2-D NMR Developments Improve Monoclonal Antibody Characterization

Ten years ago, industry commentators noted the increasing importance of biologics, and monoclonal antibodies (mAbs), in particular. At the time, just four biologic license applications were approved by the FDA; however, big pharma was buying in, with landmark deals such as the 2007 acquisition of Therapeutic Human Polyclonals (THP) by Roche. By 2014/15, the “new wave” had arrived, and of the 51 new drugs approved by the FDA in 2015, 20 were biologics.1,2

Today there is much interest in biosimilars (generic alternatives to the innovative early biotherapeutics) and biobetters. Early in 2014, the European Medicines Agency (EMA) approved 18 biosimilar drugs; in March 2015, the FDA followed, with its first approval for a biosimilar product in the United States.

Innovative products are still breaking new ground—of the 41 new drugs approved by the FDA in 2014, more than 40% were designated “first in class.” In addition, major companies continue to innovate and develop new products rather than make their own biosimilars to supplement any loss of market share. With the global biopharmaceuticals market forecast to reach a total of $497.9 billion by 2020,3 most large pharmaceutical companies now devote around 40% or more of their R&D budget to biopharmaceuticals.4

Data-driven insight and approvals

Against this commercial and regulatory background, improvements in the content and quality of data on biologics as they move through R&D can deliver significant benefits. Monoclonal antibodies are complex biological macromolecules, and accurate characterization early in the development process is crucial. The analytical methods used must provide robust and reliable results. Techniques employed in biopharmaceutical research have typically Characterizationincluded a range of hyphenated liquid chromatography approaches. However, nuclear magnetic resonance (NMR) spectroscopy offers the most direct method of characterizing the structure of proteins in solution.5 Used alone, 1-D NMR can be challenging, primarily due to the large size of protein molecules and the requirements of isotope labeling.6,7 With a size of around 150 kDa, mAbs produce data that can be limited in information content. Significantly, the nature of the data results in line broadening, which reduces sensitivity and causes overlapping resonances that are difficult to interpret.

Two-dimensional NMR approaches are beginning to augment 1-D NMR and allow much more thorough mAb characterization. In practice, both methods will likely be used together. The 1-D NMR method has been widely applied and offers easy distinction between “good” and “bad” materials, which is especially useful as a quick assessment of various formulation conditions. Despite being more complex, the information gained from 2-D NMR methods adds significant value—showing, for example, the exact position of any alteration to an mAb.

Structure and function

Monoclonal antibody-based therapeutics have already proven themselves as exceptionally valuable treatment options in the fields of gastroenterology, rheumatology, oncology, hematology and transplantation, and newer mAb-based therapeutics are currently in clinical trials. The development of mAb-based therapeutics is a lengthy and complex process with multiple challenges, such as size and charge variation, numerous post-translational modifications, long half-lives and the potential for inducing immunogenicity.8 Additionally, mAbs have several possible functional domains within a single molecule, and each individual molecule can present a unique analysis profile.

The structure of an mAb is very closely related to its function. Correct folding of mAbs is critical for drug efficacy; incorrect folding impacts safety because it causes unwanted immune, or off-target, responses. Successful development of an mAb therefore necessitates accurate characterization of the 3-D structure.9

2-D NMR applications

In a recent study by Arbogast et al.,9 methyl residues were used to “fingerprint” the structure of a candidate mAb (RM8670). The researchers investigated the methyl resonances of RM8670 because it allowed them to take advantage of the greater natural abundance of the 13C isotope (1.1%) over 15N (0.37%). This approach contrasts with the more conventional approach of mapping the nitrogen-rich amide backbone of the protein.5 Methyl groups were selected as an alternative because they are well distributed throughout proteins and are good reporters of 3-D structure.

Following sample preparation of Fc and Fab fragments, experiments were performed with- out isotopic labeling using either a 600- or 900-MHz Avance III spectrometer  (Bruker, Billerica, Mass.) equipped with triple-resonance CryoProbes (Bruker). Performing 2-D NMR on intact samples of the mAb using gradient-selected, sensitivity-enhanced heteronuclear single quantum coherence (gsHSQC) provided spectra of high enough quality to assign and analyze the spectrum peaks (Figure 1a). This experiment took 12 hours to run and it was repeated on mAb fragments (Figure 1b) that were then cleaved into constituent Fc and Fab domains to reduce the time to 4.5 hours (Figures 1c and d).

 Figure 1 – A) 2-D 13C NMR fingerprints of full-length mAb using standard gsHSQC at 900 MHz, obtained over 12 hours. B) 2-D 13C NMR fingerprints of a mixture of Fab/Fc fragments from immobilized papain cleavage using standard gsHSQC at 900 MHz, recorded in around 4.5 hours. C) 2-D 13C NMR fingerprints of Fc fragment, using standard gsHSQC at 900 MHz, recorded in around 4.5 hours. D) 2-D 13C NMR fingerprints of Fab fragment, using standard gsHSQC at 900 MHz, recorded in around 4.5 hours. Figures 1A–D reprinted with permission from Arbogast et al., 2015. Copyright 2015 American Chemical Society.

The researchers were able to show that the spectra produced from these fragments closely resembled those produced for the intact mAb, suggesting that cleaved protein fragments can act as a good proxy for the intact protein in NMR experiments. Experiment time could be further reduced by combining the rapid acquisition techniques—which helped to increase the speed of natural isotopic abundance spectroscopy—with nonuniform sampling. Of six combinations tested, the fastest time was 34 minutes using the rapid SOFAST (band-selective optimized-flip-angle short-transient)-HMQC acquisition technique with 50% nonuniform sampling.

The authors9 suggest these results demonstrate that 2-D NMR fingerprinting using 13C isotopes is a feasible option for structurally assessing mAbs in situations in which isotope labeling is not possible. Additionally, NMR provides readouts of primary, secondary, tertiary and higher-order protein structure at atomic resolution. Two-dimensional NMR could therefore find use in the biotherapeutics industry for protein structure assessment.

Conclusion

Monoclonal antibody-based therapeutics continues to grow in importance for the treatment of chronic and life-threatening diseases. Because they are complex biological macromolecules and their structure is closely related to function, it is vital to successfully characterize the 3-D structure of mAbs during the development process. NMR techniques have previously been seen as challenging, but recent advances in NMR spectroscopy have pushed its boundaries, and it can now be more readily adopted as an option for mAb characterization.

References

  1. Owens, J. 2006 Drug approvals: finding the niche. Nat. Rev. Drug Discov. 2007, 6, 99–101.
  2. Munos, B. http://www.forbes.com/sites/bernardmunos/2016/ 01/04/2015-new-drug-approvals-hit-66-year-high/3/#244edcdf47b3 (last accessed July 8, 2016).
  3. King, M. http://www.researchandmarkets.com/research/mdkxf2/biopharmaceuticals (last accessed Apr 13, 2016).
  4. Langer, E.S. The future of biopharma. BioPharm. Int. 2013, 26(1), 22.
  5. Aubin, Y.; Freedberg, D.I. et al. One and two dimensional NMR techniques for biopharmaceuticals. In Biophysical Characterisation of Proteins in Developing Biopharmaceuticals; Houde, D.J., Berkowitz, S.A., Eds; Elsevier, pp 341–83.
  6. Poppe, L.; Jordan, J.B. et al. Profiling formulated monoclonal antibodies by 1H NMR spectroscopy. Anal. Chem. 2013, 85(2), 9623–9.
  7. Poppe, L.; Jordan, J.B. On the analytical superiority of 1D NMR for fingerprinting the higher order structure of protein therapeutics compared to multidimensional NMR methods. Anal. Chem. 2015, 87, 5539–45.
  8. World Health Organization. http://www.who.int/biologicals/mAb_1st_draft_KG_IK_1_March_2016_clean.pdf
  9. Arbogast, L.W.; Brinson, R.G. et al. Mapping monoclonal antibody structure by 2D 13C NMR at natural abundance. Anal. Chem. 2015, 87, 3556–61.

Kimberly L. Colson, Ph.D., is business development manager, Bruker Biospin, 15 Fortune Dr., Billerica, Mass. 01821-3991, U.S.A.; tel.: 978-667-9580; e-mail: [email protected]www.bruker.com

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