Novel Process Analytic Technology for Continuous Bioprocessses

AbstractThis proposal aims to demonstrate how real-time measurement of protein product qualityattributes and rapid assessment of adventitious agent contamination can be used formanufacturers? risk-based decision making in continuous protein production. While Quality byDesign has been a successful approach for maintaining product quality, its adoption is abyproduct of difficulties in measuring product quality in real time. We will experimentallydemonstrate two novel platforms for in-line protein quality assessment (1) a micro/nanofluidicplatform for assessing high- and low-molecular weight protein impurities, protein structure andbinding, and glycosylation and (2) swept source Raman (SS-Raman) ? using a novel approachof a tunable laser and low-cost, fixed-wavelength detector instead of a traditional, expensivespectrometer or interferometer ? to measure process parameters and product quality at all pointsof the process. We have previously demonstrated the use of micro- and nano-fluidic assays tomeasure protein quality attributes off-line, constructed an automated sampling system to feedbioreactor supernatant to the micro/nanofluidic assays, and used Raman spectroscopy to identifyand quantify therapeutic proteins dissolved in buffer solutions at concentrations found in drugproduct and at volumes equivalent to or less than one dose. This work will be the firstdemonstration of these technologies for real-time protein quality assessment. Additionally, thiswork will develop novel approaches for rapid adventitious agent detection and demonstrate theiruse in CHO cell culture. We will demonstrate the use of Raman spectroscopy to obtain metabolicfingerprints (intracellular metabolites) and footprints (extracellular metabolites) of proteinproducing CHO cells when challenged by various pathogens and classify these fingerprints andfootprints as either normal or anomalous, indicating the presence of a potential infection. We willalso evaluate rapid Next Generation Sequencing and bioinformatic approaches for their suitabilityto rapidly detect virus contaminations of continuous CHO cell culture. Finally, this proposal willinvestigate the impact of integrating these novel technologies into a continuous monoclonalantibody manufacturing process. At its completion this work will improve (a) product and processknowledge by demonstrating the use of real-time protein attribute measurements (b) processcontrol by enabling direct measurement of critical quality attributes during manufacturing andfeedback or feedforward control strategies to be tested, and (c) safety through development ofrapid adventitious agent assays to more rapidly assure sterility of biopharmaceuticals which canimprove patient safety, assure supply of medicines, and reduce business risk.