Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow–derived multipotent mesenchymal stromal cells

doi:10.1016/j.jcyt.2016.05.012

Cytotherapy

Volume 18, Issue 9, September 2016, Pages 1197-1208

https://doi.org/10.1016/j.jcyt.2016.05.012 Get rights and content

Abstract

Background

Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)–derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use.

Methods

BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10⁶ viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining.

Results

A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10⁹ viable NC were used as starting material, yielding 38.8 ± 5.3 × 10⁶ viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases.

Discussion

Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use.

Introduction

Recent scientific advances preclude that regenerative medicine will become an essential component of medical care in the near future [1], [2]. The high number and scope of currently active clinical trials will likely make new treatments available to patients in a growing wave of medicinal cell-based products [3], [4]. As a consequence of developments in this emerging field, a specific regulatory framework introducing new concepts such as Advanced Therapy Medical Products (ATMP) has developed on methodologies taken from traditional pharmaceutical drugs [5], which are governed in Europe by the Directive 2001/83/EC and Regulation 726/2004, amended by Regulation 1394/2007.

Among all adult somatic cells proposed for clinical application, multipotent mesenchymal stromal cells (MSC) have reached a major prominence. In particular, MSC isolated from bone marrow (BM) display a number of biological properties that qualify them for clinical use, either based on their stem or stromal properties [6], [7]. These include migratory, homing and differentiation potential toward mesodermal lineages [8], [9], [10], as well as a powerful paracrine activity involving regulation of inflammation, immune response and tissue regeneration [11], [12]. Such pleiotropic activity has served as rationale in several hundred clinical trials worldwide, mostly in the autologous therapy setting. However, the major weakness is the lack of common standards in cell production processes and their quality controls, thus challenging the reliability of meta-analyses conducted on data from those clinical trials. In this sense, adherence to white papers and compliance with voluntary accreditation schemes and mandatory Good Scientific Practice (GxP) regulations may play a major role in the understanding and comparability of clinical results when using MSC-based medicines [13], [14], [15], [16].

In the present work, we described the design and validation of a Good Manufacturing Practice (GMP)-compliant manufacture process within a small academic laboratory and the subsequent production of 48 consecutive GMP-compliant batches of BM-MSC for use in a Phase I/IIa clinical trial for the treatment of chronic osteoarthritis and compassionate treatments [17], thus confirming the feasibility, consistency and reproducibility of the bioprocess, and safety of the ATMP. It includes a description of quality controls, key technical aspects and areas of potential improvement on standardization and quality assurance.

Section snippets

Production of clinical-grade BM-MSC

Within the context of a prospective, open-label, single-dose, single-arm Phase I–IIa clinical trial (Eudra-CT, 2009-016449-24; ClinicalTrials.gov identifier, NCT01227694) conducted by Institut de Teràpia Regenerativa Tissular (ITRT) at the Hospital Quirón Teknon (HQT) from October 2010 to June 2012, and compassionate treatments related to the clinical study, a cell-based therapy product for clinical use was manufactured in GMP-certified facilities (Banc de Sang i Teixits) [18], [19].

Definition and validation of the bioprocess

A bioprocess aimed at producing BM-MSC yields in the range of 80–100 × 10⁶ was designed to supply enough cells for clinical use and performance of Quality Controls (QC). This was achieved by seeding cells at low density in a primary culture after gradient separation of NC from BM, and further expansion through a second passage. The culture time spanned for approximately 21 days and 8–13 CPD (Figure 1; Table II). No evidence of turbidity was observed in any step of the 3 runs assayed in the

Discussion

A bioprocess for the manufacture of BM-MSC was successfully designed, validated and used in the production of 48 consecutive GMP-compliant batches for clinical application. This approach is compatible with small GMP facilities, as the ones that may be typically found in academic institutions or hospitals. As far as we are concerned, this is one of the few articles describing the complete procedure including validation, manufacturing and quality assessment of MSC-based products for clinical

Acknowledgments

The authors would like to acknowledge A. Pla, J.J. Cairó, F. Gòdia, R. Soler and L. Orozco for their support and advice; M. Caminal, L.Vidal, N. de la Fuente and S. Barbosa for technical assistance; and M. Rosal, A. Rojo, C. León and E. Morón from “Estabulari de l'Institut de Recerca” (Hospital de la Vall d'Hebron, Barcelona, Spain) for their careful assistance with animal management. This work was supported by “Ministerio de Economía y Competitividad” (grant number IPT-300000-2010-0017), “

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The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice–compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements.
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In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.
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Wharton's Jelly (WJ)-derived Mesenchymal Stromal Cells (MSC) are currently in the spotlight for the development of innovative MSC-based therapies due to their ease of sourcing, high proliferation capacity and improved immunopotency over MSC from other tissue sources. However, the short time window for derivation from donated fresh umbilical cord (UC) tissue fragments does not allow to consider biological features of the donor beyond serological safety testing. This limits the scope of MSC banking to rapid, prospective derivation of MSC, WJ lines without considering biological and genetic characteristics of the donor that may influence their suitability for clinical use (e.g. HLA type, inherited gene variants). In the present study, we describe a simple, efficient and reproducible approach for the cryopreservation of UC tissue fragments, compatible with established workflows in existing public frameworks for cord blood and tissue collection while guaranteeing pharmaceutical grade of starting materials for further processing under GMP standards. Herein we demonstrated the feasibility of time and cost-saving methods for cryopreservation of unprocessed UC tissue fragments directly at reception of the donated tissues using 10% Me₂SO-based cryosolution and a commercial clinical-grade defined cryopreservation medium (Cryostor®), showing the preservation of all Critical Quality Attributes in terms of identity, potency and kinetic parameters. In summary, our study provides evidence that cryopreservation of large unprocessed UC tissue fragments (5–13.5 cm) supports subsequent progenitor cell isolation and derivation of MSC,WJ, preserving their viability, identity, proliferation rates and potency.
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Cell manufactureDesign and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow–derived multipotent mesenchymal stromal cells

Abstract

Background

Methods

Results

Discussion

Introduction

Section snippets

Production of clinical-grade BM-MSC

Definition and validation of the bioprocess

Discussion

Acknowledgments

Cell Stem Cell

J Surg Res

Cytotherapy

Cytotherapy

Cytotherapy

Immunol Lett

Cytotherapy

Therapeutic human cells: manufacture for cell therapy/regenerative medicine

Adv Biochem Eng Biotechnol

Will regenerative medicine replace transplantation?

Cold Spring Harb Perspect Med

The global landscape of stem cell clinical trials

Regen Med

Challenges with advanced therapy medicinal products and how to meet them

Nat Rev Drug Discov

Marrow stromal cells as stem cells for nonhematopoietic tissues

Science

Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers

Cell Tissue Kinet

Concise review: mesenchymal stem cells and translational medicine: emerging issues

Stem Cells Transl Med

Cell manufacture
Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow–derived multipotent mesenchymal stromal cells