title: Elemental screening of cell culture media using microwave plasma atomic emission spectroscopy
Creators: name: Grant, Shane Richard
Publisher: name: University of Galway
Year: 2024
Subjects: subject: Biological and Chemical Sciences
subject: Chemistry
subject: Microwave Plasma Atomic Emission Spectroscopy
subject: Cell culture media analysis
subject: Lot to lot screening
subject: Biopharmaceutical raw materials
subject: Process Analytical Technology

description: Biopharmaceutical manufacturing is critical for a range of therapeutic and diagnostic applications and in recent years has experienced major growth aimed at enhancing process understanding and optimizing productivity. To address the growing demand for high quality biopharmaceuticals, the industry heavily relies on optimizing cell culture media formulations. Chemically defined media have become increasingly popular due to their ability to minimize process variability arising from undefined components. However, the persistence of media variability is an ongoing concern indicating the inadequacy of existing methods for characterization and control. Inorganic salts are the largest component by weight in media, containing elements ranging from high concentrations (1 to &gt;1000 mg/L) like Na, K, Ca, and Mg, down to low concentrations (1 mg/L) like Fe, Cu, Mn, Ni, Mn, and Zn. The effects of low abundance transition element variation are well known, which can impact the performance of bioprocesses and affect the quality and yield of biopharmaceutical products. High abundance elements also serve important roles in bioprocess regulation, although the effects of their variation on bioprocesses are under-reported. Thus, elemental screening is essential in both bioprocess development and quality control. Microwave Plasma Atomic Emission Spectroscopy (MP-AES) is introduced as a cost-effective and efficient elemental analysis method. Although not yet widely adopted in industry, MP-AES offers a promising alternative or complement to the established inductively coupled plasma methods. The goal of this work was to develop a method that ensures high reproducibility while simplifying sample preparation and analysis procedures. Special attention was given to mitigating the effects of easily ionized elements which have the potential to influence analyte emission properties. Systematic evaluations were carried out on sample concentration, instrumental settings, and calibration methods, including external calibration, standard additions method, internal standardization, and intrinsic plasma molecular species. Cr applied as an internal standard effectively addressed observed interferences which were likely related to freeze/thaw cycles. The results demonstrated that a 2% w/w nitric acid dilution using a high dilution (chemically defined media concentration of 1 g/L), provided acceptable levels of reproducibility (intra-batch variation &lt; inter-batch variation for independent experiments) and trueness (70 &lt; x &lt; 130% for spike recovery) for the determination of various elements, achieving analysis times of &lt; 15 minutes per sample for ten analytical and six internal standard emission lines. Sensitivity enhancement can be achieved for low abundance elements through dilution factor control (demonstrated for chemically defined media concentrations up to 5 g/L). This work suggests that further refinements in MP-AES instrumentation, such as the capability for simultaneous measurement of multiple elements, could enhance its performance in biopharmaceutical manufacturing. By adopting MP-AES, biopharmaceutical manufacturers can potentially reduce the substantial costs associated with inductively coupled plasma-based methods for the routine elemental screening of media. Overall, this thesis work highlights the significance of addressing the challenge of process variability within biopharmaceutical manufacturing, primarily arising from lot-to-lot variations in raw materials. This emphasises the need to monitor lot-to-lot elemental variability comprehensively and explores the potential of MP-AES as a practical and cost-effective solution. Through early detection of elemental variability in media, this approach enables media users to proactively improve process consistency and productivity associated with cell culture processes.