Orbitrap Technology Streamlines Analysis of Composite Antibody Mixtures

One of the primary benefits of monoclonal antibody (mAb)-based therapeutics is high target specificity. This increases the efficacy of the treatment while minimizing the provocation of an immune response in the patient.¹ The industry has witnessed a recent surge in the research and development of these therapeutics, with more than 30 mAb-based products already approved for clinical use and additional products going through clinical trials.^2,3

The production process for mixtures of recombinant mAbs requires manufacturers to either generate each mAb individually before mixing or to produce single-vessel mixtures. The first option presents financial and temporal challenges while the second requires the characterization of large, structurally similar biochemicals.^4,5 Thompson et al. recently turned to an Orbitrap platform (Thermo Scientific) with high-resolution native mass spectrometry (MS) to characterize mixtures of up to 15 therapeutic antibodies drawn from a single production vessel, with a maximum of 8 minutes acquisition time.⁶

The researchers composed three mixtures containing 6, 10, or 15 antibody species, with masses ranging from 144,381.84 Da to 146,965.22 Da. The ion signals in the mass spectra hovered around 6,000 m/z and presented a narrow charge distribution (22+ to 26+) and well-resolved peaks. The researchers unambiguously identified each mAb, including the six high-mass antibodies with an average mass difference of only 50 Da. The two mAbs that differed by only 20.94 Da could not be baseline resolved because their size difference approached the physical limit for resolution; however, the researchers were able to differentiate between them for purposes of identification.

Thompson et al. posit that the enhanced performance of the Orbitrap-based instrumentation is not a function of higher resolving power but instead results from more efficient desolvation with decreased adduct formation. They observe that this efficiency also yields greater mass accuracy, as evidenced by the wider peaks and up-shifted mass for the spectra obtained using time-of-flight platforms. The mass accuracy was an average of 7 ppm. The average mass error was 7.5 ppm, suggesting that the errors in mass accuracy result from random inaccuracies in technique. The researchers also report less than 1.2% error in the quantification of relative amounts for each baseline-resolved species in the mixtures using Protein Deconvolution 2.0 (Thermo Scientific).

To further demonstrate the capability of the Orbitrap platform, the researchers analyzed a 6-antibody mixture before and after deglycosylation. They found that, while the deglycosylated samples produced more resolved peaks, the glycosylated samples were readily distinguishable. In terms of relative abundance, the researchers obtained equal results for both the glycosylated and deglycosylated samples. This suggests that attached glycans do not affect relative ion intensities and that it is possible to eliminate this step in routine analysis.

Overall, Thompson et al. assert that native MS performed on an Orbitrap-based platform provides the necessary versatility, accuracy and reproducibility for the characterization of antibody mixtures. They believe this technology will allow producers to perform batch-to-batch characterization and mixture screening in a way that may promote the development of industry-wide guidelines and open the door for widespread therapeutic applications.

References

1. Chan, A.C., and Carter, P.J. (2010) “Therapeutic antibodies for autoimmunity and inflammation,” National Review of Immunology, 10 (pp. 301–16).

2. Reichert, J.M. (2012) “Marketed therapeutic antibodies compendium,” MAbs, 4 (pp. 413–15).

3. Strickland, I. (2012) “World Preview 2018: Embracing the Patent Cliff,” EvaluatePharma, available at http://info.evaluatepharma.com/WP2018_ELS_LP.html.

4. de Kruif, J., et al. (2010) “Generation of stable cell clones expressing mixtures of human antibodies,” Biotechnology Bioengineering, 106 (pp. 741–50).

5. Rasmussen, S.K., et al. (2012) “Recombinant antibody mixtures: Production strategies and cost considerations,” Archives of Biochemistry and Biophysics, 526 (pp. 139–45).

6. Thompson, N., et al. (2014) “Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native orbitrap mass spectrometry,” MAbs, 6(1) (pp. 1–7).

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.