAM-1 Expression)

The principle of the Enbrel (etanercept) neutralization bioassay is that the relevant cell line is prepared in cell plates. A dilution series of Enbrel (etanercept) is then prepared and added to an optimized concentration of TNF-Alpha and incubated to allow neutralization to occur, prior to addition to the cell plate. The cell plate is then incubated for a further period of time prior before the assay is completed using the relevant detection methodology. Sample results can be reported as a per cent of inhibition of cell death or as a relative potency measurement against the reference standard.