Fig. 4

The development of a transversal unbiased method for mAbs icIEF by studying the resolution function. We performed a multivariate icIEF experiment where 4 input variables are changing together, while we measure the resolution of the methods along the pH gradient. In each injection a sample consisting of a mix of ten pI-markers (5.85, 6.14, 6.61, 7.05, 7.40, 7.90, 8.40, 9.00, 9.50, 9.99, 10.17) is run under an array of different input variables: (i) total ampholytes percentage (4%, 6%, 8%); (ii) narrow range pH 5–8/pH 8–10.5 ampholytes ratio (0.33, 0.66, 1.00, 1.66, 3.00); (iii) separation times (9.0’, 10.5’, 12.0’). Due to the presence of the pI-markers in each injection, we performed an internal optimized calibration allowing us to define the shape of the pH that was present in the capillary at the end of the measure. This pH is independent of the measure since the pI-markers are only flags of the pH gradient that affected the experiments. (A,C,E) Based on the calibration curves, we computed the resolutions every 0.05 ΔpH units from pH 6 to pH 10, for each array of variables, these data are presented as scatter plots of the different arrays of variables presented in two at a time. The remaining input variables are reported as different color datasets superimposed on the same graph. The effects of the migration times can be observed by a direct comparison of panels (A,C,E). (B,D,F) These data show the average resolution in simultaneous variations of total ampholyte percentage and narrow range pH 5–8/pH 8–10.5 ampholytes ratio, in defined slots of pH. These scatterplots demonstrate that pH and ampholyte ratio strongly affect the resolution of the methods. The effects of the migration times can be observed by a direct comparison of panels B, D, F. All the scatter plots were interpolated by non-linear polynomial surfaces to obtain each corresponding-colored mesh, these are the 3D representations of the resolution functions. The Colormaps highlight the resolution values by color-coded scales.