Mast cells are highly responsive cells found in the skin, which can very rapidly secrete an array of inflammatory mediators. It is possible that severe injection-site pain, which can be seen with some TNF inhibitors, could be linked to the mediators released upon degranulation by mast cells. Certolizumab pegol (CZP) is the only PEGylated Fab' anti-TNF-α for the treatment of Crohn's disease and has been shown to have low levels of injection-site pain in clinical trials.[1] One unique aspect of the structure of CZP is the site-specific attachment of 40 kDa of PEG. The aim of these experiments was to determine the effect of PEG on non-immune-stimulated mast cell degranulation. Mast cells were cultured in vitro from stem cells using the method of Saito et al.[2] over an 8-12 week period. The expression of relevant mast cell markers were tested by flow cytometry. Degranulation, measured by β hexosaminidase release, was stimulated by the addition of the compound 48/80, which is a known non-immune activator of mast cells. Titrations of CZP, PEG and a mixture of PEG and naked Fab' at a PEG concentration of 45 mg/mL were incubated with the mast cells and a fixed amount of compound 48/80 to determine the effect on mast cell degranulation. This is a physiological concentration range at the site of injection, as the CZP is injected at a concentration of 200 mg/mL. The viability of the mast cells at the end of the experiment was assessed using the Promega CellTiter 96 AQueous One Solution Cell Proliferation Assay. The cultured human mast cells expressed markers such as CD117, CD203c and CD32, which are characteristic of tissue mast cells. Compound 48/80 mediated a good level of degranulation, as measured by β hexosaminidase release, although the absolute level varied between cell preparations. CZP, PEG alone and a mixture of naked Fab' and PEG all inhibited the majority of the degranulation in a dose-dependent fashion. No effect on cell viability with any of the reagents tested was seen at the end of the assay. PEG inhibited the degranulation of mast cells stimulated by compound 48/80 at high concentrations. The concentrations at which an effect is observed are what might be expected at the injection site but not systemically. The exact mechanism behind this activity is unclear and needs to be investigated further. This beneficial effect of PEG could explain the low level of injection-site pain observed with CZP in clinical trials.