Flow cytometric assays for identity, safety and potency of cellular therapies

Campbell, John; Fraser, Alasdair R.

doi:10.1002/cyto.b.21735

Cited by 15 publications

(16 citation statements)

References 48 publications

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“…The analysis of each drug's concentration requires the development and validation of bioanalytical methods, which are specific to each species and matrix as defined in regulatory guidance documents [1,2]. Small-molecule drug candidate methods are predominantly based on LC-MS/MS, while proteins and novel drug modality methods span numerous technology platforms from ligand-binding assays to Meso Scale Discovery to high-resolution mass spectrometry and more recently the adoption of techniques such as qPCR and quantitative flow cytometry for cell and gene therapy [3][4][5].…”

mentioning

confidence: 99%

Quantification of Abemaciclib and Metabolites: Evolution of Bioanalytical Methods Supporting a Novel Oncolytic Agent

Wickremsinhe

Lee

2021

Bioanalysis

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Aim: Bioanalytical methods undergo many revisions and modifications throughout drug development to meet the objectives of the study and development program. Results: Validated LC–MS/MS methodology used to quantify abemaciclib and four metabolites in human plasma is described. The method, initially validated to support the first-in-human study, was successfully modified to include additional metabolites as in vitro and in vivo information about the activity and abundance of human metabolites became available. Consistent performance of the method over time was demonstrated by an incurred sample reanalysis passing rate exceeding 95%, across clinical studies. An overview of the numerous methods involved during the development of abemaciclib, including the quantification of drugs evaluated as combination regimens and used as substrates during drug–drug interaction studies, is presented. Conclusion: Robust bioanalytical methods need to be designed with the flexibility required to support the evolving study objectives associated with registration and post-registration trials.

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mentioning

confidence: 99%

Quantification of Abemaciclib and Metabolites: Evolution of Bioanalytical Methods Supporting a Novel Oncolytic Agent

Wickremsinhe

Lee

2021

Bioanalysis

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“…The modifications to protocol, reagents and culture process were assessed principally using flow cytometry, which provides a rapid and quantitative method for analysis. Robust, validated flow cytometric assays are a cornerstone of effective reproducible cell therapy manufacture [21]. The use of flow cytometric analysis and functional profiling of EBV-specific T cells through cytokine expression in this study resulted in improved characterization of both start material and final product, and effective assessment of inprocess culture optima, which has been used for analysis of other T cell therapeutics including a SARS-CoV-2 T cell product for COVID-19 treatment [37].…”

Section: Discussionmentioning

confidence: 99%

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Cooper

Kowalczuk

Wilkie

et al. 2021

Preprint

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Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved GMP-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analyzing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL: T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analyzed using t-Stochastic Neighbour Embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using IL-2, TNF-α and IFN-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

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“…The modifications to protocol, reagents and culture process were assessed principally using flow cytometry, which provides a rapid and quantitative method for analysis. Robust, validated flow cytometric assays are a cornerstone of effective reproducible cell therapy manufacture [ 23 ]. The use of flow cytometric analysis and functional profiling of EBV‐specific T cells through cytokine expression in this study resulted in improved characterization of both start material and final product and effective assessment of in‐process culture optima, which has been used for analysis of other T cell therapeutics, including a SARS‐CoV‐2‐specific T cell product for COVID‐19 treatment [ 24 ].…”

Section: Discussionmentioning

confidence: 99%

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy

Cooper

Kowalczuk

Wilkie

et al. 2021

Clinical and Experimental Immunology

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Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoproteinfree GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multiparameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

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Flow cytometric assays for identity, safety and potency of cellular therapies

Cited by 15 publications

References 48 publications

Quantification of Abemaciclib and Metabolites: Evolution of Bioanalytical Methods Supporting a Novel Oncolytic Agent

Quantification of Abemaciclib and Metabolites: Evolution of Bioanalytical Methods Supporting a Novel Oncolytic Agent

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy

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