Mass Spectrometric Analysis of Antibodies

Antibody Characterization using Middle-level LC-MS

Monoclonal antibodies (mAbs) and other IgG-based biopharmaceuticals are increasingly entering clinics and improving patients’ quality of life. The inherent size, 150 kDa, and heterogeneity caused by post translational modifications (PTMs), necessitates detailed structural characterization. This can be achieved in a variety of ways with liquid chromatography and mass spectrometry (LC-MS) being extremely popular. An essential aspect of an LC-MS workflow is timely and proper sample preparation where rapid and specific enzymes can play a crucial role.

Three Established Approaches

There are three established approaches to analyzing complex biopharmaceutical molecules by LC-MS: bottom-up, top-down and middle level analysis (Fig. 1). While bottom-up peptide mapping is still the gold standard for the analysis of critical quality attributes, such an approach is resource and time intensive in terms of both data acquisition and analysis. Top-down on the other hand allows for high throughput but suffers from low resolution MS.


Three established strategies LC-MS

Figure 1. The three established approaches to analyzing complex biopharmaceuticals by LC-MS.

SmartEnzymes: Fast & Convenient

Subunit analysis LC-MS

SmartEnzymes can shorten samples preparation times while avoiding unspecific digestion. One such SmartEnzyme, FabRICATOR (IdeS), has gained widespread popularity for middle level analysis. FabRICATOR digests IgG just below the hinge at one specific site (2), and after reduction of the disulfide bonds yields antibody fragments of 23-25 kDa that are highly amenable to high-resolution mass spectrometry. A FabRICATOR-based middle-level LC-MS workflow is not only fast and specific but enables the analysis of multiple quality attributes such as glycosylation, oxidation, glycation and C-terminal lysine clipping as well as sequence confirmation.


Find out more about antibody subunit generation and digestion optimisation


Peptide map LC-MS

The complexity of LC-MS data generated, and subsequent ease of analysis can also be significantly impacted by the sample preparation approach. Specific digestion yields homogeneous samples with easy to interpret chromatograms and spectra. In comparison, peptide mapping produces heterogeneity since the enzymes are non-specific resulting in complex data that is challenging to analyze. This puts an additional requirement on the expertise of the operator with respect to data processing and reporting (Fig. 2).


Figure 2. Comparison of subunit workflow with peptide map approach.

What Critical Quality Attributes can be addressed?

Understanding and monitoring the post translational modifications that biologics may undergo and the critical quality attributes (CQA) associated with them is fundamental for their success. In one example, high resolution mass spectrometry instruments, for example quadrupole time of flight (Q-TOF), offer the possibility to achieve monoisotopically resolved peaks for the mAb subunits (Jabs et al.). This enables rapid and accurate amino acid confirmation without proteolytic digestion using enzymes such as trypsin. A review by Beck et al. also highlights the advantages of using a FabRICATOR based middle down strategy for MAb characterization. A range of other CQAs can be addressed with middle level LC-MS, see the following pages to learn more:


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