Orbitrap Illustration

Intact Antibody Characterization

Protein therapeutics, including monoclonal antibodies, are increasingly being developed and utilized for the diagnostic and therapeutic treatment of diseases including diabetes and cancer. As therapeutics, they have advantages over small-molecule drugs: they are highly specific and generally have fewer side effects.

All therapeutic proteins produced from current technologies have heterogeneities in the final product because various modifications occur during different stages of production. Due to these heterogeneities, thorough characterization is necessary for the reproducible and safe production of therapeutic proteins. Complete characterization of the quality attributes of these molecules requires sophisticated techniques.

Mass spectrometry (MS), especially high-resolution and accurate-mass MS, has become widely used in virtually all phases of protein drug development. Modern MS technologies provide accurate information on various protein properties, such as intact molecular mass, glycosylation forms, amino acid sequence, post-translational modifications, and minor impurities due to sample processing and storage, as well as higher-order structural information, such as protein conformational changes upon modifications, noncovalent interactions between protein drugs and receptor proteins, and protein aggregation caused by misfolding. Mass analysis at the intact protein level is usually the first step of the structural characterization.

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Workflow Overview for Intact Monoclonal Antibody (mAb) Characterization

The major challenges in intact mAb characterization are accurately and reproducibly measuring intact mass, determining the primary sequence at the intact protein level, and identifying and quantifying glycoforms. After production and purification of monoclonal antibodies, comprehensive characterization can be performed at the intact protein or sub-structure level on an Orbitrap-based mass spectrometer using an LC-MS workflow solution developed for robust, accurate, and comprehensive mAb characterization.
The intact antibody workflow presented here generates the following information for mAbs:

  1. Accurate intact molecular mass
  2. Identification of glycoforms
  3. Relative abundance of major glycoforms
  4. Identification of low mass modifications on heavy and light chains
  5. Extensive amino acid sequence coverage of intact or reduced mAbs


Intact Monoclonal Antibody Characterization Using a Bench-Top Orbitrap Mass Spectrometer

Hao Z, Zhang Y, Horn D, Sharma S, Wu S, Lin I, Pan Y, Yang Y, Huhmer A.
ASMS 2012 Oral Presentation

Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Michalski A, Damoc E, Lange O, Denisov E, Nolting D, Müller M, Viner R, Schwartz J, Remes P, Belford M, Dunyach JJ, Cox J, Horning S, Mann M, Makarov A.
Mol Cell Proteomics. 2012 Mar;11(3):O111.013698.