Impact considerations of post-production processes on cell and gene drug products.

Cell and gene therapies are demonstrating clinical efficacy, but prohibitive product costs and operational complexity bottlenecks may limit expanded patient access to these innovative and transformative products. An initial survey and subsequent article published through the International Society for Cell & Gene Therapy in 2017 presented a roadmap on how specific steps, from tissue procurement and material acquisition to facility operation and production, contribute to the high cost of cell and gene therapies. Herein the authors expanded the investigation to provide considerations to better understand how post-production procedures can impact a product's accessibility to patients. The administration of a drug product to and follow-up in a patient involve key decisions in several post-production process areas, such as product storage, distribution and handling logistics and compliance, across the value chain through integrated data management solutions. Understanding as well as carefully evaluating these specific components is not widely considered during early process development but is critical in developing a viable product life cycle.

Transitioning from development to commercial: risk-based guidance for critical materials management in cell therapies.

A key hurdle to ensuring patient access to cell and gene therapies (CGTs) and continued growth of the industry is the management of raw materials. The combination of rapid growth, individual product and process complexity and limited industry-specific guidance or awareness presents non-obvious risk mitigation challenges for transitioning from development to clinical application. Understanding, assessing and mitigating the varied raw material risks for CGT products during product and clinical development are critical for ensuring smooth transitions into commercialization and for preventing interruption of product supply to patients. This article presents a risk-based approach driven by concerns for patient safety that can help focus and coordinate efforts to address the most critical risk factors. Highlighted are some of the highest risk materials common to the manufacture of many CGTs, including the primary starting material, culture media, reagents and single-use components. Using a hypothetical gene-edited cell therapy as an example, we describe the general manufacturing process and subsequently incorporate the described methodology to perform a sample risk assessment. The practical approach described herein is intended to assist CGT manufacturers and suppliers in actively assessing materials early in development to provide a basic starting point for mitigating risks experienced when translating CGT products for clinical and long-term commercial application.

Modulating cell state to enhance suspension expansion of human pluripotent stem cells.

The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.

Chemically controlled aggregation of pluripotent stem cells.

Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical-based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension-based manufacturing of PSC cultures that are critical for the translation of regenerative medicine strategies to clinical products.

A roadmap for cost-of-goods planning to guide economic production of cell therapy products.

Cell therapy products are frequently developed and produced without incorporating cost considerations into process development, contributing to prohibitively costly products. Herein we contextualize individual process development decisions within a broad framework for cost-efficient therapeutic manufacturing. This roadmap guides the analysis of cost of goods (COG) arising from tissue procurement, material acquisition, facility operation, production, and storage. We present the specific COG considerations related to each of these elements as identified through a 2013 International Society for Cellular Therapy COG survey, highlighting the differences between autologous and allogeneic products. Planning and accounting for COG at each step in the production process could reduce costs, allowing for more affordable market pricing to improve the long-term viability of the cell therapy product and facilitate broader patient access to novel and transformative cell therapies.

Achieving Efficient Manufacturing and Quality Assurance through Synthetic Cell Therapy Design.

New methods to manipulate gene and cell state can be used to engineer cell functionality, simplify quality assessment, and enhance manufacturability. These strategies could help overcome unresolved cell therapy manufacturing challenges and complement frameworks to design quality into these complex cellular systems, ultimately increasing patient access to living therapeutics.

Quality cell therapy manufacturing by design.

Transplantation of live cells as therapeutic agents is poised to offer new treatment options for a wide range of acute and chronic diseases. However, the biological complexity of cells has hampered the translation of laboratory-scale experiments into industrial processes for reliable, cost-effective manufacturing of cell-based therapies. We argue here that a solution to this challenge is to design cell manufacturing processes according to quality-by-design (QbD) principles. QbD integrates scientific knowledge and risk analysis into manufacturing process development and is already being adopted by the biopharmaceutical industry. Many opportunities to incorporate QbD into cell therapy manufacturing exist, although further technology development is required for full implementation. Linking measurable molecular and cellular characteristics of a cell population to final product quality through QbD is a crucial step in realizing the potential for cell therapies to transform healthcare.

CD24 tracks divergent pluripotent states in mouse and human cells.

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

Methods for evaluation of helium/oxygen delivery through non-rebreather facemasks.

BACKGROUND: Inhalation of low-density helium/oxygen mixtures has been used both to lower the airway resistance and work of breathing of patients with obstructive lung disease and to transport pharmaceutical aerosols to obstructed lung regions. However, recent clinical investigations have highlighted the potential for entrainment of room air to dilute helium/oxygen mixtures delivered through non-rebreather facemasks, thereby increasing the density of the inhaled gas mixture and limiting intended therapeutic effects. This article describes the development of benchtop methods using face models for evaluating delivery of helium/oxygen mixtures through facemasks. METHODS: Four face models were used: a flat plate, a glass head manikin, and two face manikins normally used in life support training. A mechanical test lung and ventilator were employed to simulate spontaneous breathing during delivery of 78/22 %vol helium/oxygen through non-rebreather facemasks. Based on comparison of inhaled helium concentrations with available clinical data, one face model was selected for measurements made during delivery of 78/22 or 65/35 %vol helium/oxygen through three different masks as tidal volume varied between 500 and 750 ml, respiratory rate between 14 and 30 breaths/min, the inspiratory/expiratory ratio between 1/2 and 1/1, and the supply gas flow rate between 4 and 15 l/min. Inhaled helium concentrations were measured both with a thermal conductivity analyzer and using a novel flow resistance method. RESULTS: Face models borrowed from life support training provided reasonably good agreement with available clinical data. After normalizing for the concentration of helium in the supply gas, no difference was noted in the extent of room air entrainment when delivering 78/22 versus 65/35 %vol helium/oxygen. For a given mask fitted to the face in a reproducible manner, delivered helium concentrations were primarily determined by the ratio of supply gas flow rate to simulated patient minute ventilation, with the inspiratory/expiratory ratio playing a secondary role. However, the functional dependence of helium concentration on these two ratios depended on the mask design. CONCLUSIONS: Large differences in mask performance were identified. With continued refinement, the availability of reliable benchtop methods is expected to assist in the development and selection of patient interfaces for delivery of helium/oxygen and other medical gases.