Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid sample, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG), and thus represents the largest protein successfully ionized by DESI to date.

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2−8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.

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