1H-NMR and MALDI-TOF MS as metabolomic quality control tests to classify platelet derived medium additives for GMP compliant cell expansion procedures

PLoS One. 2018 Sep 6;13(9):e0203048. doi: 10.1371/journal.pone.0203048. eCollection 2018.

Abstract

Introduction: Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples.

Aims: We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation.

Methods: We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS.

Results: Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro.

Discussion: MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets
  • Calcium Chloride
  • Cell Proliferation
  • Cells, Cultured
  • Culture Media* / chemistry
  • Humans
  • Manufacturing Industry
  • Metabolomics* / methods
  • Proton Magnetic Resonance Spectroscopy*
  • Quality Control*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*

Substances

  • Culture Media
  • Calcium Chloride

Grants and funding

The present study was funded by Progetto di Ricerca Finalizzata (n°: J32I14001830001; Recipient: M. Mazzucato). The funder provided support for research materials and in the form of salaries for authors (F.A. and M.B.), but did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. GEMFORLAB SrL and BRACCO Imaging Italia SrL were not funders of this study. A free, scientific partnership agreement between CRO Aviano National Cancer Institute and Bioindustry Park Silvano Fumero S.p.A. - Colloretto Giacosa (Turin, Italy), was officially ruled by the General Director of the CRO Aviano National Cover Letter Cancer Institute on 26th of May, 2015. The Bioindustry Park Silvano Fumero encloses GEMFORLAB SrL and BRACCO Imaging Italia SrL. The partnership agreement was principally aimed to accomplish the present pilot study without any financial reward and reciprocally sharing personnel competences and obtained results. Collection and analysis of metabolomic data of the present study were performed by personnel of the GEMFORLAB SrL and BRACCO Imaging Italia SrL. Specimens, collected at CRO Aviano National Cancer Institute, were provided to metabolomic laboratories as blind samples: this was done to completely avoid potential biases on results. Growth factor concentration measurements and analysis as well as cell culture experimental procedures and data analysis were performed by personnel of the CRO Aviano National Cancer Institute.