Abstract
Charge variant analysis is widely performed to assess changes in product quality during the manufacture of monoclonal antibodies (mAbs) and can be achieved using techniques such as capillary isoelectric focusing (cIEF), cation exchange chromatography (CEX) and mass spectrometry (MS). As large therapeutic biomolecules (~150kDa), mAbs can be challenging to characterise due to their heterogeneity, which can result from post-translational modifications and alternative disulphide bond connectivities, which alter higher order structure. Coupling of ion mobility to high resolution native mass spectrometry (IM-MS) presents a powerful tool for conformational characterisation of intact mAbs, mAb subunits and associated fragments. In this work, Cyclic IM-MS (cIM-MS) technology was used to compare the ion mobility behaviour and conformation of F(ab')2 fragments from IgG1 preparations which had exhibited heterogeneity during charge variant analysis.
IgG1 samples were separated and fractionated into lower acidic and higher acidic species using CEX chromatography. Enriched fractions were cleaved using FabRICATOR protease (Genovis AB, Sweden) below the hinge region to generate F(ab')2 and Fc/2 fragments. F(ab')2 fragments were isolated by subtractive affinity purification with Protein A magnetic beads (Promega, UK) and were desalted and buffer exchanged into 100 mM ammonium acetate, pH 6.8 using Zeba™ Spin Desalting Columns 7K MWCO, 0.5ml, (ThermoScientific, UK). Native MS and IM-MS experiments were performed using off-line nanoflow-ESI delivery to directly infuse the desalted samples onto a SELECT SERIESTM Cyclic IMS (Waters Corporation, UK), with subsequent analysis of the data within MassLynxTM v4.2.
Comparative native MS analysis of F(ab)'2 samples showed that higher, less protonated m/z species were detected for the higher acidic F(ab)'2 sample, whilst lower, more protonated m/z species were observed with the lower acidic F(ab)'2 sample. In principle, the degree of protonation was consistent with the charge characteristics that the samples exhibited during prior CEX analysis.
Examination of ion mobility peaks for given charge states within the native MS spectra demonstrated that the higher acidic F(ab)'2 sample had faster drift times, reflecting a more compact conformation in the gas phase. Conversely the lower acidic F(ab)'2 species had slower drift times, indicative of a less compact conformation. These results demonstrate the capability of cIM-MS to resolve mAb fragment conformers, providing a novel analytical tool for investigation of structural differences relating to charge heterogeneity of mAbs.
IgG1 samples were separated and fractionated into lower acidic and higher acidic species using CEX chromatography. Enriched fractions were cleaved using FabRICATOR protease (Genovis AB, Sweden) below the hinge region to generate F(ab')2 and Fc/2 fragments. F(ab')2 fragments were isolated by subtractive affinity purification with Protein A magnetic beads (Promega, UK) and were desalted and buffer exchanged into 100 mM ammonium acetate, pH 6.8 using Zeba™ Spin Desalting Columns 7K MWCO, 0.5ml, (ThermoScientific, UK). Native MS and IM-MS experiments were performed using off-line nanoflow-ESI delivery to directly infuse the desalted samples onto a SELECT SERIESTM Cyclic IMS (Waters Corporation, UK), with subsequent analysis of the data within MassLynxTM v4.2.
Comparative native MS analysis of F(ab)'2 samples showed that higher, less protonated m/z species were detected for the higher acidic F(ab)'2 sample, whilst lower, more protonated m/z species were observed with the lower acidic F(ab)'2 sample. In principle, the degree of protonation was consistent with the charge characteristics that the samples exhibited during prior CEX analysis.
Examination of ion mobility peaks for given charge states within the native MS spectra demonstrated that the higher acidic F(ab)'2 sample had faster drift times, reflecting a more compact conformation in the gas phase. Conversely the lower acidic F(ab)'2 species had slower drift times, indicative of a less compact conformation. These results demonstrate the capability of cIM-MS to resolve mAb fragment conformers, providing a novel analytical tool for investigation of structural differences relating to charge heterogeneity of mAbs.
Original language | English |
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Number of pages | 1 |
Publication status | Published - 17 Jul 2023 |
Event | BSPR-EuPA Annual Scientific Meeting: Next Generation Proteomics - Newcastle University, Newcastle Upon Tyne, United Kingdom Duration: 17 Jul 2023 → 20 Jul 2023 |
Conference
Conference | BSPR-EuPA Annual Scientific Meeting |
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Country/Territory | United Kingdom |
City | Newcastle Upon Tyne |
Period | 17/07/23 → 20/07/23 |