Analytical and Bioanalytical Chemistry

, Volume 410, Issue 13, pp 3197–3207 | Cite as

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control

  • Simone Albrecht
  • Christian Kaisermayer
  • David Reinhart
  • Monica Ambrose
  • Renate Kunert
  • Anna Lindeberg
  • Jonathan BonesEmail author
Research Paper


The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MSE was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 106 dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control.

Graphical abstract

Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death


Biomanufacturing Cell death CHO Proteomics LC-MRM-MS Targeted proteomics 



The authors acknowledge support from Enterprise Ireland under the Innovation Partnership funding program, with grant reference IP/2014/0309, co-funded by the European Union through the European Regional Development Fund (ERDF) 2014-2020 program.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1029_MOESM1_ESM.pdf (4 kb)
ESM 1 (PDF 4 kb)
216_2018_1029_MOESM2_ESM.xlsx (605 kb)
ESM 2 (XLSX 605 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Simone Albrecht
    • 1
  • Christian Kaisermayer
    • 2
  • David Reinhart
    • 3
  • Monica Ambrose
    • 2
  • Renate Kunert
    • 3
  • Anna Lindeberg
    • 2
  • Jonathan Bones
    • 1
    • 4
    Email author
  1. 1.NIBRT Characterisation and Comparability LaboratoryNational Institute for Bioprocessing Research and TrainingCo. DublinIreland
  2. 2.BioMarin International LimitedCo. CorkIreland
  3. 3.Vienna Institute of Biotechnology, Department of BiotechnologyUniversity of Natural Resources and Life Sciences, Vienna (BOKU)ViennaAustria
  4. 4.School of Chemical and Bioprocess EngineeringUniversity College DublinDublin 4Ireland

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