A targeted liquid chromatography mass spectrometry method for routine monitoring of cell culture media components for bioprocess development.

The analysis of cell culture media (CCM) components is critical for understanding cell growth kinetics and overall product quality during biomanufacturing. Given the diverse physical and chemical nature of CCM compounds present at a wide range of concentrations, there is an increasing demand for single-platform analytical assays with exceptional specificity and sensitivity. This study presents a targeted LC-MS/MS method for the identification and quantitation of 110 CCM analytes is presented, where target metabolites are monitored over an 20-min gradient. The analyte panel constitutes amino acids, vitamins, organic acids, nucleic acids, carbohydrates, and lipids. The method employs isotopically labeled standards to enable specific and accurate relative quantitation of CCM compounds based on physicochemical properties and retention time. Quantitation is performed on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. The method demonstrates strong linearity with an R2 of ≥0.99 with three orders of linear dynamic range and inter-day and intra-day precision with a%CV of <10% for spiked-in quality control samples. We also present three case studies to demonstrate method applicability in the bioprocessing space for developing vaccines and biologics.

Low-molecular-weight impurity in Poloxamer 188 responsible for atypical cell culture performance for mAb production.

During a recent manufacturing campaign for a monoclonal antibody using a fed-batch process, poor cell culture performance was observed across two manufacturing sites with similar scales and equipment. Root cause analysis indicated that the poor cell culture performance was linked to the production basal media. Genealogy of the precursor raw materials used in the media revealed that a particular lot of Poloxamer 188 (P188) was the common link to the poor-performing media lots. P188 serves a critical role in protecting cells against shear in cell culture bioprocesses. However, the small-scale studies suggested that the poor cell culture performance was cytostatic in nature rather than being caused due to lack of shear protection. Several P188 lots were tested analytically using SEC-MS and RP-LC-MS methods and a unique low molecular weight species was identified in the suspect lot of poloxamer. The impurity was identified to be polypropylene oxide (PPO), a reaction intermediate in P188 synthesis. Spiking studies with PPO further confirmed its cytostatic nature. This case study highlights yet another scenario where lot-to-lot variability continues to impact bioprocesses and re-emphasizes the need for robust analytical and cell-culture raw material screening methods.