Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.
An untargeted LC-MS/MS platform was implemented for monitoring variations in CHO cell culture media upon exposure to high temperature short time (HTST) treatment, a commonly used viral clearance upstream strategy. Chemically defined (CD) and hydrolysate-supplemented media formulations were not visibly altered by the treatment. The absence of solute precipitation effects during media treatment and very modest shifts in pH values observed indicated sufficient compatibility of the formulations evaluated with the HTST-processing conditions. Unsupervised chemometric analysis of LC-MS/MS data, however, revealed clear separation of HTST-treated samples from untreated counterparts as observed from analysis of principal components and hierarchical clustering sample grouping. An increased presence of Maillard products in HTST-treated formulations contributed to the observed differences which included organic acids, observed particularly in chemically defined formulations, and furans, pyridines, pyrazines, and pyrrolidines which were determined in hydrolysate-supplemented formulations. The presence of Maillard products in media did not affect cell culture performance with similar growth and viability profiles observed for CHO-K1 and CHO-DP12 cells when cultured using both HTST-treated and untreated media formulations.
A new high performance chelation ion chromatography method was developed for the direct separation and detection of selected transition metals in shellfish (mussel) tissue digest samples. Using two different bonded silica monolithic chelating stationary phases, exhibiting differing selectivities for metals of interest, were applied within the method. The bonded chelating chemistries were N-hydroxyethyliminodiacetic (HEIDA) and N-(2-hydroxyethyl)-N-(2-[phosphonomethyl)amino]acetic (HEPMA) acids. Quantitative determination of Mn(II), Cd(II) and Zn(II) concentrations within shellfish tissue (Mytilus edulis), following microwave assisted acid digestion. Post-column detection was achieved using spectophotometric detection at 510 nm after reaction with 4-(2-pyridylazo) resorcinol. The eluents employed were 0.5 M KNO 3 , pH 2.4, for the HEIDA-bonded stationary phase, and 0.1 M KNO 3 , pH 2.6, for the HEPMA-bonded monolithic column. The HEPMA phase provided improved resolution between Mn(II) and interfering alkaline earth metal cations compared to the HEIDA-bonded phase. Concentrations of metals were determined using standard addition, and Cd(II), Mn(II), and Zn(II) cations were detected at <10 mg g À1 , <50 mg g À1 and <700 mg g À1 , respectively. Sample analysis using sector field inductively coupled plasmamass spectrometry (ICP-MS) was carried out to generate comparative data to that obtained using the chromatographic method.
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