September 1, 2017 (Vol. 37, No. 15)

To Characterize the Inherent Microheterogeneity of mAbs,
One Must Consider the Surrounding mAb Environment

Monoclonal antibodies (mAbs) have steadily been garnering interest since their initial introduction as approved and commercially available therapeutics more than twenty years ago. The first major milestone in the development of mAbs was an article published in Nature in 1975 by Georges J.F. Köhler and César Milstein1 on the fusion of myeloma cell lines with B cells to create hybridomas that could produce antibodies specific to known antigens; the authors were awarded the Nobel Prize in Medicine 10 years later for this discovery. The second milestone came in response to a clinical failure due to the high dissimilarity of the murine antibodies and the human immune system.

The disparity between animal models caused some drastic reactions, such as anaphylactic shock. The first approach to resolve the dissimilarities was the production of chimeric antibodies, in which murine variable regions were grafted onto human constant regions. The second to rectify model disparities approach was described, again in Nature, by Greg Winter and coauthors in 1988.2 The researchers successfully humanized mAbs by grafting murine hypervariable regions onto amino acid domains of human antibodies, a technique that eliminates most immunogenic reactions and produces molecules of ~95% human origin.

The benefits of therapeutic antibodies include their ability to be inherently more selective than traditional small-molecule pharmaceuticals through specific binding activities, lower toxicities (related directly to the better selectivity of mAbs and the reduction of off-target effects), higher efficacy, and of course, the large potential revenues that are associated with the successful commercialization of biologics. The FDA has approved 68 antibody-based therapeutics up to the beginning of 2017,3 and more than 50 antibody therapeutics are currently being evaluated in late-stage clinical studies4 for a range of indications.

As mAbs are produced from cell lines and fermentation processes, they are fundamentally less pure than traditional chemically synthesized products, and can also be subjected to a number of possible degradation events and chemical modifications throughout their manufacturing and storage (Figure 1). This lack of purity seen with mAb variants and the microheterogeneity inherent to the nature of the molecule—due to things such as glycosylation—requires significant and robust characterization, not only during discovery and research but throughout the drug-development process, as well as during quality control (QC) exercises and lot-release testing.

Antibody-drug conjugates (ADCs) are a newer class of antibody-based biopharmaceuticals that entered the market in 2001. They are complex molecules composed of an antibody targeting a certain tumor marker linked to a small-molecule cytotoxin. There are different chemistries available to attach the cytotoxin to the antibody, and the coupling results in a distribution with varying numbers of drugs attached to a single antibody molecule, requiring the need to monitor the drug-to-antibody ratio (DAR).

There are a vast number of analytical methodologies that can be applied during the development process of mAbs and ADCs, from chromatographic techniques such as size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), traditional reversed-phase (RP) liquid chromatography (LC) and capillary electrophoresis (CE); to enzyme-linked immunosorbent assays (ELISAs). High-resolution accurate-mass–mass spectrometry (HRAM–MS) can be added to several of these chromatographic techniques to provide increased confidence in detection.

Figure 1. An illustration of some examples of modifications that can occur with mAb products; the microheterogeneity of mAbs makes their analysis challenging.

Intact Mass Analysis under Native Conditions

The analysis of protein therapeutics at the intact level can involve sample preparation, such as treatment with glycosidases for mass determination of the plain protein, or in the case of ADCs, it may be necessary to simplify the sample significantly for the accurate determination of the DAR. Additionally, depending on the choice of chromatographic separation conditions, the three-dimensional protein structure of a mAb is either preserved or disrupted:

• Use of aqueous buffers at near-neutral pH results in the detection of fewer and lower charge state; these results appear at the higher-mass end of the m/z scale (Figure 2A, Figure 2B). This pH fosters native or native-like conditions.

• Organic solvents, such as acetonitrile in the presence of acids (e.g., formic acid)—typically used in reversed-phase chromatography—disrupt protein folding. The structure opens up and provides a larger surface, enabling more protons to attach, leading to the detection of extra and higher charge states, and therefore, results appear on the lower end of the m/z scale. This is an example of intact mass analysis under denaturing conditions.

Analysis under native conditions involves instrumental requirements regarding mass-detection capabilities towards 8,000 m/z to capture the entire charge envelope, as well as capabilities to provide sufficient desolvation, which is more challenging when electrospraying purely aqueous solvents without any acid additive. Moreover, the efficient transfer of the lower-charged ions of large proteins or even protein complexes requires optimization. Higher resolution power of the mass spectrometer is also crucial to handle complex profiles, such as the mAbs that incorporate multiple glycosylation patterns (Figure 2C).5

Figure 2. The analysis of infliximab under native conditions. A) Raw spectrum showing the charge envelope at higher m/z with fewer charge states than under denaturing conditions; inset shows the size-exclusion chromatography separation of infliximab and the salt peak. B) Zoom of the most intense charge state, z=26. C) Deconvolution result obtained from native liquid chromatography–mass spectrometry analysis showcasing infliximab’s complex pattern due to various glycoforms and varying levels of C-terminal lysine clipping.

Recently, there have been several major advances in analytical instrumentation and separation techniques that allow for the analysis of protein therapeutics, mixtures thereof,6,7 and large protein assemblies and complexes up to a megadalton in molecular weight under native-like conditions.8

The main benefits of analysis under native conditions are twofold:

1. Non-covalent interactions are preserved, enabling ligand binding studies,antibody receptor studies,10 the analysis of Cys-linked ADCs, and the analysis of protein complexes.11

2. The detection of lower-charge states at higher m/z values, providing higher spatial resolution and often simplified peak patterns due to less overlap of charge states when compared with denatured analysis (Figure 3).

Figure 3. Liquid chromatography–mass spectrometry (LC–MS) analysis of the antibody-drug conjugate (ADC) trastuzumab emtansine demonstrating how native mass spectrometry (bottom) provides greater m/z separation of coeluting species charge states compared with analysis under denaturing conditions (top). High-quality spectra obtained in both cases clearly show there is no need for deglycosylation.

Instrument Vendors Address Analytical Challenges

Recent significant innovations in Orbitrap-based mass spectrometry have added the capability to perform native MS analysis with mass detection of up to 8,000 m/z without compromising the performance of normal operation modes. These enhanced capabilities—together with controlled-source desolvation, spectral resolution, and the use of accurate deconvolution algorithms—are necessary for the simplified and rapid analysis of mAbs on the intact level under native conditions.

To further increase the applicability of analysis under native conditions, traditional intact separations such as SEC,12 IEC,13 hydrophobic-interaction chromatography (HIC), and CE14 can now be routinely performed using buffers that are both mass-spectrometry compatible and nondenaturing; this allows HRAM–MS to be added to these commonly used chromatographic analyses, to both increase the amount of information that can be obtained in a single experiment and provide greater confidence in the results.

Figure 4 demonstrates the increased information that can be obtained when a complex biotherapeutic sample, such as a random lysine-conjugated ADC, is analyzed under native conditions using SEC coupled to mass spectrometry. Through the use of powerful informatics, the DAR can easily be calculated in addition to observing the glycosylation profile, which would have been lost if the ADC were deglycosylated. So, if complex mAbs such as ADCs can be analyzed with unprecedented detail using native LC–MS to determine not only the glycoforms that are present but also the DARs that exist (Figure 4), how far can this technique be pushed?

Heck et al.7 wrote that complex mixtures containing multiple antibodies could be analyzed using native mass spectrometry to provide unambiguous qualitative and quantitative results. Notably, Dr. Heck noted that while “complete glycan profiling cannot be performed using native MS, the assessment of glycan heterogeneity, as well as quantitation of the various glycoforms, can conveniently be performed in parallel with the quantitation of the different antibody components in a single analytical characterization.” The Heck paper also pointed out that “high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization” and “as a complementary tool in product characterization and screening of biotherapeutics.”15

Figure 4. Deconvolution results (right) of the liquid chromatography–mass spectrometry (LC–MS) analysis of the antibody-drug conjugate trastuzumab emtansine under native conditions; showing the complex pattern due to varying drug loads and different glycoforms. For drug-antibody ratio (DAR) calculation, a single glycoform or multiple glycoforms can be considered (left).


With the significant technological advancements of high-resolution mass spectrometers, as well as improvements for the separation of therapeutics when combined with mass spectrometric analyses, researchers can obtain more in-depth structural insight more quickly, with less effort, than ever before. Native LC–MS, therefore, is becoming increasingly useful for researchers undertaking the characterization of protein therapeutics who require a greater understanding of complex samples—and for those who need information that cannot necessarily be obtained under traditional denaturing conditions.

Simon Cubbon, Ph.D., is vertical marketing manager, Thermo Fisher Scientific, U.K.; Aaron O. Bailey, Ph.D., is mass spectrometry specialist, Thermo Fisher Scientific, U.S.; and Kai Scheffler, Ph.D., is biopharma support expert, Thermo Fisher Scientific, Germany.

1. G. Köhler and C. Milstein, “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256(5517), 495–497 (1975).
2. L. Riechmann, M. Clark, H. Waldmann, and G. Winter, “Reshaping Human Antibodies for Therapy,” Nature 332(6162), 323–327 (1988).
3. H.H. Cai, “Therapeutic Monoclonal Antibodies Approved by FDA in 2016,” MOJ Immunol. 5(1), 00145 (2016).
4. J.M. Reichert, “Antibodies to Watch in 2017,” mAbs 9(2), 167–181 (February/March, 2017).
5. S. Rosati, E.T. van den Bremer, J. Schuurman, P.W. Parren, J.P. Kamerling, and A.J. Heck, “In-Depth Qualitative and Quantitative Analysis of Composite Glycosylation Profiles and Other Micro-Heterogeneity on Intact Monoclonal Antibodies by High-Resolution Native Mass Spectrometry Using a Modified Orbitrap,” mAbs 5(6), 917–924 (2013). 
6. Y. Yin, G. Han, J. Zhou, M. Dillon, L. McCarty, L. Gavino, D. Ellerman, C. Spiess, W. Sandoval, and P.J. Carter, “Precise Quantification of Mixtures of Bispecific IgG Produced in Single Host Cells by Liquid Chromatography-Orbitrap High-Resolution Mass Spectrometry,” mAbs, 8(8), 1467–1476, (2016).
7. N.J. Thompson, L.J. Hendriks, J. de Kruif, M. Throsby, and A.J.R. Heck, “Complex Mixtures of Antibodies Generated from a Single Production Qualitatively and Quantitatively Evaluated by Native Orbitrap Mass Spectrometry, mAbs 6(1), 197–203, (2014).
8. M. van de Waterbeemd, J. Snijder, I.B. Tsvetkova, B.G. Dragnea, J.J. Cornelissen, and A.J. Heck, “Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry,” J. Am. Soc. Mass Spectrom. 27(1000Y1009), (2016).
9. H.J. Maple, O. Scheibner, M. Baumert, M. Allen, R. Taylor, R. Garlish, M. Bromirski, and R. Burnley, “Application of the Exactive Plus EMR for automated Protein–Ligand Screening by Non-Covalent Mass Spectrometry,” Rapid Commun. Mass Spectrom. 28, 1561–1568 (2014).
10. R. Gahoual, A. Heidenreich, G.W. Somsen, P. Bulau, D. Reusch, M. Wuhrer, and M. Haberger, “Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry,” Analyt. Chem. 89(10), 5404–5412 (2017).
11. J. Marcoux, T. Champion, O. Colas et al., “Native Mass Spectrometry and Ion Mobility Characterization of Trastuzumab Emtansine, a Lysine-Linked Antibody-Drug Conjugate,” Protein Science?: A Publication of the Protein Society, 24(8), 1210–1223 (2015).
12. S. Lin, H. Wang, Z. Hao, P. Bennett, and X. Liu, Thermo Fisher Scientific, San Jose, CA, USA, “Analysis of Monoclonal Antibodies and Their Fragments by Size-Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer,” White Paper, (2016).
13. A.O. Bailey et al., “Native Ion-Exchange Chromatography Directly Coupled to Orbitrap Mass Spectrometry Allows Surface Charge Discrimination and Online Detection of Intact Proteins,” Poster Presentation, Thermo Fisher Scientific,.
14. S. Houel et al., “Evaluation of a Microfluidic Electrophoresis Device Coupled to an Orbitrap Mass Spectrometer for the Characterization of Biotherapeutics Proteins,” Poster Presentation, Thermo Fisher Scientific.
15. N J Thompson, L J Hendriks, J de Kruif, M Throsby & A J R Heck. Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry, mAbs, 6:1, 197-203, (2014).

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