Insulin therapy for people with diabetes encompasses an enormous number of different choices for formulations of insulin and its analogs. For quality assurance of biopharmaceutical products, reliable and validated analytical methods are required. Biological assays play an important role for determining the potency of hormones like insulin in living subjects. Concerning insulin bioassay requirements, regulations differ especially between European and US pharmacopoeias, where for the latter animal based so-called bioidentity testing assures the potency of insulins. The European pharmacopoeia does not demand for a bioidentity test. Their quality assurance is solely based on a high-performance liquid chromatography method quantifying total insulin but not necessarily discriminating from, e.g., misfolded fractions. Recently, a replacement of the US pharmacopoeia “rabbit blood sugar method” by an in vitro cell test has been suggested. Depending on various factors such as pH, temperature, and shear forces, insulins in pharmaceutical formulations can undergo a continuous process of degradation after manufacturing, during storage, and delivery until it is used by patients. Especially, changes in secondary structure of the protein take place, with the consequence of a decrease in biological potency due to partial misfolding of the monomers and subsequent aggregation to fibrils. For quality assurance, infrared spectroscopy has been suggested for insulin characterization, as it is possible to discriminate between native and misfolded structural isomers. However, the correlation between partially misfolded insulins and biological activity was not evaluated systematically until now. For such a study, online monitoring of substrates and metabolites in the culture medium of the insulin-dependent human monocytic cell line (MONO-MAC-6; https://www. dsmz.de/collection/catalogue/details/culture/ACC-124) represents an option. A linear relationship between biological activity and glucose metabolism rate of these cells within the exponential growth phase has been assumed. By analyzing the time-dependent glucose uptake, the biological potency of tested insulins can be assessed without the need for glucose clamp in humans or animal experiments. Based on a linear dependency between insulin potency and glucose metabolism rate of these human monocytes, the medium glucose, lactate, and other compounds of molecular masses less than the microdialysis membrane cut-off (20 kDa) were measured. For microdialysis, a commercial sterile catheter was submerged into the cell culture, operated at a perfusate flow rate of 1 μL/min. The isotonic perfusate contained a known concentration of mannitol as internal standard and losses were used for dialysis recovery rate calculation. Dialysate infrared spectra were continuously measured in a 30-μm flow-through cell. Multivariate analyte quantification was applied using data within the spectral interval between 1600 and 980 cm including consideration of the dialysis recovery rates to be independent from membrane fouling (see Figure 1); for further experimental details, see also Vahlsing et al. For standardization, two parameters are essential, ie, growth rate and number of cells after 60 hours with the onset of the exponential growth phase. By determining the normalized slope of the time-dependent glucose utilization until reaching the stationary glucose level within the cell culture (multiplication of the glucose concentration slope by the ratio of growth rate and number of cells after 60 hours), the potency of each tested insulin can be assessed when compared to certified insulin samples (see table in Figure 1). The bioassay has been found to be independent from formulation excipients such as phenol or m-cresol. Assuming a linear dependency, halving of the insulin units in the cell culture medium leads to an average normalized slope of (–1.45 ± 0.03) × 10 (58 %), which is slightly larger than expected, when using the first trial results for benchmarking. The presented monocytic cell-based bioassay could be a promising method for insulin potency assessment as an alternative to 895541 DSTXXX10.1177/1932296819895541Journal of Diabetes Science and TechnologyDelbeck et al. letter2019