Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

Abstract Background A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). Results The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. Conclusions These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

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Process Development and Validation of Expanded Regulatory T Cells for Prospective Applications: An Example of Manufacturing a Personalized Advanced Therapy Medicinal Product

10.21203/rs.3.rs-675062/v1 ◽

2021 ◽

Author(s):

Cristiana Lavazza ◽

Silvia Budelli ◽

Elisa Montelatici ◽

Mariele Viganò ◽

Francesca Ulbar ◽

...

Keyword(s):

Clinical Trials ◽

Cell Therapy ◽

Process Development ◽

Solid Organ Transplantation ◽

Good Manufacturing Practice ◽

Cell Number ◽

Favorable Effect ◽

Solid Organ ◽

Advanced Therapy Medicinal Product ◽

Advanced Therapy

Abstract BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cells transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotyping).RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function.CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

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Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: Off-the-shelf GMP-manufactured donor-independent ATMP

10.21203/rs.3.rs-47993/v1 ◽

2020 ◽

Author(s):

Seda Ballikaya ◽

Samar Sadeghi ◽

Elke Niebergall-Roth ◽

Laura Nimtz ◽

Jens Frindert ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Manufacturing Process ◽

Ex Vivo ◽

Good Manufacturing Practice ◽

Process Data ◽

Advanced Therapy Medicinal Product ◽

Drug Products ◽

Advanced Therapy ◽

Immunomagnetic Isolation

Abstract Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes.Methods: We developed and validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium and immunomagnetic isolation of the ABCB5+ cells from the mixed culture.Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product.Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

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Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data

Cytotherapy ◽

10.1016/j.jcyt.2020.08.012 ◽

2020 ◽

Cited By ~ 1

Author(s):

Andreas Kerstan ◽

Elke Niebergall-Roth ◽

Jasmina Esterlechner ◽

Hannes M. Schröder ◽

Martin Gasser ◽

...

Keyword(s):

Medicinal Product ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Good Manufacturing Practice ◽

Clinical Use ◽

Advanced Therapy Medicinal Product ◽

Process Validation ◽

Human Data ◽

Advanced Therapy

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Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: off-the-shelf GMP-manufactured donor-independent ATMP

Stem Cell Research & Therapy ◽

10.1186/s13287-020-01987-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Seda Ballikaya ◽

Samar Sadeghi ◽

Elke Niebergall-Roth ◽

Laura Nimtz ◽

Jens Frindert ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Manufacturing Process ◽

Ex Vivo ◽

Good Manufacturing Practice ◽

Process Data ◽

Advanced Therapy Medicinal Product ◽

Drug Products ◽

Advanced Therapy ◽

Immunomagnetic Isolation

Abstract Background Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes. Methods We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5+ cells from the mixed culture. Results Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product. Conclusion We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

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Therapeutic options in post-transplant lymphoproliferative disorders

Therapeutic Advances in Hematology ◽

10.1177/2040620711412417 ◽

2011 ◽

Vol 2 (6) ◽

pp. 393-407 ◽

Cited By ~ 20

Author(s):

Heiner Zimmermann ◽

Ralf Ulrich Trappe

Keyword(s):

T Cell ◽

Cell Therapy ◽

Phase Ii ◽

Phase Ii Trial ◽

Solid Organ Transplantation ◽

Lymphoproliferative Disorders ◽

Treatment Modalities ◽

Cytotoxic T Cell ◽

Solid Organ ◽

T Cell Therapy

Post-transplantation lymphoproliferative disorders (PTLD) are the second most frequent malignancies after solid organ transplantation and cover a wide spectrum ranging from polyclonal early lesions to monomorphic lymphoma. Available treatment modalities include immunosuppression reduction, immunotherapy with anti-B-cell monoclonal antibodies, chemotherapy, antiviral therapy, cytotoxic T-cell therapy as well as surgery and irradiation. Owing to the small number of cases and the heterogeneity of PTLD, current treatment strategies are mostly based on case reports and small, often retrospective studies. Moreover, many studies on the treatment of PTLD have involved a combination of different treatment options, complicating the evaluation of individual treatment components. However, there has been significant progress over the last few years. Three prospective phase II trials on the efficacy of rituximab monotherapy have shown significant complete remission rates without any relevant toxicity. A prospective, multicenter, international phase II trial evaluating sequential treatment with rituximab and CHOP-based chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisone) is ongoing and preliminary results have been promising. Cytotoxic T-cell therapy targeting Epstein–Barr virus (EBV)-infected B cells has shown low toxicity and high efficacy in a phase II trial and will be a future therapeutic option at specialized centers. Here, we review the currently available data on the different treatment modalities with a focus on PTLD following solid organ transplantation in adult patients.

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Immunomodulative cell therapy after solid organ transplantation

Current Opinion in Organ Transplantation ◽

10.1097/mot.0b013e32834f02de ◽

2012 ◽

Vol 17 (1) ◽

pp. 33 ◽

Cited By ~ 2

Author(s):

Marc-Hendrik Dahlke

Keyword(s):

Cell Therapy ◽

Organ Transplantation ◽

Solid Organ Transplantation ◽

Solid Organ

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Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: Off-the-shelf GMP-manufactured donor-independent ATMP

10.21203/rs.3.rs-47993/v2 ◽

2020 ◽

Author(s):

Seda Ballikaya ◽

Samar Sadeghi ◽

Elke Niebergall-Roth ◽

Laura Nimtz ◽

Jens Frindert ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Manufacturing Process ◽

Ex Vivo ◽

Good Manufacturing Practice ◽

Process Data ◽

Advanced Therapy Medicinal Product ◽

Drug Products ◽

Advanced Therapy ◽

Immunomagnetic Isolation

Abstract Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes.Methods: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium and immunomagnetic isolation of the ABCB5+ cells from the mixed culture.Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product.Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

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Measures to minimize cross-contamination risks in Advanced Therapy Medicinal Product manufacturing

ScienceOpen Research ◽

10.14293/s2199-1006.1.sor-life.aejrv9.v1 ◽

2014 ◽

Author(s):

Livia Roseti ◽

Marta Serra ◽

Brunella Grigolo

Keyword(s):

Medicinal Product ◽

Main Product ◽

Good Manufacturing Practice ◽

Cross Contamination ◽

Medicinal Products ◽

Advanced Therapy Medicinal Product ◽

Advanced Therapy ◽

European Regulations ◽

Product Manufacturing

Current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. These quality requirements are generally thought to be elaborate and costly. However they ensure three main product characteristics: safety, consistency and absence of cross-contamination. The term cross-contamination is used to indicate misidentification of one cell line or culture by another. The Good Manufacturing Practice Guidelines suggest some recommendations in order to prevent cross-contaminations and require a demonstration that the implemented actions are effective. Here we report some practical examples useful both to minimize cross-contamination risks in an Advanced Therapy Medicinal Product production process and to evaluate the efficacy of the adopted measures.

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MSC Manufacturing for Academic Clinical Trials: From a Clinical-Grade to a Full GMP-Compliant Process

Cells ◽

10.3390/cells10061320 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1320

Author(s):

Chantal Lechanteur ◽

Alexandra Briquet ◽

Virginie Bettonville ◽

Etienne Baudoux ◽

Yves Beguin

Keyword(s):

Large Scale ◽

Raw Materials ◽

Good Manufacturing Practice ◽

Phase Iii ◽

Scale Production ◽

Clinical Grade ◽

Advanced Therapy Medicinal Product ◽

Long Term Storage ◽

Advanced Therapy ◽

New Variant

Following European regulation 1394/2007, mesenchymal stromal cell (MSCs) have become an advanced therapy medicinal product (ATMP) that must be produced following the good manufacturing practice (GMP) standards. We describe the upgrade of our existing clinical-grade MSC manufacturing process to obtain GMP certification. Staff organization, premises/equipment qualification and monitoring, raw materials management, starting materials, technical manufacturing processes, quality controls, and the release, thawing and infusion were substantially reorganized. Numerous studies have been carried out to validate cultures and demonstrate the short-term stability of fresh or thawed products, as well their stability during long-term storage. Detailed results of media simulation tests, validation runs and early MSC batches are presented. We also report the validation of a new variant of the process aiming to prepare fresh MSCs for the treatment of specific lesions of Crohn’s disease by local injection. In conclusion, we have successfully ensured the adaptation of our clinical-grade MSC production process to the GMP requirements. The GMP manufacturing of MSC products is feasible in the academic setting for a limited number of batches with a significant cost increase, but moving to large-scale production necessary for phase III trials would require the involvement of industrial partners.

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