An improved capillary isoelectric focusing-mass spectrometry method for high-resolution characterization of monoclonal antibody charge variants

Routine and high-resolution characterization of monoclonal antibody (mAb) charge variants is vital for controlling mAb quality as therapeutics. Capillary isoelectric focusing-mass spectrometry (cIEF-MS) has emerged as a powerful tool for...

A Mini Review on Capillary Isoelectric Focusing-Mass Spectrometry for Top-Down Proteomics

Mass spectrometry (MS)-based top-down proteomics (TDP) requires high-resolution separation of proteoforms before electrospray ionization (ESI)-MS and tandem mass spectrometry (MS/MS). Capillary isoelectric focusing (cIEF)-ESI-MS and MS/MS could be an ideal method for TDP because cIEF can enable separation of proteoforms based on their isoelectric points (pIs) with ultra-high resolution. cIEF-ESI-MS has been well-recognized for protein characterization since 1990s. However, the widespread adoption of cIEF-MS for the characterization of proteoforms had been impeded by several technical challenges, including the lack of highly sensitive and robust ESI interface for coupling cIEF to MS, ESI suppression of analytes from ampholytes, and the requirement of manual operations. In this mini review, we summarize the technical improvements of cIEF-ESI-MS for characterizing proteoforms and highlight some recent applications to hydrophobic proteins, urinary albumin variants, charge variants of monoclonal antibodies, and large-scale TDP of complex proteomes.

Rapid determination of full and empty adeno-associated virus capsid ratio by capillary isoelectric focusing

: Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.