Abstract This proposal aims to demonstrate how real-time measurement of protein product quality attributes and rapid assessment of adventitious agent contamination can be used for manufacturers’ risk-based decision making in continuous protein production. While Quality by Design has been a successful approach for maintaining product quality, its adoption is a byproduct of difficulties in measuring product quality in real time. We will experimentally demonstrate two novel platforms for in-line protein quality assessment (1) a micro/nanofluidic platform for assessing high- and low-molecular weight protein impurities, protein structure and binding, and glycosylation and (2) swept source Raman (SS-Raman) – using a novel approach of a tunable laser and low-cost, fixed-wavelength detector instead of a traditional, expensive spectrometer or interferometer – to measure process parameters and product quality at all points of the process. We have previously demonstrated the use of micro- and nano-fluidic assays to measure protein quality attributes off-line, constructed an automated sampling system to feed bioreactor supernatant to the micro/nanofluidic assays, and used Raman spectroscopy to identify and quantify therapeutic proteins dissolved in buffer solutions at concentrations found in drug product and at volumes equivalent to or less than one dose. This work will be the first demonstration of these technologies for real-time protein quality assessment. Additionally, this work will develop novel approaches for rapid adventitious agent detection and demonstrate their use in CHO cell culture. We will demonstrate the use of Raman spectroscopy to obtain metabolic fingerprints (intracellular metabolites) and footprints (extracellular metabolites) of protein producing CHO cells when challenged by various pathogens and classify these fingerprints and footprints as either normal or anomalous, indicating the presence of a potential infection. We will also evaluate rapid Next Generation Sequencing and bioinformatic approaches for their suitability to rapidly detect virus contaminations of continuous CHO cell culture. Finally, this proposal will investigate the impact of integrating these novel technologies into a continuous monoclonal antibody manufacturing process. At its completion this work will improve (a) product and process knowledge by demonstrating the use of real-time protein attribute measurements (b) process control by enabling direct measurement of critical quality attributes during manufacturing and feedback or feedforward control strategies to be tested, and (c) safety through development of rapid adventitious agent assays to more rapidly assure sterility of biopharmaceuticals which can improve patient safety, assure supply of medicines, and reduce business risk.