Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins

Understanding and managing peak shape for basic solutes in reversed-phase high performance liquid chromatography

“…These have a relatively thin porous coating on an inert non-porous core, providing a reduced diffusion distance and thus better mass transfer and efficiency for these slow-diffusing large molecules (but see also above). 43 Of these 10 additives, only 6 gave acceptable results although considerable differences were obtained in selectivity, peak shape and MS sensitivity. These differences were highlighted when subunit analysis of Trastuzumab with these 6 acid additives was performed after (partial) digestion using immunoglobulin degrading enzyme of Streptococcus pyogenes (IDES).…”

“…Peptides are readily analysed by RP techniques, whereas the analysis of intact proteins can alternatively be carried out by Size Exclusion Chromatography (SEC) or by ion exchange chromatography (IX). 43,44 The latter techniques have the advantage of being applicable to the analysis of ''native'' (i.e. non denatured) proteins as the use of organic solvents is not essential.…”

Fractionation and online mass spectrometry based on imaged capillary isoelectric focusing (icIEF) for characterizing charge heterogeneity of therapeutic antibody

“…The most common methods for charge variant monitoring in the biotechnology industry include ion exchange chromatography (IEX), traditional isoelectric focusing (IEF) and imaged capillary isoelectric focusing (icIEF). [10][11][12][13][14][15][16][17][18][19][20][21] Specically, icIEF technology based on isoelectric point (pI) differentiation is becoming the gold standard across the pharmaceutical industry for protein charge variant characterization. Furthermore, subsequent MS analysis would provide a powerful and comprehensive analysis of the charge variants and their structure.…”

Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques

“…Imaged capillary isoelectric focusing (icIEF) and ion-exchange chromatography (IEX) are two essential techniques routinely used for charge variant analysis of therapeutic mAbs during their development and in quality control. − These two techniques separate mAb charge variants based on different mechanisms and therefore have varying separation selectivities. However, the conditions commonly used in these two methods contain nonvolatile substances and usually cannot be used in conjunction with the most powerful means of mass spectrometry (MS) for protein identification.…”