Intact mass analysis is the measurement and determination of the molecular weight of the intact antibody protein. It may measure the antibody protein in its native form, or it may measure the heavy and light chains separately after reduction. In the case of antibody fragments, it may also measure the fragments, such as Fab or VH+CH1.
When the antibody’s primary sequence is known, intact mass can provide extra information to confirm the primary sequence of the antibody protein. However, certain sequence variations, such as Leucine/Isoleucine mutation and the swap of two amino acids, cannot be validated this way. More importantly, intact mass analysis can help derive the relative ratios of expressed glycoforms conferring biological activity or resulting in immunogenicity.
The general steps of a typical intact mass analysis is as follows,
- LC-MS on the purified antibody protein or its fragments to generate a full MS spectrum
- Deconvolution of the high charged full MS spectrum
- Data analysis and interpretation
The image below illustrates a highly charged envelope of a target protein (B) as well as the corresponding deconvoluted spectrum (C).
(Image credit: http://www.genengnews.com/gen-articles/analysis-of-ptms-via-capillary-lc-coupled-with-hram-mass-spec/5722)