Tuning metabolic efficiency for increased product yield in high titer fed-batch Chinese hamster ovary cell culture.

High demand in manufactured biologics drives the continued need for increased productivity. In this study elevated lactate metabolization resulted in improved metabolic efficiency and cellular productivity for a readily intensified high titer fed-batch process. Scheduled base or lactate feeds during the stationary growth phase led to increased titers (+9% and +8% respectively) without impacting the overall growth performance. The higher lactate consumption induced by either feed strategy substituted for glutamate catabolism and consequently reduced ammonia build-up. Direct correlation between increased titers and reduced ammonia levels was shown. Product quality attributes were impacted by both feeding strategies but could be matched with the control process by shortening the cell culture duration while maintaining titer constant.

Characterization of Polysorbate 80 by Liquid Chromatography-Mass Spectrometry to Understand Its Susceptibility to Degradation and Its Oxidative Degradation Pathway.

A liquid chromatography-mass spectrometry (LC-MS) method was developed to provide a fingerprint of polysorbate 80 (PS80) subspecies that enables identification of PS80 degradation pathway. The developed method demonstrates unique monoester peak profile of PS80 from different vendors, attributed by differences in relative abundance of the fatty acid monoesters. The LC-MS method was also applied to examine the susceptibility of PS80, at different grades, to auto-oxidation and hydrolysis. PS80 oxidative degradation induced by iron or occurred in open bottle without nitrogen overlay was found to follow the same pathway, but at a much faster rate in the former scenario. The oxidation preferentially occurs at the double bond of fatty acid chains, thus providing explanation on the faster degradation observed in PS80 at Chinese Pharmacopia (ChP) grade than at multi-compendial (MC) grade. In contrast, the difference in susceptibility of MC and ChP grade PS80 against esterase-induced hydrolysis in placebo was not pronounced. The method was also able to provide a fingerprint to identify both PS80 hydrolysis and oxidation in mAb drug product stability samples, but it required a solid phase extraction step to remove protein prior to the analysis.

Colorimetric and Physico-Chemical Property Relationships of Chemically Defined Media Powders Used in the Production of Biotherapeutics.

Growth of mammalian cells in the production of biotherapeutics often require the benefits of chemically defined media (CDM). Storage, handling and stability advantages of CDM powders govern the preponderance of their use across the industry. Physico-chemical property lot-to-lot variation of these multicomponent powders, however, continues to be a challenge. Process imposed degradation of amino acids and vitamins, for example, can influence cell density, specific titer, and the quality profile of the molecule expressed due to the lack of process understanding and suitable mitigation controls. Such degradation can materialize in either their manufacture or in downstream media dissolution steps. Colorimetry, in lieu of visual appearance, can be an effective surveillance method for the direct assessment of CDM quality as color change is indicative of chemical-physical variations. This work describes a series of studies aimed to establish relationships between quantitative color change and physico-chemical attribute variation of glucose-free and glucose-based powders. The results illustrate color change is indicative of amino acid glycation, vitamin degradation and particle size shifts. These relationships enable a colorimetric control strategy for the sensitive and rapid detection of relevant CDM variation to drive additional targeted assessments to improve the productivity and robustness of cell culture processes.

Enabling Robust and Rapid Raw Material Identification and Release by Handheld Raman Spectroscopy.

A fast, reproducible, non-destructive method to confirm raw material identification in real-time upon material receipt within a warehouse environment is desired. Current practices in pharmaceutical manufacturing often employ compendia methods for raw material identification tests, which require sample preparation prior to time-consuming chemical analysis and often employ subjective spectral comparisons. We have developed, qualified, and validated a rapid objective identity method ("Rapid ID") by Raman spectroscopy using the Bruker BRAVO handheld Raman spectrometer for 46 common raw materials used in upstream and downstream biopharmaceutical cell culture-based processes. Materials in the Raman identification library include amino acids and other solid neat organic chemicals, liquid organics, polyatomic salts, polymers, emulsifiers, peptides, aqueous solutions, and buffers. Selection of reference spectra and hit quality index limit(s) was based upon a comprehensive spectral survey across multiple suppliers and lots to account for normal cause spectral variation. Method repeatability and reproducibility, selectivity, and robustness against various operational and environmental factors (e.g., instrumental variance, material packaging, and thermal effects) were evaluated. Benefits of a handheld Raman Rapid ID approach include significant reduction of the time for raw material quality release from weeks to minutes, enhanced objectivity, and robust data integrity via autonomous electronic reporting. In addition, routine collection of rich spectroscopic data on raw materials can be leveraged to support further continuous improvement initiatives, including routine monitoring of method performance, continuous improvement of the library, proactive detection of shifts in raw material properties, and provision of data for investigations focused on raw materials. Rapid ID methods are consistent with the move toward the principles of Pharma 4.0-high automated processes with continuous process verification and a holistic control strategy.LAY ABSTRACT: A fast, reproducible, non-destructive method is desired to confirm raw material identification in real time upon receipt within a warehouse environment. We have developed, qualified and validated a rapid objective identity method ("Rapid ID") by Raman spectroscopy using the Bruker BRAVO handheld Raman spectrometer for 46 common raw materials used in upstream and downstream biopharmaceutical cell culture-based processes. Benefits of a handheld Raman Rapid ID approach include significant time reduction of raw material quality release from weeks to minutes, enhanced objectivity, and robust data integrity via autonomous electronic reporting. Rapid ID methods are consistent with the move toward the principles of Pharma 4.0: high automated processes with continuous process verification and a holistic control strategy.