Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

Abstract

BACKGROUND Chemically defined media (CDM) are used routinely in industrial settings for the production of biopharmaceuticals from mammalian cells, so attention has shifted to understanding their chemical behavior and impact on process robustness. In this context, one particular problem is the risk of cross reactivity and instability in complex mixtures of chemicals at high concentrations. RESULTS In order to characterize potentially unstable medium compounds in feed media, we developed and validated a novel liquid chromatography tandem mass spectrometry (LC‐MS/MS) method to simultaneously quantify the majority of medium compounds. We used this application advantage to provide the following insights into molecular processes occurring in CDM prepared at different temperatures and during storage: Concentration profiles of critical compounds in CDM are independent of preparation temperature and storage material but show significant alterations over time. In particular l‐cysteine, l‐proline, vitamin B6, thiamine and cyanocobalamin were found to be rather unstable. Using mixed mode chromatography allows expansion of the method to cover additional analytes of interest, as demonstrated by inclusion of seven additional CDM reaction products. CONCLUSION The successful development of a new LC‐MS/MS method allowed us to show the impact of storage duration on the stability of CDM compounds. Furthermore, the results suggest that essential parameters for medium preparation (e.g. temperature) and storage upscaling (e.g. vessel material) are not impacting upon the chemical composition of CDM. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Combined NMR and Computational Study of Cysteine Oxidation during Nucleation of Metallic Clusters in Biological Systems

“…or in the mixture with NaBH 4 . Contrary to earlier reports, 107 we observed earlier (Figure S5 in the SI) and confirmed again that spontaneous oxidation of CYS (10 mM) in water under neutral pH condition did not occur during 7 days (Figure 5, black spectrum), while basic pH conditions (addition of NaOH) led to very slow oxidation process (Figure 5, blue spectrum), at least on the time scale (120 min) that corresponds to the standard preparation of AgNPs or AuNPs. The presence of AgNO 3 (10 mM) at neutral pH catalyzed oxidation of CYS (10 mM), but the process was slower than in basic aqueous solution as only a very small change was observed in 1 H NMR spectrum for 24 h (see Figure S6 in the SI).…”

Application of metabolic modeling for targeted optimization of high seeding density processes

“…LC‐MS/MS was applied to determine vitamin concentrations and additional metabolites such as 2‐hydroxybutyrate, indole‐3‐lactic acid, indole‐3‐carboxylic acid, and 2‐deoxycitidine. LC‐MS/MS samples were measured using an InfinityLab Poroshell Hydrophilic interaction chromatography (HILIC)‐Z column (Agilent Technologies) as described previously (Krattenmacher et al, 2018). Tandem mass spectrometry was performed on a QTRAP® 6500 + triple quadrupole mass spectrometer (Sciex) equipped with an ESI source and coupled to an Agilent 1260 series binary HPLC system (Agilent Technologies).…”

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

“…The impact of preparation parameters on media quality was investigated in a comprehensive DoE approach. Due to the fact that not all media batches could be prepared at the same time and since media storage is known to have an effect on media characteristics , the factor storage time was included as a covariate in the DoE.…”