Subunit middle-down analysis
Monoclonal antibody (mAb) subunit analysis involves the antibody fragmentation through digestion and reduction of IgG antibody molecules. The cysteine protease produced by S.pyogenes, known as IdeS, is highly specific. IdeS digestion followed by reduction generates three subunits that are readily separated by reversed-phase (RP) UHPLC using Thermo Scientific MAbPac RP columns. Deconvolution of subunit mass spectrometry (MS) spectra is easy with powerful Thermo Scientific BioPharma Finder software.
Subunit mass analysis workflow
Fab Preparation Kit
Thermo Scientific Pierce Fab Preparation Kit uses immobilized papain protease to digest human or mouse IgG antibodies to make separate Fab and Fc antibody fragments and subsequently to purify the Fab using Protein A agarose.
Subunit mass analysis categories
Use Thermo Scientific MAbPac RP columns for high-performance, reversed-phase HPLC and LC/MS characterization of mAbs, antibody fragments, charge variants, antibody drug conjugates (ADCs), PEGylated proteins, and bispecific proteins.
The innovative architecture of the new Thermo Scientific Vanquish Flex UHPLC system is tailored to the specific and demanding analytical requirements that these essential drugs impose. Vanquish Flex UHPLC has been designed and manufactured to exacting engineering standards to be truly built for biopharma.
Protein reduction
Choose between research grade dithiothreitol (DTT) or Tris-(2-Carboxyethyl)phosphine, (TCEP) for antibody reduction.
The Thermo Scientific Orbitrap Exploris 240 mass spectrometer provides a complete BioPharma characterization solution. The extreme resolving power of the Thermo Scientific Orbitrap mass analyzer ensures isotopic resolution of monoclonal antibody subunits and facilitates middle-down sequencing.
The sliding window method and Thermo Scientific Xtract deconvolution algorithm within Thermo Scientific™ BioPharma Finder™ software enables monoisotopic mass determination of monoclonal antibody subunits.
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