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Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

ABSTRACT: The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic-inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.

SUBMITTER: Murisier A

PROVIDER: S-EPMC9311719 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

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Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

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