Skip to main content
SpringerLink
Log in

Selective suppression of excipient signals in 2D 1H–13C methyl spectra of biopharmaceutical products

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

While the use of 1H–13C methyl correlated NMR spectroscopy at natural isotopic abundance has been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies, spectral interference from aliphatic excipients remains a significant obstacle to its widespread application. These signals can cause large baseline artifacts, obscure protein resonances, and cause dynamic range suppression of weak peaks in non-uniform sampling applications, thus hampering both traditional peak-based spectral analyses as well as emerging chemometric methods of analysis. Here we detail modifications to the 2D 1H–13C gradient-selected HSQC experiment that make use of selective pulsing techniques for targeted removal of interfering excipient signals in spectra of the NISTmAb prepared in several different formulations. This approach is demonstrated to selectively reduce interfering excipient signals while still yielding 2D spectra with only modest losses in protein signal. Furthermore, it is shown that spectral modeling based on the SMILE algorithm can be used to simulate and subtract any residual excipient signals and their attendant artifacts from the resulting 2D NMR spectra.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

CP:

Contact pulses

HOS:

Higher order structure

HSQC:

Heteronuclear single quantum coherence

NUS:

Non-uniform sampling

PCA:

Principal component analysis

PFG-STE:

Pulsed-field gradient stimulated echo

RF:

Radio frequency

RMS:

Root mean squared

SIERRA:

Selective excipient reduction and removal

SMILE:

Sparse multidimensional iterative lineshape enhancement

SOFAST:

Selective optimized flip angle short transient

S/N:

Signal to noise

References

  • Arbogast L, Majumdar A, Tolman JR (2013) Unraveling long range residual dipolar coupling networks in strongly aligned proteins. J Magn Reson 235:26–31

    Article  ADS  Google Scholar 

  • Arbogast LW, Brinson RG, Marino JP (2015) Mapping monoclonal antibody structure by 2D 13 C NMR at natural abundance. Anal Chem 87:3556–3561

    Article  Google Scholar 

  • Arbogast LW, Brinson RG, Marino JP (2016a) Application of natural isotopic abundance 1H–13C- and 1H–15N-correlated two-dimensional NMR for evaluation of the structure of protein therapeutics. Methods Enzymol 566:3–34

    Article  Google Scholar 

  • Arbogast LW, Brinson RG, Formolo T, Hoopes JT, Marino JP (2016b) 2D 1HN, 15N correlated NMR methods at natural abundance for obtaining structural maps and statistical comparability of monoclonal antibodies. Pharm Res 33:462–475

    Article  Google Scholar 

  • Arbogast LW, Delaglio F, Schiel JE, Marino JP (2017) Multivariate analysis of two-dimensional 1H, 13C Methyl NMR spectra of monoclonal antibody therapeutics to facilitate assessment of higher order structure. Anal Chem 89:11839–11845

    Article  Google Scholar 

  • Aubin Y, Gingras G, Sauvé S (2008) Assessment of the three-dimensional structure of recombinant protein therapeutics by NMR fingerprinting: demonstration on recombinant human granulocyte macrophage-colony stimulation factor. Anal Chem 80:2623–2627

    Article  Google Scholar 

  • Aubin Y, Jones C, Freedberg DI (2010) Using NMR spectroscopy to obtain the higher order structure of biopharmaceutical products: simple methods can characterize polysaccharide vaccines and recombinant cytokines at high resolution. Biopharm Int 23:28–34

    Google Scholar 

  • Braun AC, Ilko D, Merget B, Gieseler H, Germershaus O, Holzgrabe U, Meinel L (2015) Predicting critical micelle concentration and micelle molecular weight of polysorbate 80 using compendial methods. Eur J Pharm Biopharm 94:559–568

    Article  Google Scholar 

  • Brinson RG, Ghasriani H, Hodgson DJ, Adams KM, McEwen I, Freedberg DI, Chen K, Keire DA, Aubin Y, Marino JP (2017) Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim. J Pharm Biomed Anal 141:229–233

    Article  Google Scholar 

  • Chen K, Park J, Li F, Patil SM, Keire DA (2018) Chemometric methods to quantify 1D and 2D NMR spectral differences among similar protein therapeutics. AAPS PharmSciTech 19:1011–1019

    Article  Google Scholar 

  • Chiarparin E, Pelupessy P, Bodenhausen G (1998) Selective cross-polarization in solution state NMR. Mol Phys 95:759–767

    Article  ADS  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Emsley L, Bodenhausen G (1990) Gaussian pulse cascades: new analytical functions for rectangular selective inversion and in-phase excitation in NMR. Chem Phys Lett 165:469–476

    Article  ADS  Google Scholar 

  • Ferrage F, Eykyn TR, Bodenhausen G (2000) Coherence transfer by single-transition cross-polarization: quantitation of cross-correlation effects in nuclear magnetic resonance. J Chem Phys 113:1081–1087

    Article  ADS  Google Scholar 

  • Ferrage F, Eykyn TR, Bodenhausen G (2004) Frequency-switched single-transition cross-polarization: a tool for selective experiments in biomolecular NMR. ChemPhysChem 5:76–84

    Article  Google Scholar 

  • Franks J, Glushka JN, Jones MT, Live DH, Zou Q, Prestegard JH (2016) Spin diffusion editing for structural fingerprints of therapeutic antibodies. Anal Chem 88:1320–1327

    Article  Google Scholar 

  • Ghasriani H, Hodgson DJ, Brinson RG, McEwen I, Buhse LF, Kozlowski S, Marino JP, Aubin Y, Keire DA (2016) Precision and robustness of 2D-NMR for structure assessment of filgrastim biosimilars. Nat Biotechnol 34:139–141

    Article  Google Scholar 

  • Japelj B, Ilc G, Marušič J, Senčar J, Kuzman D, Plavec J (2016) Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies. Sci Rep 6:32201

    Article  ADS  Google Scholar 

  • Pelupessy P, Chiarparin E (2000) Hartmann-Hahn polarization transfer in liquids: an ideal tool for selective experiments. Concepts Magn Reson 12:103–124

    Article  Google Scholar 

  • Poppe L, Jordan JB, Lawson K, Jerums M, Apostol I, Schnier PD (2013) Profiling formulated monoclonal antibodies by 1 H NMR spectroscopy. Anal Chem 85:9623–9629

    Article  Google Scholar 

  • Rajagopalan S, Chow C, Raghunathan V, Fry CG, Cavagnero S (2004) NMR spectroscopic filtration of polypeptides and proteins in complex mixtures. J Biomol NMR 29:505–516

    Article  Google Scholar 

  • Schleucher J, Schwendinger M, Sattler M, Schmidt P, Schedletzky O, Glaser SJ, Sørensen OW, Griesinger C (1994) A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. J Biomol NMR 4:301–306

    Article  Google Scholar 

  • Singh SM, Bandi S, Jones DNM, Mallela KMG (2017) Effect of polysorbate 20 and polysorbate 80 on the higher-order structure of a monoclonal antibody and Its Fab and Fc fragments probed using 2D nuclear magnetic resonance spectroscopy. J Pharm Sci 106:3486–3498

    Article  Google Scholar 

  • Wishart DS (2013) Characterization of biopharmaceuticals by NMR spectroscopy. TrAC Trends Anal Chem 48:96–111

    Article  Google Scholar 

  • Ying J, Delaglio F, Torchia DA, Bax A (2017) Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data. J Biomol NMR 68:101–118

    Article  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the help and support of Jinfa Ying for assistance with modifying the SMILE algorithm. We further acknowledge the support of the NIST Biomanufacturing Initiative and W.M. Keck for support of the biomolecular NMR instrumentation.

Author information

Authors and Affiliations

  1. Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology, 9600 Gudelsky Dr., Rockville, MD, 20850, USA

    Luke W. Arbogast, Frank Delaglio & John P. Marino

  2. Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD, 21218, USA

    Joel R. Tolman

Authors
  1. Luke W. Arbogast
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank Delaglio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joel R. Tolman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John P. Marino
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luke W. Arbogast.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1772 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Arbogast, L.W., Delaglio, F., Tolman, J.R. et al. Selective suppression of excipient signals in 2D 1H–13C methyl spectra of biopharmaceutical products. J Biomol NMR 72, 149–161 (2018). https://doi.org/10.1007/s10858-018-0214-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-018-0214-1

Keywords

Search

Navigation