**Abstract**

Mesenchymal stem cells (MSCs) are advanced therapy medicinal products used in cell therapy applications. Several MSC products have already advanced to phase III clinical testing and market approval. The manufacturing of MSCs must comply with good manufacturing practice (GMP) from phase I in Europe and phase II in the US, but there are several unique challenges when cells are the therapeutic product. Any GMP-compliant process for the production of MSCs must include the expansion of cells *in vitro* to achieve a sufficient therapeutic quantity while maintaining high cell quality and potency. The process must also allow the efficient harvest of anchorage-dependent cells and account for the influence of shear stress and other factors, especially during scale-up. Bioreactors are necessary to produce clinical batches of MSCs, and bioprocess development must therefore consider this specialized environment. For the last 10 years, we have investigated bioprocess development as a means to produce high-quality MSCs. More recently, we have also used bioreactors for the cocultivation of stem cells with other adult cells and for the production of MSC-derived extracellular vesicles. This review discusses the state of the art in bioprocess development for the GMP-compliant manufacture of human MSCs as products for stem cell therapy.

**Keywords:** bioreactors, quality-by-design, critical process parameters, stem cell potency, standardization

## **1. Manufacturing cell therapy products**

Cell therapy is a growing clinical research and healthcare sector in which living cells are introduced into a patient in an attempt to ameliorate or cure a disease. Stem cell therapy is one of the most promising fields within this sector because the introduced cells have the capacity to differentiate, allowing the repopulation of diseased organs with healthy cells, or to allow even complete organ regeneration. This chapter will focus on one specific type of stem cell (MSCs), which are variously defined as mesenchymal stem cells, mesenchymal stromal cells, or (most recently) medicinal signaling cells [1]. These various definitions reflect the controversial origin and functionality of MSCs and uncertainty about their clinical potential [2, 3]. Following encouraging initial results, the unclear or disappointing outcomes of some MSC clinical trials have clouded the picture [4], but the pioneers of this approach still regard MSCs as a promising therapeutic option [5]. One of the key issues in the deployment of MSCs is ensuring they are safe and effective, which requires a well-characterized manufacturing process.

In order to provide enough MSCs for cell therapy, donor cells must be isolated from tissue and then expanded *in vitro* to reach a population of 1–9 × 108 cells, which is the typical dose for adult treatment [6]. The success or failure of MSC therapy depends on this *in vitro* expansion process, which was first studied in detail following the failure of the MSC product Prochymal in phase III trials for graft versus host disease (GvHD) [4], whereas a similar product succeeded in phase II. One reason proposed for the contrasting outcomes of each trial was the substantial differences in the MSC expansion step at the manufacturing scale, highlighting the specialized and complex nature of MSCs [4].

#### **1.1 Definition of MSCs and current approved products**

MSCs are classified as advanced therapeutic medicinal products (ATMPs) under regulations in Europe and the US. Many countries follow the regulations laid down by the US Food and Drug Administration (FDA), which defines MSCs as cell therapy products, whereas the European Medicines Agency (EMA) defines MSCs as cell-based medicinal products and distinguishes between somatic cell therapy medicinal products (SCTMPs) and tissue engineered products (TEPs) [7]. This means that clinical studies and drug approval are covered by a specific regulatory framework applied at the national or regional level. Manufacturing must therefore be compliant with good manufacturing practice (GMP) regulations that have been tailored for ATMPs, following strict criteria for product specification and release for clinical use. However, the regulatory framework for MSC manufacturing is confounded by ambiguous product definitions reflecting regional differences in the way the regulations are implemented. For example, the EMA requires GMP compliance and manufacturing authorization for phase I material, whereas the FDA does not apply this requirement until phases II and III, and in Canada, GMP compliance is not strictly required at any phase [8]. Even so, various MSC products have been manufactured under these different regulatory jurisdictions and have proceeded through clinical development, in some cases gaining market authorization from the local regulatory agency [9]. Most of these products are allogenic, which means that MSCs from one or more healthy donors are expanded, processed, and stored and then applied to patients as an off-the-shelf product (**Table 1**). In 2016, the allogenic MSC product TEMCELL (developed by Mesoblast) was licensed to JCR Pharmaceuticals, which received market authorization in Japan under a fast-track protocol for patients with steroid-refractory acute GvHD. Mesoblast also conducted a phase III trial with this product in the US, involving 60 patients of the same indication, achieving the primary endpoints (NCT02336230). In 2018, ALOFISEL (Takeda Pharma), an expanded allogenic adipose-derived MSC product, was approved by the EMA to treat complex perianal fistula in patients with Crohn's disease. This was supported by a placebo-controlled trial involving 212 patients [10]. Stempeucel (Stempeutics), an expanded allogenic MSC product, received market authorization from the Drug Controller General of India to treat limb ischemia in patients with Buerger's disease. However, it is limited to 200 patients on a cost-recovery basis, and a postmarket surveillance study is required. Ninety patients have already received an injection of this MSC product in a phase II trial, achieving a significantly better outcome than standard care [11]. CARTISTEM (Medipost) is an allogenic culture-expanded umbilical cord blood MSC product to treat knee articular cartilage defects in patients with osteoarthritis, grade IV, and following approval for the South Korean market in 2012, its clinical outcomes have remained

**205**

drug by the EMA and FDA.

regulatory approval for a small number of products.

**1.2 The therapeutic properties of MSCs**

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

Exemplary products ALOFISEL Queencell

Cell source Adipose tissue Adipose tissue

modulation

batch)

Product storage Frozen, off-the-shelf No storage

Batch size Large (min. 100–1000 doses per

*0.003% of all citizens (741 million) in Europe are putative patients.*

*Indication and properties of MSC products impact their manufacturing.*

Patients per year 23,000 (in EU)\* n.d.

**Product 1 Product 2**

MSCs 7 × 107\*\*

tissue

MSCs

parallel

Regeneration, anti-apoptosis

Small (1 dose per batch)

Indication Crohn's disease, perianal fistula Regeneration of subcutaneous

Cell type Allogenic MSCs Autologous, patient-specific

Anti-inflammation, immune

Scalability of production Scale up Scale out, several batches in

Stability under storage Stable >6 month, frozen Fresh, stable max. 24 hours

*\*\*Stromal vascular fraction contains MSCs and other cell types such as preadipocytes, endothelial progenitor cells,* 

*\*\*\*Following both products have different critical quality attributes (CQAs) and the manufacturing processes have* 

Manufacturing type Bulk manufacturing Patient-specific batch

stable over 7 years of follow-up studies [12]. Several autologous MSC products have also been approved in South Korea, meaning that the MSCs are isolated from the patient's own tissue and then manipulated/expanded in a patented process and re-injected into the patient 4–6 weeks later. NEURONATA-R (Corestem) and Cellgram-AMI (Pharmicell) are autologous bone marrow-derived MSCs indicated for amyotrophic lateral sclerosis and acute myocardial infarction, respectively. Two other MSC products derived from adipose-tissue have been approved (Anterogen): a mixture of autologous adipose-derived MSCs with other cells for subcutaneous tissue defects (Queencell) and a pure adipose-derived MSC product for Crohn's fistula treatment (Cupistem) [9]. NEURONATA-R has been designated as an orphan

This brief survey of the market shows that the promise of MSC therapy is materializing, with positive efficacy data in controlled clinical trials followed by

Although MSCs have been used in cell therapy applications for many years, the fundamental biology of these cells and their precise therapeutic properties are not fully understood. MSCs were initially isolated from bone marrow (bm-MSCs) based on their plastic adherence, but today they are usually isolated from adipose tissue (ad-MSCs) or umbilical cord blood (uc-MSCs), which are more accessible [13]. MSCs are also found in various other adult, fetal, and perinatal tissues [14]. Regardless of their origin, MSCs are heterogeneous and polyclonal cells, with at least three

*DOI: http://dx.doi.org/10.5772/intechopen.90029*

Cells per dose 1.2 × 108

Therapeutic relevant cell

*pericytes, mast cells, and fibroblast.*

*n.d. not determined.*

*different critical process parameters (CPPs).*

properties\*\*\*

*\**

**Table 1.**

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products DOI: http://dx.doi.org/10.5772/intechopen.90029*


*\* 0.003% of all citizens (741 million) in Europe are putative patients.*

*\*\*Stromal vascular fraction contains MSCs and other cell types such as preadipocytes, endothelial progenitor cells, pericytes, mast cells, and fibroblast.*

*\*\*\*Following both products have different critical quality attributes (CQAs) and the manufacturing processes have different critical process parameters (CPPs).*
