A scalable refold for human neurotrophin-4 was developed as part of a manufacturing process required for the production of supplies for preclinical and clinical studies. The process redox system, chaotrope, solubilization additives, pH, temperature and protein concentration were optimized. The limited availability of suitable material for experimentation during concurrent downstream process development led to the approach described in the present paper: a combination of OFAT (one factor at a time) and multivariate DOE (design of experiments) to identify appropriate conditions. The optimized refold conditions included the use of sulfonated protein, raw materials utilized in other process operations and an inexpensive redox system. The conditions were found to be robust and were demonstrated from the millilitre scale to the 300 litre pilot scale. A process control procedure that utilized an RPC (reversed-phase chromatography) quantitative assay to monitor the percentage conversion into oxidized protein was developed. Refold conversions of 80-90% were obtained under ambient temperature and atmospheric conditions, with reaction times of approx. 18 h.