LC-MS Characterization of Therapeutic Proteins



Proteins developed for therapeutic treatment or diagnostic testing are large, complex molecules that require specialized data analysis techniques. These analyses can provide a protein’s basic sequence and can be used to indicate post translational modifications (PTM’s), internal disulfide linkages, overall protein conformation, and binding activity to other molecules. The information presented in this paper will focus on four analytical techniques typically employed for the analysis of monoclonal antibodies (mAb’s).

 1.  Intact Analysis, including glycoform differentiation
2.  Peptide Mapping
3.  Internal Disulfide Linkages
4.  Glycopeptide Analysis

Figure 1. Full MS1 spectra of a mAb sample


Most protein samples are not completely homologous and the protein itself may consist of multiple proteoforms. Performing an intact analysis of an ~150kD protein, with multiple populations, requires a high-resolution MS detector and a software package capable of differentiating the proteoforms without artifacts.

Antibody proteins routinely have multiple glycosylated forms, of varying population. Eurofins EAG Materials Science analyzed a denatured (non-native) confirmation of a monoclonal antibody (mAb) sample with multiple glycoforms, which are protein variants that differ with respect to number or types of attached glycans. Intact samples were analyzed using a high-resolution LC/MS system, a Thermo Q-Exactive. The data was processed utilizing Byos® software from Protein Metrics. The data as seen in Figure 1, appears as a complex spectrum. This is due to the multiple charge states of a large protein and the multiple glycoforms. Even with this complexity, a high-resolution detector is able to resolve the multiple isotopes, allowing for the deconvolution and identification of the glycoform populations. Spectral data is highly complex due to the large size, multiple charge states and multiple glycoforms present. High resolution MS and advanced software are critical for performing analyses of complex molecular samples such as this.

Figure 2. Zoomed view of high-resolution data of a mAb sample

A zoomed view of the spectrum can be seen in Figure 2. Isotopic resolution of individual charge states is shown even above m/z 3000, with nearly baseline resolution. This allows for the positive identification of the glycoforms present.

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