Abstract
This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.
Highlights
Based on the therapeutic and commercial success of monoclonal antibodies, many new antibody-based drug formats have been developed in the last decade. [1] As result of their production using recombinant DNA technology and expression in living systems, antibody-based drugs are prone to several enzymatic and chemical post-translational modifications (PTMs) throughout any stage of their life cycle up to the formulated product
The commercial IonHance CX-mass spectrometry (MS) pH concentrate A and B were diluted 10 times with Milli-Q water to reach a composition of 50 mM ammonium acetate with 2% of acetonitrile, titrated to pH 5.0 and 160 mM ammonium acetate with 2% of acetonitrile, titrated to pH 8.5
Further evaluation of the different buffer systems showed that charge variants of infliximab and elotuzumab were better separated when using the MES, Yan or In-House buffer compared to the Commercial buffer
Summary
Based on the therapeutic and commercial success of monoclonal antibodies (mAbs), many new antibody-based drug formats have been developed in the last decade. [1] As result of their production using recombinant DNA technology and expression in living systems, antibody-based drugs are prone to several enzymatic and chemical post-translational modifications (PTMs) throughout any stage of their life cycle up to the formulated product. [16] the strategy of using MS-compatible mobile phases for IEX separation has emerged This allows a direct coupling of IEX to MS that can potentially allow unbiased and straightforward characterization of charge variants within a relatively short analysis time. [4,17] Here, we demonstrate a straightforward method to couple CEX to a compact benchtop MS system consisting of a high-resolution Time of Flight (ToF) mass analyser, offering a resolving power of 10,000. The goal was to obtain the best achievable resolution with a ToF instrument and the highest signal intensities of the tested mAbs in native conditions Thanks to this new CEX-MS setup, straightforward and rapid analysis of charge variants was made possible (in less than 10 min), allowing the separation and identification of several charge variants with a new level of throughput
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