Imaged capillary isoelectric focusing: Applications in the pharmaceutical industry and recent innovations of the technology

Post‐translational modifications (PTMs) of adeno‐associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low‐concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.

show abstract

“…The analysis of AAV9 by icIEF native fluorescence was performed as described previously [10] using the Maurice instrument (ProteinSimple).…”

Section: Methodsmentioning

confidence: 99%

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity

Ramı́rez

Haitjema

et al. 2023

Electrophoresis

Self Cite

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Wang

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

Imaged capillary isoelectric focusing (icIEF) and ionexchange chromatography (IEX) are two essential techniques that are routinely used for charge variant analysis of therapeutic monoclonal antibodies (mAbs) during their development and in quality control. These two techniques that separate mAb charge variants based on different mechanisms and IEX have been developed as front-end separation techniques for online mass spectrometry (MS) detection, which is robust for intact protein identification. Recently, an innovative, coupled icIEF-MS technology has been constructed for protein charge variant analysis in our laboratory. In this study, icIEF-MS developed and strong cation exchange (SCX)-MS were optimized for charge heterogeneity characterization of a diverse of mAbs and their results were compared based on methodological validation. It was found that icIEF-MS outperformed SCX-MS in this study by demonstrating outstanding sensitivity, low carryover effect, accurate protein identification, and higher separation resolution although SCX-MS contributed to higher analysis throughput. Ultimately, integrating our novel icIEF-HRMS analysis with the more common SCX-MS can provide a promising and comprehensive strategy for accelerating the development of complex protein therapeutics.

show abstract

Imaged capillary isoelectric focusing: Applications in the pharmaceutical industry and recent innovations of the technology

Cited by 25 publications

References 65 publications

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity

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

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

Contact Info

Product

Resources

About