Imaged capillary isoelectric focusing employing fluorocarbon and methylcellulose coated fused silica capillary for characterization of charge heterogeneity of protein biopharmaceuticals

Abstract

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.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…Neutral coated capillaries are used to suppress the EOF and allow for focusing in specified positions. Available coatings are fluorocarbon (FC)-based [278] or contain immobilized methylcellulose [279]. For CIEF, in many cases, the neutral coated capillary from SCIEX is used in conjunction with the aforementioned CIEF gel [240,272,280].…”

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…Dynamic coatings are based on reversible (dynamic) adsorption of small ions, for example, oligoamines and detergents (SDS, CTAB), or hydrophilic polymers and cellulose derivatives, for example, methylcellulose [108] or hydroxypropylcellulose (HPC) [109] added to the BGE to the capillary or microchannel wall. Static (permanent) modifications of the inner capillary or microchannel wall include covalent bonding or strong physical adsorption of the synthetic and natural polymers, for example, neutral linear polyacrylamide (LPA) [110], PVA [111], poly(vinyl pyrrolidone) [92], poly(ethylene oxide) (PEO) [112], PEG [113], fluorocarbon [114], and linear and grafted cellulose derivatives [108,114], or charged polymers, such as cationic polybrene (PB) (hexadimethrine bromide) [94], poly(diallyldimethylammonium chloride) (PDADMAC) [113], and PEI [115], and anionic poly(sodium styrene sulfonate) (PSS) [113], and dextran sulfate [110].…”