Abstract
Background aims
Cell therapies are costlier to manufacture than small molecules and protein therapeutics
because they require multiple manipulations and are often produced in an autologous
manner. Strategies to lower the cost of goods to produce a cell therapy could make
a significant impact on its total cost.
Methods
Borrowing from the field of bioprocess development, the authors took a design of experiments
(DoE)-based approach to understanding the manufacture of a cell therapy product in
pre-clinical development, analyzing main cost factors in the production process. The
cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus
(AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune
dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.
Results
Using gene editing efficiency as the response variable, an initial screen was conducted
for other variables that could influence the editing frequency. The multiplicity of
infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant
factors used for the optimization step to generate a response contour plot. Cost analysis
was done for multiple points in the design space to find cost drivers that could be
reduced. For the range of values tested (50 000–750 000 vg/cell AAV and 0.8–4 μg sgRNA),
editing with the highest MOI and sgRNA yielded the best gene editing frequency. However,
cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV
and 1.78 μg sgRNA.
Conclusions
The authors used DoE to define key factors affecting the gene editing process for
a potential investigational therapeutic, providing a novel and faster data-based approach
to understanding factors driving complex biological processes. This approach could
be applied in process development and aid in achieving more robust strategies for
the manufacture of cellular therapeutics.
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References
- What does cell therapy manufacturing cost? A framework and methodology to facilitate academic and other small-scale cell therapy manufacturing costings.Cytotherapy. 2020; 22: 388-397
- Design of experiments.British Medical Journal. 1936; 1: 554
- Design of experiments (DOE)—history, concepts, and relevance to in vitro culture.In Vitro Cellular and Developmental Biology - Plant. 2016; 52: 547-562
- Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.Cytotechnology. 2008; 57: 251-261
- Bioprocess optimization using design-of-experiments methodology.Biotechnology Progress. 2008; 24: 1191-1203
- Defined Serum-Free Medium for Bioreactor Culture of an Immortalized Human Erythroblast Cell Line.Biotechnol. J. 2018; 13: 1-12
- Microplates with integrated oxygen sensing for medium optimization in animal cell culture.Cytotechnology. 2004; 46: 1
- Design of experiments with small-scale bioreactor systems: Efficient bioprocess development and optimization.Bioprocess Int. 2014; 12
- The Landscape of Cellular and Gene Therapy Products: Authorization, Discontinuations, and Cost.Hum. Gene Ther. Clin. Dev. 2019; 30: 102-113
Goodwin, M., Lee, E., Lakshmanan, U., Shipp, S., Froessl, L., Barzaghi, F., ... & Bacchetta, R. CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells. Science advances 2020, 6(19), eaaz0571.
- CRISPR/Cas9 genome editing in human hematopoietic stem cells.Nat. Protoc. 2018; 13: 358-376
- Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.J. Exp. Med. 2018; 215: 985-997
- User guide to MODDE - Version 12.Sartorius Stedim Data Analytics AB. 2017;
- Double-strand break end resection and repair pathway choice.Annu. Rev. Genet. 2011; 45: 247-271
- Good Manufacturing Practices (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs.Cytotherapy. 2013; 15: 362-383
- Estimation of manufacturing development costs of cell-based therapies: a feasibility study.Cytotherapy. 2021; 23: 730-739
- Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.Cell Stem Cell. 2019; 24: 821-828.e5
- Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.Sci. Transl. Med. 2021; 13
- Gene correction for SCID-X1 in long-term hematopoietic stem cells.Nat. Commun. 2019; 10
- CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.Nature. 2016; 539: 384-389
- Targeted genome editing in human repopulating haematopoietic stem cells.Nature. 2014; 510: 235-240
- Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.Cell Rep. 2018; 23: 2606
- CD4+ T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer.Sci. Transl. Med. 2013; 5: 1-11
- Design of CAR-T Cell Manufacturing Process.Penn Scholarly Commons. 2020;
- Clinical use of lentiviral vectors.Leuk. 2018 327. 2018; 32: 1529-1541
Schuster SJ, et al. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma. 380, 45–56 (2018).
Neelapu SS, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. 377, 2531–2544 (2017).
Article info
Publication history
Published online: February 26, 2022
Accepted:
January 27,
2022
Received:
October 12,
2021
Identification
Copyright
© 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.
