Benefits of Middle-level Analysis of Antibodies using FabRICATOR®

Performance Activity

Generation of defined IgG subunits via single-site, below hinge cdigestion, enabling high-resolution middle-level LC-MS and improved PTM characterization.

The high specificity of FabRICATOR – with a single digestion site below the hinge – and the fact that it exhibits activity on a wide range of species and subclasses of IgG make it an ideal tool for efficient characterization of a range of different molecules. This example shows FabRICATOR being used for middle-level characterization of IgG and a comparison, using data, to intact mass analysis of the human IgG1 adalimumab.

Following FabRICATOR digestion and reduction of the disulfide bonds, the scFc, LC and Fd’ are efficiently separated by reversed-phase LC in under 10 minutes. Digestion and reduction of the antibody generates subunits of around 25 kDa which is ideal for high resolution mass spectrometry. Conversely, at the intact level, the mass spectrum is much more complex, and while the masses of the major species can be determined, there are many overlapping species, many of which are difficult to unequivocally identify.

Higher quality mass spectra of antibody subunits compared to intact analysis

Some modifications, such as oxidation, lead to mass shifts which are quite small and not possible to identify at the intact level. Additionally, any modifications that can be determined cannot be localized to a specific subunit, which is important for in-depth characterization of therapeutic antibodies. Middle-level analysis following FabRICATOR digestion not only allows many more post-translational modifications to be identified, but they can be localized to an individual subunit.

Domain specific modification information at the subunit level

FabRICATOR is a highly efficient enzyme, and often, digestions can be carried out in 30 minutes or less. This, combined with a short LC separation, means that accurate monoisotopic mass of subunits, identification and site-specific localization of modifications can be determined simultaneously. This generates a much greater amount of relevant information than can be achieved at the intact level, which is even more apparent when the antibodies are more complex and heterogeneous.

Simultaneous mass determination and analysis of PTMs

Middle-level analysis compared to intact analysis of antibodies. Reversed-phase LC separation of reduced adalimumab scFc, LC and Fd’ subunits following FabRICATOR digestion (left); deconvoluted mass spectra of scFc, LC and Fd’ adalimumab subunits (middle) and deconvoluted mass spectra of intact adalimumab (right). Chromatographic separation of the reduced subunits can be achieved in less than 10 minutes and there is much improved mass spectral resolution when analysing the indiviual subunits than at the intact level.

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