Characterization of a protein, biosimilar, antibody, and their isoforms is usually done by peptide mapping analysis of a digested mixture using bottom-up sequencing. This is a powerful technique for obtaining complete coverage, including complex modifications. Unfortunately, in the process, the connectivity and quantitative relationships of isoforms and modifications are lost. An alternate approach is top-down and middle-down analysis of the protein. In top-down analysis, the whole protein is analyzed on the mass spectrometer for molecular weight and limited fragmentation. This allows the analysis of the quantitative aspects of modification to be examined. Middle-down analysis first involves breaking the antibody into light and heavy chains using reduction or top and bottom using specific enzymes. The resulting pieces weighing typically 25–50 kDa are easier to fragment with high coverage. When the analysis is done by liquid chromatography, it is possible to separate and estimate the number of isoforms present. This analysis requires a high-resolution mass spectrometer such as the Thermo Scientific™ Q-Exactive™ Exploris 480, or Thermo Scientific ™ Orbitrap Fusion™ Lumos Eclipse. This protocol describes a short liquid chromatography mass spectrometry (LC-MS) separation of intact proteins and mAbs.
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