In Situ Microscopy for Real‐Time Visualization of Microcarrier Cell Cultures for Live Virus Vaccine Process Development

In microcarrier (MC) cell culture processes for live virus vaccine (LVV) production, a variety of process phenomena (i.e., cell surface adhesion, cell aggregation, MC aggregation, cell lysis, cell death, surface detachment, and accumulation of cellular debris) exist that significantly underlie the performance of the process itself. Nonetheless, it remains difficult to directly characterize these critical phenomena during a cell culture process. Process analytical technology (PAT) offers a unique opportunity to potentially overcome these challenges in a manner that provides real-time information via directly interfacing analytical technology with the bioprocess itself. In this work, we propose the utilization of in situ microscopy as a real-time, in-line PAT to directly visualize and characterize both cell and microcarrier behavior simultaneously using two commercially available probe-based technologies. To the best of our knowledge, this is the first report of in situ microscopy applied to an upstream LVV cell culture process, providing direct visualization and characterization of key process phenomena in real-time. Cell growth, cell death, surface detachment, accumulation of cellular debris, and aggregation of MCs are directly elucidated via our proposed in situ technology. Notably, we observe significant differences in the in situ microscopy data relative to offline microscopy with regards to aggregation of MCs. MC aggregation is observed to be highly prevalent in the LVV-based process studied herein, particularly during viral replication. Significant MC aggregation is not observed in offline analysis, suggesting that manual sampling may disrupt MC-aggregate structures present in the bioreactor culture, and as such, highlights the abundant need for in situ observation to enable accurate and representative process analysis and modeling. Our observations of MC aggregation carry implications for cell growth, cell death, and viral infection in MC-based LVV cell cultures, indicating this elucidated phenomena may be much more prevalent in LVV cell culture processes than previously believed. In situ microscopy thus provides a novel and powerful PAT methodology, with easy to interpret direct visualization for readout, for characterizing upstream LVV processes in real time, in which we can now significantly advance our process understanding beyond what can be achieved using traditional offline characterization methods.