Dataset Information

Quality Control of Therapeutic Peptides by 1H NMR HiFSA Sequencing.

ABSTRACT: Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and for pharmaceutical product safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance. This study builds on quantum mechanical 1H iterative Full Spin Analysis (HiFSA) to establish NMR peptide sequencing methodology that overcomes the intrinsic limitations of principal compendial methods in identifying small structural changes or minor impurities that affect effectiveness and safety. HiFSA sequencing yields definitive identity and purity information concurrently, allowing for API quality assurance and control (QA/QC). Achieving full peptide analysis via NMR building blocks, the process lends itself to both research and commercial applications as 1D 1H NMR (HNMR) is the most sensitive and basic NMR experiment. The generated HiFSA profiles are independent of instrument or software tools and work at any magnetic field strength. Pairing with absolute or 100% qHNMR enables quantification of mixtures and/or determination of peptide conformer populations. Demonstration of the methodology uses single amino acids (AAs) and peptides of increasing size, including the octapeptide, angiotensin II, and the nonapeptide, oxytocin. The feasibility of HiFSA coupled with automated NMR and qHNMR for use in QC/QA efforts is established through case-based examples and recommended procedures.

SUBMITTER: Choules MP

PROVIDER: S-EPMC6452441 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Quality Control of Therapeutic Peptides by <sup>1</sup>H NMR HiFSA Sequencing.

Choules Mary P MP Bisson Jonathan J Gao Wei W Lankin David C DC McAlpine James B JB Niemitz Matthias M Jaki Birgit U BU Franzblau Scott G SG Pauli Guido F GF

The Journal of organic chemistry 20190222 6

Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and for pharmaceutical product safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance. This study builds on quantum mechanical <sup>1</sup>H iterative Full Spin Analysis (HiFSA) to establish NMR peptide sequencing methodology that overcomes the intrinsic limitations of principal compendial methods in identifying small structural changes ...[more]

PMID: 30793905

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Project description: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.

Dataset Information

Quality Control of Therapeutic Peptides by 1H NMR HiFSA Sequencing.

Publications

Quality Control of Therapeutic Peptides by <sup>1</sup>H NMR HiFSA Sequencing.

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