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

Abstract

IntroductionEx 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.AimsWe 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.MethodsWe 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.ResultsMedium 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.DiscussionMALDI-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.

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“…We demonstrated that to obtain stable SRGF batches, that equally stimulate MSC proliferation rate, at least 16 different SRGF products derived from single donors must be mixed together[ 69 ]: To achieve this aim, we adopted a predictive mathematical approach, followed by “wet biology” validation. In order to identify, in compliance with GMP requirements, a reliable and comprehensive quality control assay for SRGF, we manufactured from platelet concentrates several medium additive types differently promoting ASC growth rate[ 70 ]. Interestingly, while integrative analysis of growth factor concentration changes was shown to be insufficiently sensitive, 1 H-NMR and MALDI-TOF MS could clearly identify differences between product isoforms.…”

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“…75 1 H NMR was recently used by Agostini et al to characterize and predict the quality of platelet-derived media supplements for industrial cell manufacturing, leveraging the ease of sample preparation and throughput of media analysis by NMR. 76 Continuous flow NMR techniques for monitoring mammalian cell culture systems have existed for decades 35…”