Background Tumour necrosis factor alpha (TNF-α) is a proinflammatory cytokine, the main mediator in inflammatory and autoimmune diseases, as well as during various attacks on cells such infections. It is therefore involved in the course of a large number of pathologies such as rheumatoid arthritis, Crohn’s disease, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis and ulcerative colitis. Infliximab (Remicade) is a chimeric monoclonal antibody (75% human, 25% murine) which acts by binding to TNF-α and blocking its effect. The cost of treatment with infliximab is quite high and the stability indicated by the manufacturer once the vial is opened is 24 hours.
Purpose The purpose of this research has been to evaluate the biological activity of infliximab when reconstituted and diluted to 10.0 mg/ml and 5.0 mg/ml in NaCl 0.9% in a long term stability study up to 15 days. A study of the drug degradation has been also tackled to cheque any remaining activity.
Materials and Methods An indirect non-competitive ELISA immunoassay was developed based on the use of ELISA plates sensitised with TNF-α. The plates were incubated ‘overnight’ at 4ºC using recombinant TNF-α from E. Coli at a concentration of 1 µg/ml. The immunoassay was validated in terms of calibration function (from 0.2 to 50.0 µg/ml), detection limit (0.06 µg/ml), precision as within-day reproducibility (relative standard deviation lesser than 10%), and accuracy as percentage of recovery (higher that 90%). The infliximab solutions of 10.0 mg/ml and 5.0 mg/ml in NaCl 0.9% were stored refrigerated at 4°C protected from daylight. The biological activity of these solutions was tested periodically up to 15 days by the ELISA method developed. The ELISA was also used to study the drug degradation in a stress study involving the exposure of samples of infliximab (50.0 mg/ml) for 24 hours to different stress conditions: basicity (NaOH 0.1M), acidity (HCl 0.1M), oxidation (H2O2 1% and 10%), temperature (50ºC) and ultraviolet light (250 w/m, 25ºC).
Results All the samples analysed showed considerable biological activity; this biological activity was surprisingly even observed in those samples subjected to strongly stressed conditions. For the reconstituted sample of 10.0 mg/ml, a remaining activity of 52% was observed. In the case of the 5.0 mg/ml sample, the remaining activity decreased to 35%.
The biological activity measured using the samples submitted to stress conditions indicated a remaining activity at least equal to the upper concentration studied in the calibration function, i.e, 50 µg/ml. These samples were analysed directly, without dilution, because they had been expected to lose their biological activity totally.
Conclusions The biological activity of infliximab solutions of 10.0 mg/ml and 5.0 mg/ml in NaCl 0.9% when stored refrigerated at 4ºC protected from the daylight was maintained at 52% and 35% respectively up to 7 days. The biological activity was also shown in infliximab samples submitted to stress conditions. More experiments are currently being conducted to confirm these results.
Acknowledgement Financial support was provided by the Project PI10/00201 (Instituto Carlos III, Ministerio de Economía y Competitividad, Spain). We also want to thank the Hospital Pharmacy Unit of the University Hospital of San Cecilio who kindly supplied all the infliximab samples.
No conflict of interest.
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