Refining proteoform characterization in biopharmaceuticals: A paradigm for the impact of pI markers and carrier ampholytes in imaged capillary isoelectric focusing tandem mass spectrometry.

Proteoforms, structurally distinct yet closely related protein isoforms, play a pivotal role in biopharmaceutical development, directly influencing therapeutic efficacy, safety, and stability. These molecular variants arise from genetic polymorphisms, alternative splicing, and post-translational modifications, necessitating advanced analytical techniques for precise characterization. Imaged capillary isoelectric focusing (icIEF) coupled with mass spectrometry (MS) has become a powerful high-resolution tool for resolving and identifying proteoforms in complex biopharmaceutical samples.

Advancing protein heterogeneity analysis: Nano-flow pressure mobilization for precise icIEF fractionation and online MS detection.

This study addresses the challenges of high-resolution protein charge variant fractionation and efficient online mass spectrometry (MS) detection in imaged capillary isoelectric focusing (icIEF)-based workflows. icIEF often faces limitations in efficiency, peak integrity, and detection sensitivity due to diffusion and uncontrolled mobilization. To overcome these, we developed a novel icIEF fractionation framework that integrates nano-flow pressure mobilization with the capillary diameter transformation technique (CDTT).

Imaged capillary isoelectric focusing and online mass spectrometry for milk whey protein characterization in dairy products.

Characterizing major bovine milk proteins, including whey and casein, is of significant interest in the dairy industry. The diverse array of protein proteoforms can be different in terms of genetic variation, breed ways, lactation stage, and animal nutritional status. Current routine methods for bovine milk protein profiling are typically based on immunological techniques, infrared spectroscopy, slab gel isoelectric focusing, capillary electrophoresis, and high-performance liquid chromatography.

Imaged capillary isoelectric focusing tandem high-resolution mass spectrometry using nano electrospray ionization (ESI) for protein heterogeneity characterization.

Recombinant monoclonal antibodies (mAbs) have been spurring the rapid growth of commercial biotherapeutics. During production their charge heterogeneity must be assessed as a critical quality attribute to ensure safety, efficacy, and potency. Although imaged capillary isoelectric focusing (icIEF) is a powerful tool for this process, it could be improved further with tandem high-resolution mass spectrometry (HRMS).