Humira TNF-Alpha Neutralisation Assays

The Humira (Adalimumab) 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 Humira (Adalimumab). BioOutsource are industry experts in the development, optimization and qualification/validation of bioassays and have applied this expertise to Humira (Adalimumab) biosimilars to offer a wide range of TNF-Alpha neutralization assays to our customers. We have provided services to support clone selection, comparability and biosimilarity activities with assays performed using our existing methodology, adapted to incorporate the customer’s reference standard of choice.

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

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

The principle of the Humira (Adalimumab) neutralization bioassay is that the relevant cell line is prepared in cell plates. A dilution series of Humira (Adalimumab) is then prepared, added to an optimized concentration of TNF-Alpha and incubated to allow neutralization to occur, prior to addition to the cell plate. The cells are then incubated with the Humira (adalimumab)/TNF-Alpha mix for a further period of time 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.