Using LC-MS to Identify Clipping in Self-Assembled Nanoparticles During Vaccine Development

  • Nicole A. Schneck
  • Vera B. Ivleva
  • Erwin Rosales-Zavala
  • Xiangchun Wang
  • Deepika Gollapudi
  • Jonathan W. Cooper
  • Q. Paula LeiEmail author
Application Note


A hemagglutinin stabilized stem nanoparticle (HA-SS-np) that is designed to provide broad protection against influenza is being developed as a potential vaccine. During an early formulation screening study, reducing gel (rCGE) analysis indicated product degradation in a few candidate buffers at the first-week accelerated stability point, whereas no change was shown in the size exclusion chromatography (SEC) measurement. A LC-MS workflow was therefore applied to investigate the integrity of this large HA-SS-np vaccine molecule (≈ 1 MDa). Application of LC-MS was critical to rationalize the conflicting results from the rCGE and SEC assays and led to the discovery that (1) an unexpected sequence clipping in the HA-SS-np subunits occurred, explaining the atypical reducing gel profile, and (2) an undisrupted disulfide bond held the two fragments together, explaining the unchanged SEC profile. This analytical case study led to a formulation buffer redesign, which mitigated the issue.


LC-MS Characterization Vaccine Nanoparticles Clipping Influenza 



The authors would like to acknowledge the Vaccine Research Center teams within the Vaccine Production Program under NIH and thank Cindy Cai, Sashikanth Banappagari, KC Cheng, and Lisa Kueltzo for their helpful discussions, as well as Jessica Bahorich, Heather Lawlor, Kevin Carlton, and Jason Gall for their project and leadership support.

Supplementary material

13361_2019_2318_MOESM1_ESM.docx (431 kb)
ESM 1 (DOCX 430 kb)


  1. 1.
    Vlasak, J., Ionescu, R.: Fragmentation of monoclonal antibodies. mAbs. 3, 253–263 (2011)CrossRefGoogle Scholar
  2. 2.
    Ivleva, V.B., Schneck, N.A., Gollapudi, D., Arnold, F., Cooper, J.W., Lei, Q.P.: Investigation of sequence clipping and structural heterogeneity of an HIV broadly neutralizing antibody by a comprehensive LC-MS analysis. J. Am. Soc. Mass Spectrom. 29, 1515–1523 (2018)CrossRefGoogle Scholar
  3. 3.
    Gao, S.X., Zhang, Y., Stansberry-Perkins, K., Buko, A., Bai, S., Nguyen, V., Brader, M.L.: Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity. Biotechnol. Bioeng. 108, 977–982 (2011)CrossRefGoogle Scholar
  4. 4.
    Dorai, H., Santiago, A., Campbell, M., Tang, M.P., Lewis, M.J., Wang, Y., Lu, Q.-Z., Wu, S.-L., Hancock, W.: Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins. Biotechnol. Prog. 27, 220–231 (2011)CrossRefGoogle Scholar
  5. 5.
    Nachbagauer, R., Krammer, F.: Universal influenza virus vaccines and therapeutic antibodies. Clin. Microbiol. Infect. 23, 222–228 (2017)CrossRefGoogle Scholar
  6. 6.
    Kanekiyo, M., Wei, C.J., Yassine, H.M., McTamney, P.M., Boyington, J.C., Whittle, J.R., Rao, S.S., Kong, W.P., Wang, L., Nabel, G.J.: Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature. 499, 102–106 (2013)CrossRefGoogle Scholar
  7. 7.
    Yassine, H.M., Boyington, J.C., McTamney, P.M., Wei, C.J., Kanekiyo, M., Kong, W.P., Gallagher, J.R., Wang, L., Zhang, Y., Joyce, M.G., Lingwood, D., Moin, S.M., Andersen, H., Okuno, Y., Rao, S.S., Harris, A.K., Kwong, P.D., Mascola, J.R., Nabel, G.J., Graham, B.S.: Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nat. Med. 21, 1065–1070 (2015)CrossRefGoogle Scholar
  8. 8.
    Impagliazzo, A., Milder, F., Kuipers, H., Wagner, M.V., Zhu, X., Hoffman, R.M.B., van Meersbergen, R., Huizingh, J., Wanningen, P., Verspuij, J., de Man, M., Ding, Z., Apetri, A., Kükrer, B., Sneekes-Vriese, E., Tomkiewicz, D., Laursen, N.S., Lee, P.S., Zakrzewska, A., Dekking, L., Tolboom, J., Tettero, L., van Meerten, S., Yu, W., Koudstaal, W., Goudsmit, J., Ward, A.B., Meijberg, W., Wilson, I.A., Radošević, K.: A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science. 349, 1301–1306 (2015)CrossRefGoogle Scholar

Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious DiseasesNational Institutes of HealthGaithersburgUSA

Personalised recommendations