Full characterization of heterogeneous antibody samples under denaturing and native conditions on the Q Exactive BioPharma mass spectrometer

Goal

To demonstrate the capabilities of the Thermo Scientific™ Q Exactive™ BioPharma mass spectrometry platform, particularly the High Mass Range (HMR) Mode for the characterization of antibody samples. The three different operational modes of the BioPharma option are described and application data are presented for the major application workflows.

Introduction

MS analysis of antibodies at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to their heterogeneity caused by various modifications that are relevant for their efficacy. Intact proteins analyzed by ESI-MS are detected in higher charge states that also provide more complexity in mass spectra.

Analysis of proteins in native or native-like conditions with zero or minimal organic solvents and neutral or weakly acidic pH can allow proteins to preserve non-covalent interactions and retain high degrees of folding. This effect has analytical benefits: greater protein folding leads to reduced charge states, increased mass separation, and increased signal at higher m/z. This strategy has been utilized for the analysis of antibodies and antibody drug conjugates present in highly complex mixtures of different antibody/drug combinations.¹ Requirements for performing native MS on antibody samples include scanning towards 8,000 m/z and increased transmission optimization for large compounds. This feature has so far only been available on the Thermo Scientific™ Exactive ™ Plus EMR mass spectrometer. Here are shown the results obtained after successful implementation of the HMR Mode as part of the BioPharma Option now available on both the Thermo Scientific™ Q Exactive™ Plus and Q Exactive HF™ mass spectrometers aimed at adding the capability to perform native MS analysis with mass detection up to 8,000 m/z without compromising performance of normal operation modes. These enhanced capabilities are necessary for the analysis of antibody samples on the intact level under native conditions requiring the detection of masses beyond the standard mass range of up to 6,000 m/z.

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