Development of a STAT5 phosphorylation assay as a rapid bioassay to assess interleukin-7 potency.

Interleukin (IL)-7 is a cytokine inducing the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. As a consequence of IL-7 activating this pathway, STAT5 is phosphorylated. In pharmaceutical quality control, the potency of biopharmaceuticals is commonly assessed by proliferation assays. This is also possible for IL-7 conjugates. However, the disadvantage of these classical "endpoint-assays" is that they require very long incubation times, up to several days, since they measure the downstream events of a cellular response. As an alternative to this, we developed a rapid intracellular phosphorylation assay, measuring IL-7 induced STAT5 phosphorylation in Kit 225 cells. The Kit 225 human T cell line expresses the IL-7 receptor and is responsive to IL-7, therefore making it a good candidate cell line for assay development. Like the Kinase receptor activation (KIRA) assay, developed by Sadick et al. [1], the STAT5 phosphorylation assay was performed using two separate microtiter plates: the first one for cell stimulation and lysis, the second one for enzyme-linked immuno sorbent assay (ELISA). The assay showed a high accuracy and precision with a mean recovery of 102% and a mean coefficient of variation of 9%. In comparison to the classical proliferation assay, the phosphorylation assay is much faster. Thus, the assay procedure time can at least be reduced from six to three days by using STAT5 phosphorylation instead of proliferation as an endpoint due to the shorter incubation time with IL-7. Moreover, the phosphorylation assay shows a wider dynamic range and higher signal to noise ratios and is thus more robust than the proliferation assay.mAs a consequence, this assay could serve as reliable, accurate, precise and fast alternative to the classical proliferation assay for IL-7. This study also serves as an example for the typical steps during development and qualification / validation of a potency assay for quality control testing.