Chromatography at-30 °C for Reduced Back-Exchange, Reduced Carryover, and Improved Dynamic Range for Hydrogen-Deuterium Exchange Mass Spectrometry

For hydrogen-deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during protein analyses should be minimized to promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 and 0 degrees C can lose >30% of the deuterium marker within 15 min of sample injection. This report describes the architecture and performance of a dual-enzyme, HDX-MS instrument that conducts liquid chromatography (LC) separations at subzero temperature, thereby reducing back-exchange and supporting longer LC separations with improved chromatographic resolution. LC separations of perdeuterated, fully reduced, iodoacetamide-treated BSA protein digest standard peptides were performed at 0, -10, -20, and -30 degrees C in ethylene glycol (EG)/H2O mixtures. Analyses conducted at -20 and -30 degrees C produced similar results. After subtracting for deuterium retained in arginine side chains, the average peptide eluted during a 40 min gradient contained approximate to 16% more deuterium than peptides eluted with a conventional 8 min gradient at 0 degrees C. A subset of peptides exhibited approximate to 26% more deuterium. Although chromatographic peaks shift with EG concentration and temperature, the apparatus elutes unbroadened LC peaks. Electrospray ion intensity does not decline with increasing EG fraction. To minimize bias from sample carryover, the fluidic circuits allow flush and backflush cleaning of all enzyme and LC columns. The system can perform LC separations and clean enzyme columns simultaneously. Temperature zones are controlled +/- 0.058 degrees C. The potential of increased sensitivity by mixing acetonitrile with the analytical column effluent was also examined.

Automated summary

To improve the reproducibility of HDX-MS in the quality control of biopharmaceuticals, researchers have developed a dual-enzyme HDX-MS instrument that conducts liquid chromatography separations at subzero temperature. This instrument reduces back-exchange and supports longer separations with improved resolution.