Imaged capillary isoelectric focusing (icIEF) technology has been proven to be robust for the characterization of protein charge heterogeneity due to its high-resolution pI discrimination and high throughput. Although high-performance...

A carrier ampholyte is a molecule containing both acid and base functionality that is critical for imaged capillary isoelectric focusing. The quality of an imaged capillary isoelectric focusing separation for protein charge variants’ characterization is highly dependent on attributes of the carrier ampholytes used including baseline signal, linearity of the pH gradient, pI discrimination, and consistency between manufactured lots. AESlytes are a high‐resolution carrier ampholyte series that have been developed for the high‐resolution and selective characterization of diverse and complex protein drugs including diverse fusion proteins, antibody‐drug‐conjugate, bi‐specific antibodies, and viral proteins. While routine commercial ampholytes usually cannot solve such challenges, AESlytes demonstrate a reduction in baseline noise and distinguishably increased consistency between lots as compared to other commercial ampholytes. Here we apply AESlytes for the imaged capillary isoelectric focusing separation of several commercial fusion proteins and biosimilars with excellent repeatability. In addition, AESlytes with narrow‐range pH were employed directly coupled to a mass spectrometer for optimizing the separation resolutions allowing more reliable and accurate protein charge variant identification. Our study demonstrates that innovative high‐resolution carrier ampholytes as critical reagents play an essential role in high‐performance imaged capillary isoelectric focussing and tandem mass spectrometry analysis the routinely commercial ampholytes cannot achieve, especially for extremely complex protein drugs.

Identifying and characterizing charge heterogeneity is essential for the successful development and production of biopharmaceuticals. Imaged capillary isoelectric focusing technology based on isoelectric point differentiation has been becoming the gold standard of quality and manufactory process control in the biopharmaceutical industry due to its high‐resolution characterization of protein charge variants with high throughput. Fluorocarbon‐coated capillaries are widely used in the imaged capillary isoelectric focusing to suppress the electroosmotic flow and minimize the protein adsorption onto the fused silica capillary inner surface. Additionally, polymers such as methylcellulose are usually added to the sample solution to serve as a dynamic capillary coating that improves the peak shape and resolution during the imaged capillary isoelectric focusing separation, especially for complex proteins. However, the addition of methylcellulose tends to result in tedious operation, “spike peaks” from bubble generation, and frequent capillary blockage during the imaged capillary isoelectric focusing separation. In addition, methylcellulose is not compatible with mass spectrometry and easily produces the contamination of mass spectrometry ion source when carrying out imaged capillary isoelectric focusing—mass spectrometry direct coupling. Recently, a new imaged capillary isoelectric focusing method was developed employing a methylcellulose‐coated capillary cartridge, to avoid the addition of methylcellulose which is then present throughout the whole analysis. When applied to protein drug characterization the established imaged capillary isoelectric focusing method demonstrated high repeatability, stable coatings, outstanding separation efficiency, and excellent isoelectric point differentiation. In addition, we compared imaged capillary isoelectric focusing separation using the methylcellulose‐coated capillary with that utilizing the routinely coated capillary such as fluorocarbon, illustrating that methylcellulose coating provided consistent results and could be seamlessly integrated into an existing drug discovery process. Finally, the methylcellulose‐coated fused silica capillary was applied to imaged capillary isoelectric focusing—mass spectrometry for characterizing protein charge variants allowing reliable identification of mass spectrometry after imaged capillary isoelectric focusing separation. This can greatly simplify the operation steps and prevent the contamination of mass spectrometry ion source that often results from using routinely coated capillaries ultimately making this an essential innovation of imaged capillary isoelectric focusing—mass spectrometry that greatly improves the imaged capillary isoelectric focusing compatibility with mass spectrometry.