Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Automated calibration and in-line measurement of product quality during therapeutic monoclonal antibody purification using Raman spectroscopy.

Current manufacturing and development processes for therapeutic monoclonal antibodies demand increasing volumes of analytical testing for both real-time process controls and high-throughput process development. The feasibility of using Raman spectroscopy as an in-line product quality measuring tool has been recently demonstrated and promises to relieve this analytical bottleneck. Here, we resolve time-consuming calibration process that requires fractionation and preparative experiments covering variations of product quality attributes (PQAs) by engineering an automation system capable of collecting Raman spectra on the order of hundreds of calibration points from two to three stock seed solutions differing in protein concentration and aggregate level using controlled mixing. We used this automated system to calibrate multi-PQA models that accurately measured product concentration and aggregation every 9.3 s using an in-line flow-cell. We demonstrate the application of a nonlinear calibration model for monitoring product quality in real-time during a biopharmaceutical purification process intended for clinical and commercial manufacturing. These results demonstrate potential feasibility to implement quality monitoring during GGMP manufacturing as well as to increase chemistry, manufacturing, and controls understanding during process development, ultimately leading to more robust and controlled manufacturing processes.

In-line product quality monitoring during biopharmaceutical manufacturing using computational Raman spectroscopy.

The implementation of process analytical technologies is positioned to play a critical role in advancing biopharmaceutical manufacturing by simultaneously resolving clinical, regulatory, and cost challenges. Raman spectroscopy is emerging as a key technology enabling in-line product quality monitoring, but laborious calibration and computational modeling efforts limit the widespread application of this promising technology. In this study, we demonstrate new capabilities for measuring product aggregation and fragmentation in real-time during a bioprocess intended for clinical manufacturing by applying hardware automation and machine learning data analysis methods. We reduced the effort needed to calibrate and validate multiple critical quality attribute models by integrating existing workflows into one robotic system. The increased data throughput resulting from this system allowed us to train calibration models that demonstrate accurate product quality measurements every 38 s. In-process analytics enable advanced process understanding in the short-term and will lead ultimately to controlled bioprocesses that can both safeguard and take necessary actions that guarantee consistent product quality.

Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.

Bioprocess Technologies that Preserve the Quality of iPSCs.

Large-scale production of induced pluripotent stem cells (iPSCs) is essential for the treatment of a variety of clinical indications. However, culturing enough iPSCs for clinical applications is problematic due to their sensitive pluripotent state and dependence on a supporting matrix. Developing stem cell bioprocessing strategies that are scalable and meet clinical needs requires incorporating methods that measure and monitor intrinsic markers of cell differentiation state, developmental status, and viability in real time. In addition, proper cell culture modalities that nurture the growth of high-quality stem cells in suspension are critical for industrial scale-up. In this review, we present an overview of cell culture media, suspension modalities, and monitoring techniques that preserve the quality and pluripotency of iPSCs during initiation, expansion, and manufacturing.

Current state of the art in continuous bioprocessing.

There is an upsurge of interest in continuous bioprocessing, but currently continuous downstream bioprocessing has not been implemented to generate clinical material. This review focusses on the current state of the art of continuous downstream processing, highlighting the key advantages over traditional batch manufacturing. This allows the identification of scenarios where continuous downstream processing may be critical for commercial manufacturing success.