(675f) Advancing Real-Time Live Cell Imaging Technologies to Support the Development of Downstream Purification Processes for Therapeutic Viruses | AIChE

(675f) Advancing Real-Time Live Cell Imaging Technologies to Support the Development of Downstream Purification Processes for Therapeutic Viruses


Gough, I., McMaster University
Steenbakkers, L., McMaster University
Sorman Paulsson, E., Sartorius
Edlund, C., Sartorius
Sjogren, R., Sartorius
Barnes, K., Sartorius
Corbett, B., McMaster University
Mhaskar, P., McMaster University
Latulippe, D., McMaster University
Oncolytic virus therapies are a promising cancer treatment that uses viruses to selectively kill cancer cells, with the first FDA approved treatment (Imlygic for melanoma) released in 2015. However, widespread adoption of these treatments is limited due to the complexity of purifying viruses. It is generally well known that the development and optimization of novel purification processes has been greatly hindered due to the practical limitations of the current standard analytical methods (e.g., Hexon staining) for assessing virus infectivity titer. These analytical methods often require a minimum of 72 to 84 hours and significant manpower due to timed antibody washes, creating a major bottleneck within virus manufacturing research activities. Therefore, there needs to be a synergistic effort to develop analytical methods for determining virus infectivity titer that can process and analyze samples at the increased throughput of cutting-edge virus purification methods. Here, we demonstrate a wash-free adenovirus infectivity assay developed based on the production and concentration requirements of an adenovirus purification process with anion-exchange membrane absorbers. The assay was developed in collaboration with Sartorius and McMaster and leverages the Incucyte® Live-Cell Analysis System to reduce required manpower and increase assay throughput.

Adenovirus samples with a GFP-transgene were initially used, with the Incucyte® Live-Cell Analysis System and the Incucyte® AI Confluence software implemented to optimise cell culture regimes and cell confluence over time to ensure assay viability. Segmentation algorithms provided by the Incucyte® Base Analysis software were used to estimate virus titer values by the appearance of fluorescently labelled cells, which were compared to estimates obtained through traditional Hexon staining. Subsequently, adenovirus samples without a GFP transgene were used to extend the Incucyte’s ability to provide label-free titer estimates using machine learning methodologies, with the results similarly validated using Hexon staining and compared. The comparative costs and labour were then tabulated, demonstrating the relative benefits of the Incucyte® based assay as the number of samples increases.

Overall, investigation of optimized virus purification techniques is essential as specialized biotherapeutics gain clinical prominence. However, development of analytical assays that can match increased purification throughput has lagged, making sample analysis the bottleneck in virus therapeutic production. The assay presented in this work has been specifically developed to handle the sampling requirements and virus titer range associated with virus membrane chromatography, demonstrating the potential of Incucyte® integrated live-cell analysis techniques in the analytical workflow to enable new separation processes.