Protein therapeutics are among the fastest growing classes of drugs. Characterization of these macro-molecular compounds is essential for their safety and their advancement as candidate drugs. Heterogeneity within these therapeutic proteins complicates characterization and quality control. Capillary isoelectric focusing (cIEF) is a technique that uses amphoteric reagents to separate molecules based on their isoelectric points (pI). CIEF has become popular for charge and variant analysis because it can separate molecules with resolutions as high as &lt;0.01 pI units. Integrating cIEF with Mass Spectrometry (MS) detection is ideal for subsequent mass characterization. MS has the capacity to quantify and identify protein modifications that cIEF alone is unable to.<br/><br/>One major obstacle to progress in the field is that the concentration of ampholytes required for high-resolution cIEF (&gt;5% m/v) is mutually exclusive with robust electrospray ionization. Ampholytes suppress ionization and contaminate the instrument, resulting in poor sensitivity and robustness. One solution is to dilute the ampholytes; which improves ionization efficiency at the expense of sensitivity, capacity and resolution. To address this challenge, we constructed and validated a novel in-line interface between cIEF and MS. The interface, shown in number 2 of the attached image, consists of Nafion tubing sheathed in a stainless steel tube in which liquid counter-flow occurs. Ampholytes permeate the membrane and are discarded while the molecules of interest continue into the mass spectrometer. This interface overcomes the principle challenge of reducing the ampholytes concentration prior to MS without substantially altering the separation itself.<br/><br/>We optimized our interface for both commercial and amino acid ampholytes (Glu, Asp, Gly, His, Lys). We next conducted automated high-resolution cIEF-ESI-MS. Peak shape, desalting efficiency, and reproducibility were assessed. The results of this work on protein and antibody standards and the development of the industry academic partnership will be discussed.

Robust mass spectrometry of isoelectric-focused separations of proteins

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