Protein Fractionation Using icIEF-Based Charge Separation 

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Why Choose MauriceFlex for icIEF-Based Protein Fractionation?

Protein fractionation with MauriceFlex lets you collect charge variants and provides significant advantages on time and capital when compared with IEX, HPLC and other existing technologies. The key advantages are: 

• Same-day fraction collection  
• High purity, MS-ready fractions with just 100 µL sample; single run often provides enough material for intact MS analysis 
• Compatibility with urea and methylcellulose 
• Ability to pool fractions for peptide mapping 
• Ability to analyze fractions with methods like SPR and mass photometry  
• Routine CE-SDS and icIEF assays on the same system 
• Eliminating the need for bridging studies otherwise required with IEX and HPLC methods 

How does icIEF Fractionation with MauriceFlex Work? 

Protein charge isoforms are first separated with icIEF and then mobilized so that each isoform (or variant) can be eluted and collected for downstream characterization.  

The MauriceFlex system allows same-day icIEF separation and icIEF-based protein fractionation for LC-MS. It also enables charge variant analysis workflows in addition to mass spectrometry, such as binding studies with SPR and size variant analysis with mass photometry. Irrespective of the downstream characterization method used, charge variants collected do not require buffer exchange or re-optimization and are typically ready to use as-is with LC-MS, SPR, and mass photometry.  

Because downstream characterization requirements can vary based on the phase of drug development, instrument, and expertise availability, fractions collected on MauriceFlex are indeed flexible and can be analyzed on a variety of MS instruments, with little to no sample prep. 

1. Fractionation for Biosimilar Characterization with LC-MS workflows (intact or subunit mass analysis) 
   The MauriceFlex and BioAccord™ LC-MS system (Waters) were used together to analyze the differences between the innovator drug Belimumab and a research-grade biosimilar¹. By directly loading the collected acidic, main, and basic fractions onto the LC-MS system for intact mass analysis, critical differences between the two molecules were observed, including the presence of C-terminal lysine, amino acid clipping, and deamidation. Another case study delves into subunit analysis of the acidic, main, and basic peaks of Mosunetuzumab, which is a bispecific antibody, and a research-grade biosimilar². In this study, subunit analysis was performed by using a digestive enzyme called FabRICATOR^® (Genovis) before loading on the BioAccord System. Most notably, LC-MS analysis clearly showed incorrect pairing of the light and heavy chains in the biosimilar, thus underscoring the need for such in-depth analysis of charge variant fractions. 
     
2. Microfluidic MS platforms like ZipChip for rapid, high-throughput characterization  
   Another collaborative study highlighted how MauriceFlex fractions can be paired with the ZipChip-MS system (908 Devices) for rapid charge variant characterization³. In this work, acidic, main, and basic fractions from an antibody-drug conjugate (ADC) parent mAb were collected using MauriceFlex and analyzed without post-run sample preparation. The ZipChip-MS workflow, which uses capillary electrophoresis coupled directly to mass spectrometry, enabled fast and sensitive intact mass analysis of individual charge variants. Results revealed major glycoforms and minor isoforms that aligned closely with reported mass data, confirming the ability of the icIEF fractionation + ZipChip-MS combination to deliver high-quality results in a fraction of the time required for traditional IEX workflows. This collaboration underscored the versatility of MauriceFlex fractions for use across different MS platforms, reinforcing its value as an MS-agnostic solution for biopharmaceutical characterization. 

Beyond Molecular Structure: Insights into Biological Function 

Importantly, charge heterogeneity doesn’t just affect molecular structure but can impact biological function too. In another collaborative study, the MauriceFlex system was used to fractionate acidic, main, and basic variants of the bispecific antibody Mosunetuzumab and a research-grade biosimilar, which were then analyzed for binding using the Alto™ Digital SPR System (Nicoya)⁴. Despite requiring only 2 µL of each fraction, the SPR assay provided detailed kinetic and affinity data for binding to CD3 and CD20 ligands. The results revealed that while most fractions displayed comparable binding across innovator and biosimilar, the acidic fraction of the biosimilar showed significantly weaker binding to CD20. These findings correlated with the aforementioned intact LC-MS study that had shown incorrect assembly in the same acidic fraction, as further evidence of how the combined icIEF fractionation and SPR workflow offers a powerful way to link structural heterogeneity with functional impact directly. 

Continued Impact on the Field 

A recent study by Wittmann et al. evaluated the suitability of the MauriceFlex platform for offline MS analysis⁵. Using Matuzumab as the analyte, charge analysis and fraction collection were completed in just 2.5 hours, with further characterization done on a QTOF MS system with an ESI source. The study offers a positive conclusion and states that “This cIEF fractionation with offline MS approach provides excellent selectivity and sensitivity for thoroughly investigating all charge variants of pharmaceutical monoclonal antibodies. The straightforward method development and reliable technical setup enable platform methods to effectively characterize these crucial substances during drug development”.  Another study published in Electrophoresis by McElroy et al. demonstrated method optimization for fractionation of AAV charge variants with MauriceFlex, making significant inroads in AAV capsid protein characterization⁶.   

Maximizing Value from a Single Instrument 

In an environment where resources, time, and expertise are often stretched thin, MauriceFlex provides a clear advantage with simplified workflows and high-quality data offering deeper insights, all through a single platform. By integrating icIEF, CE-SDS, and icIEF fractionation, and enabling seamless connections to MS and SPR, MauriceFlex empowers scientists with a multi-attribute analysis tool that characterizes biotherapeutics with precision and efficiency, surpassing the capabilities of traditional workflows.  

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