SMART sensors for cell growth monitoring in closed system G-Rex for scalable, cost-conscious cell and gene therapy manufacturing.

Abstract

Cell and gene-modified cell therapy (CGT) includes creating engineered cancer killing cells ex vivo and then subsequently putting them into the patient's body to selectively attack cancerous cells. An immune cell manufacturing device called G-Rex (for "gas permeable rapid expansion"), is widely used for production in current clinical trials and commercially approved drug products. G-Rex is uniquely simple as it eliminates the need for pumps and flow circuits to deliver oxygen and nutrients and instead relies on passive convection, greatly minimizing complexity and allowing for scalable manufacturing. The elimination of the need to pump media allows concurrent manufacturing without requiring additional capital equipment investments, thereby decreasing cycle times and increasing utilization rates of existing equipment and space. To further simplify G-Rex based manufacturing operations, it would be ideal to eliminate any need of physical intervention to assess cell quantity. Alternative CGT drug product manufacturing technologies require invasive sampling of media or cell suspension to determine cell quantity or monitor cell growth characteristics. With that goal in mind, herein we describe integration of a "single-use metabolite absorbing resonant transducer" in G-Rex (SMART G-Rex) and an accompanying contact-free reader (SMART Reader). The SMART G-Rex is wirelessly interrogated by the SMART Reader to determine the extent of cell expansion in the G-Rex at any given time. As cell quantity increases, the SMART G-Rex absorbs cell secreted metabolites (organic compounds such as eicosanoids) which cause a change in resonant frequency (measured as % of frequency change, or Skroot Growth Index (SGI)). Results: By placing SMART G-Rex in proximity of the SMART Reader, whether in or out of an incubator, changes in resonant frequency are detected and translated into cell quantity with high reliability (<1.5% coefficient of variation across replicates). For example, using 45 sensors across multiple donor cell samples, we established 99% confidence in the SMART system's accuracy at signaling a >60X expansion (3×10⁹ viable cells) at harvest. Benchtop reader measurements required approximately 15 minutes to read 11 vessels (average of 1 minute 20 seconds per vessel), representing a >50% reduction in time compared to conventional manual sampling and a complete elimination of invasive sampling altogether. The SMART G-Rex provides a rapid, noninvasive, and scalable means for real-time quantification of the viable cell count in a CGT drug production process and informs manufacturers when a CGT drug product has reached the desired quantity of cells necessary to proceed with formulation, fill, and finish.