[
    {
        "id": "authors:6b7dc-75086",
        "collection": "authors",
        "collection_id": "6b7dc-75086",
        "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210528-141159173",
        "type": "article",
        "title": "Intensified Production of Vaccinia-Based Oncolytics in the High Density Cell Respirator (HDCR) Bioreactor Improves Vaccine Logistics and Economics",
        "author": [
            {
                "family_name": "Cook",
                "given_name": "Colin A.",
                "orcid": "0000-0002-6283-5105",
                "clpid": "Cook-Colin-A"
            },
            {
                "family_name": "Kang",
                "given_name": "Seonah",
                "clpid": "Kang-Seonah"
            },
            {
                "family_name": "Lu",
                "given_name": "Jianming",
                "clpid": "Lu-Jianming"
            },
            {
                "family_name": "Tai",
                "given_name": "Yu-Chong",
                "orcid": "0000-0001-8529-106X",
                "clpid": "Tai-Yu-Chong"
            },
            {
                "family_name": "Chatterjee",
                "given_name": "Saswati",
                "clpid": "Chatterjee-Saswati"
            },
            {
                "family_name": "Fong",
                "given_name": "Yuman",
                "clpid": "Fong-Yuman"
            }
        ],
        "abstract": "Intensification of cell-based production processes is key to improving oncolytic vaccine logistics and economics by saving on GMP space, time, labor, and feedstock. Many promising vectors, including those based on vaccinia virus (e.g. CF33, JX-594/Pexa-Vec) are still produced using flask-based culture due to the cost, effort, and uncertainty involved in adapting to stirred-tank or perfusion processes. Based on prior success culturing adherent cells (e.g. HEK293, A549, CV-1) to high densities (107-8 cells/mL) using the scalable high-density cell respirator (HDCR) bioreactor, we hypothesized that the platform could support orders-of-magnitude-intensified production of replication competent viruses. Here we report on CF33 virus production as a proof-of-concept for oncolytic virotherapy. Bioreactor cartridges were produced based on a 4-stack of 600 cm\u00b2 HDCR membranes, featuring a proprietary gas perfusable and permeable microarchitecture that optimally oxygenates cells even at high densities. Media perfusion was feedback-controlled based on glucose measurements in the waste stream. A549 cells were seeded into the bioreactor on microcarriers, expanded 10-fold to mid-10\u2077 cells/mL densities, infected with CF33 virus (MOI of 0.1), and harvested 48 hours post-infection. CF33 virus was gradient purified and titered for functional virus by plaque forming assay. Multiple production runs using different strains of CF33 virus validated the reproducibility of the process, as summarized in Table 1. Importantly, cell specific titers (PFU/cell) remained comparable to conventional flask-based production, leading to significant intensification due to the higher cell densities supported in the HDCR bioreactor. Volumetric productivity is on the order of 100\u00d7 that of cell factories. The efficient usage of media due to the gas-media decoupled operation of the HDCR bioreactor enables gradient-purified virus costs of around $500/1010 PFU. This is significantly below the $10,000/1010 PFU charged by CMOs and supports our mission of democratizing access to life saving medicines. The straightforward adaptation of CF33 virus production from a flask-based to a high yield, intensified process highlights the logistical and economical advantage of the HDCR platform for oncolytics.",
        "doi": "10.1016/j.ymthe.2021.04.019",
        "issn": "1525-0016",
        "publisher": "American Society of Gene & Cell Therapy",
        "publication": "Molecular Therapy",
        "publication_date": "2021-04-27",
        "series_number": "4, S1",
        "volume": "29",
        "issue": "4, S1",
        "pages": "397-398"
    },
    {
        "id": "authors:0fchp-ejx43",
        "collection": "authors",
        "collection_id": "0fchp-ejx43",
        "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-102736884",
        "type": "article",
        "title": "A Universal High Density Cell Respirator (HDCR) Bioreactor for Intensified Production of Gene Therapy Vectors",
        "author": [
            {
                "family_name": "Cook",
                "given_name": "Colin A.",
                "orcid": "0000-0002-6283-5105",
                "clpid": "Cook-C-A"
            },
            {
                "family_name": "Boyoglu",
                "given_name": "Cemil",
                "clpid": "Boyoglu-C"
            },
            {
                "family_name": "Bugga",
                "given_name": "Lakshmi",
                "clpid": "Bugga-L"
            },
            {
                "family_name": "Liu",
                "given_name": "Yen-Hsi",
                "clpid": "Liu-Yen-Hsi"
            },
            {
                "family_name": "Lu",
                "given_name": "Jianming",
                "clpid": "Lu-Jianming"
            },
            {
                "family_name": "Pang",
                "given_name": "Ka Ming",
                "clpid": "Pang-Ka-Ming"
            },
            {
                "family_name": "Sivanandam",
                "given_name": "Venkatesh",
                "clpid": "Sivanandam-V"
            },
            {
                "family_name": "Tai",
                "given_name": "Yu-Chong",
                "orcid": "0000-0001-8529-106X",
                "clpid": "Tai-Yu-Chong"
            },
            {
                "family_name": "Fong",
                "given_name": "Yuman",
                "clpid": "Fong-Yuman"
            },
            {
                "family_name": "Chatterjee",
                "given_name": "Saswati",
                "clpid": "Chatterjee-S"
            }
        ],
        "abstract": "AAV-based gene therapy vectors are under intense investigation and rapidly becoming established as clinical therapy for gene-based diseases including inherited diseases. Despite their great promise, the single biggest limitation to their widespread use is the inability to manufacture sufficient quantities of high quality AAV vectors rapidly particularly at low cost. This represents the single biggest limitation to the development of clinical AAV gene therapy. \n\nThe intensification of cell-based production processes holds great promise for increasing the capacity and speed of AAV vector manufacturing while also reducing cost, by saving on space, time, labor, and resources. While much effort to date has focused on improving cell specific productivity (vector genomes (vg)/cell), little progress has been made on increasing cell density, which remain at 10\u2076\u207b\u2077 cells/mL due to limitations of either surface area or gas exchange and shear forces (e.g. stirred tank reactors, perfusion reactors). With total productivity being the product of specific cell productivity and cell density, there exists substantial untapped potential in high-density culture. Recognizing the enormous discrepancy in the necessary gaseous exchange rate compared to soluble nutrient/waste exchange rate for most cells (e.g. 90\u00d7 for HEK293), we hypothesized that 1) decoupling mass transport through novel bioreactor architecture would enable higher cell densities and that 2) the optimized cell niche afforded by this architecture would maintain cell specific productivity even at high cell densities, with an overall impact of significantly elevated production capacity. \n\nWith the goal of improving AAV vector production capacities and to address issues of cell growth and densities, we designed a high-density cell respirator (HDCR). The HDCR is a scalable bioreactor comprised of stackable, gas and media perfusable membranes, which achieves an oxygen mass transfer coefficient k_La of > 40/hr via membrane permeation to support high density culture >10\u2078 cells/mL. The micro-architecture of the membranes has been engineered using integrated finite element modeling to guarantee that all cells receive sufficient gas, nutrient, and waste exchange and are protected from shear forces. We present cell growth curves demonstrating the system is compatible with suspension (e.g. CHO-S), adherent (e.g. HEK293, A549), and microcarrier (e.g. CV-1 on Cytodex-3) cultures to >10\u2078 cells/mL making it a universal platform. Importantly, we demonstrate that the HDCR bioreactor is compatible with AAV vector production, including the steps of cell seeding, expansion, transfection, feeding, and harvesting. We show for the first time that the optimized cell growth niche in the HDCR maintains productivity (within 80% vg/cell of low-density culture) despite increasing cell density by 400% for a total increase in AAV production of 320%. The results of ongoing optimization studies for AAV production in the HDCR will be presented. From these initial empirical results and modelling, we predict that a scaled HDCR bioreactor fitting inside a standard 160L tissue culture incubator would support the expansion of >2\u00d710\u00b9\u00b2 cells and production of up to 10\u00b9\u2077 vector genomes of AAV. By matching mass transport with cellular demands AAV production can be intensified, unlocking the potential for researchers to screen AAV candidates faster and for clinical vectors to be affordably manufactured.",
        "doi": "10.1016/j.ymthe.2020.04.019",
        "issn": "1525-0016",
        "publisher": "American Society of Gene & Cell Therapy",
        "publication": "Molecular Therapy",
        "publication_date": "2020-04-28",
        "series_number": "4",
        "volume": "28",
        "issue": "4",
        "pages": "30-31"
    }
]