Washout Kinetics of Viral Vectors from Cultured Mammalian Cells

Bagutti, Claudia; Schmidlin, Martin; Müeller, Matthias; Brodmann, Peter

doi:10.1177/153567601201700404

Cited by 3 publications

(1 citation statement)

References 22 publications

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“…Usually this is accomplished via manifold washing of the cell suspension during the post-transduction cultivation. 50,51 Such washing procedures can be integrated into the activity matrix of the Prodigy device. In this context, methods should be implemented to check residual vector concentration in supernatants (Jang et al .…”

Section: Discussionmentioning

confidence: 99%

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

Oberschmidt

Morgan

Huppert³

et al. 2019

Human Gene Therapy Methods

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In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS ® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 10 9 leukocytes (including 1.1–2.3 × 10 9 NK cells) collected by small-scale lymphapheresis ( n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log −3.6 after CD3 depletion and log −3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult ® chamber of Prodigy resulted in approximately 4.2–8.5-fold NK cell expansion rates by adding of NK MACS ® basal medium containing NK MACS ® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redire...

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Section: Discussionmentioning

confidence: 99%

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

Oberschmidt

Morgan

Huppert³

et al. 2019

Human Gene Therapy Methods

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Biosafety Recommendations on the Handling of Animal Cell Cultures

Herman

Pauwels

2014

Cell Engineering

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Biosafety Considerations for Viral Vector Gene Therapy: An Explanation and Guide for the Average Everyday-Hero Pharmacist

Hernandez

2022

Journal of Pharmacy Practice

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Purpose An overview of the multi-faceted biosafety points that must be taken into consideration by pharmacists and pharmacies in order to provide viral vector gene therapy to their practice site. Summary As science and medicine evolves, pharmacists and other healthcare workers are continually faced with unique challenges in the workplace. They are expected to be informed and proficient on new therapies and standards of practice, and be able to apply this knowledge appropriately for their patients. One such advancement that seems to be picking up speed in recent years is gene therapy, which is often achieved with the assistance of a viral vector. As these viral vector doses move closer to mainstream medicine, a host of issues and concerns for the pharmacists, nurses, and caregivers that are involved in the process begin to rise to the surface, often rooted in the critical concern: “How do we dispense, utilize, and administer these doses safely?” Unfortunately, there is no singular, concise source of information for addressing biosafety with viral vector products, and guidance must be gathered from a variety of resources in order to mesh together a reasonable working process. Conclusion: While this may seem to be a daunting task, facilities that already meet USP 797 and USP 800 guidelines are well on their way to being ready to provide viral vector doses. By incorporating additional steps and reviewing biosafety specific resources, these sites can easily adapt to provide these new and novel therapies for their patient population.

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Washout Kinetics of Viral Vectors from Cultured Mammalian Cells

Abstract: Abstract

Cited by 3 publications

References 22 publications

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering

Biosafety Recommendations on the Handling of Animal Cell Cultures

Biosafety Considerations for Viral Vector Gene Therapy: An Explanation and Guide for the Average Everyday-Hero Pharmacist

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