Figure 8.

Reproducibility of ‘Balanced Method’ optimized for both selectivity and dynamic range. (a) Deconvoluted spectrum shows a different glycosylation pattern for a second batch of trastuzumab analyzed using a method which was optimized for both specificity and dynamic range (Orbitrap resolution setting = 45,000, Sliding Window Deconvolution Mass/Merge Tolerance = 3.3 ppm, LC gradient = 1–8% B in 30 min). (b) XDC traces plotted at 3.3 ppm mass/merge tolerance demonstrate reproducibly sufficient separation in both mass and RT domains to distinguish deconvoluted G0F/G1F isoforms as being deamidated (LC-N30) or otherwise unmodified. Doublet peaks are observed for both the deamidated and main peak isoforms of G0F/G1F, presumably due to isomerization at HC-D102 as previously identified by Harris et al.²³