Raman spectroscopy is an information-rich, label-free, non-invasive, non-destructive analytical technique with many recent applications in biological systems. It has been used to distinguish cancer cells, stem cell differentiation, immune cell classification and activation. Raman spectroscopy has already been used as a process analytical technology (PAT) for therapeutic protein manufacturing and is being developed for cell therapy manufacturing applications. Regulatory T-cells (Tregs) effectively suppress immune response, such that clinical trials are investigating the potential for Treg therapies to treat a range of autoimmune diseases. We work with Tregs isolated from pediatric thymuses that are a byproduct of open heart surgery. We have developed a bioprocess for these Tregs to be expanded ex vivo, allowing for the allogeneic treatment of multiple patients from a single donor. We investigated the suitability of Raman spectroscopy as a tool to monitor thymic Tregs expanded over a 21-day culture. Raman spectra were collected from dry-fixed samples. Univariate analyses were used to establish a profile for expanding Tregs, which showed notably consistent trends in Raman bands associated with cellular proteins and lipids, that varied predictably with stimulation timing. Raman markers indicating poor culture outcomes, including cell death, were identified. Multivariate analyses revealed that Tregs could be distinguished from conventional T-cells isolated from the same donor, enabling the monitoring of unwanted cell types. Off-the-shelf Raman spectroscopy probes are commonly used in biopharmaceutical manufacturing to monitor process parameters but are not yet widely employed in the cell therapy context. Adaptation of these probes to therapeutic cell manufacturing standards and validation of previous results in an active culture setting should enable the development of non-invasive, non-destructive tools for process monitoring.

Raman spectroscopy as a process analytical technology for regulatory T-cell manufacturing

ACS Fall 2021

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