*n.d. not determined.*

#### **Table 1.**

*New Advances on Fermentation Processes*

requires a well-characterized manufacturing process.

the specialized and complex nature of MSCs [4].

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

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

In order to provide enough MSCs for cell therapy, donor cells must be isolated

cells,

from tissue and then expanded *in vitro* to reach a population of 1–9 × 108

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

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

**204**

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

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 drug by the EMA and FDA.

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 regulatory approval for a small number of products.

#### **1.2 The therapeutic properties of MSCs**

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

subpopulations defined based on morphology. Type I MSCs are spindle-shaped proliferating cells resembling fibroblasts. Type II MSCs are large, flat, epithelial-like cells, which are more senescent than type I cells and feature visible cytoskeletal structures and granules. Finally, type III MSCs are small round cells with a high capacity for self-renewal [15]. The heterogeneity of MSCs can be considered beneficial in that it ensures that some therapeutically active cells are present, but it reduces the maximum potential efficacy because some of the cells are inactive. However, even monoclonal MSCs become heterogeneous during expansion [16].

Despite the heterogeneity described above, the International Society of Cell Therapy has published a set of minimal criteria that must be met before cells can be defined as MSCs. Such cells must (i) show plastic adherence; (ii) be able to differentiate into cartilage, bone, and fat tissue *in vitro*; and (iii) express the cluster of differentiation (CD) surface markers CD73, CD90, and CD105, but not CD11b, CD14, CD19, CD34, CD45, or HLA-DR [17]. However, this standard set of markers does not distinguish between MSCs and fibroblasts or nonstem mesenchymal cells [18]. Several other markers may be more specific but are only detected in certain MSC isolates or subpopulations. These include stage-specific embryonic antigen-4 (SSEA-4), stem cell antigen-1 (SCA1), nestin, CD44, CD146, CD166, and CD271 [19]. A unique MSC surface marker has yet to be identified.

It is important to note that MSCs cannot be defined merely as a collection of surface markers because this says nothing about their therapeutic effect (**Figure 1**). Initially, the therapeutic potential of MSCs was believed to reflect their ability to migrate into damaged tissues, differentiate *in situ*, and replace damaged or dead cells. However, although MSCs can differentiate *in vitro*, their ability to differentiate *in viv*o has never been confirmed [20]. Current opinion is that MSCs migrate to injury sites and secrete chemoattractants that recruit tissue-specific stem cells,

#### **Figure 1.**

*Properties of MSCs and their mode of action. MSCs modulate the host immune systems, e.g., by secreting various trophic factors. Thereby, they reduce inflammation, promote neoangiogenesis, and prevent apoptosis and fibrosis. Further, they stimulate local stem cells to develop new tissue. TSG-6, tumor necrosis factorinducible gene 6 protein also known as TNF-stimulated gene 6 protein; STC1, stanniocalcin 1; IL-4/6/10, interleukins 4, 6 and 10; CCL20, macrophage inflammatory protein-3; IDO, indoleamine 2,3-dioxygenase; PGE2, prostaglandin E2; VEGF, vascular endothelial growth factor; FGF-2, basic fibroblast growth factor; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor 1; CXCL12, stromal cell-derived factor 1; MMP1/2/9, matrix metalloproteinase-1/2/9.*

**207**

*1.3.1 Identity*

*1.3.2 Potency*

in the identity attributes.

*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

without evidence of transformation into tumor cells [23].

**1.3 The critical quality attributes of MSCs**

dependent on the therapeutic indication.

which in turn generate new tissues or exert positive immunomodulatory effects [1]. The MSC secretome comprises a pool of cytokines, chemokines, growth factors, and extracellular vesicles (carrying proteins, lipids, and various forms of RNA). This secretome differs widely among MSC isolates and subpopulations and can be used to functionally distinguish between several MSC types (e.g., type I, II, and III cells), revealing that the self-renewable type III cells are therapeutically the most

The immunomodulatory properties of MSCs and their secretion of antiinflammatory molecules and extracellular vesicles are an important therapeutic functionality [14]. MSCs are therefore logical candidates for the treatment of immune disorders, including GvHD, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and diabetes [21]. MSCs also secrete peptides and factors that promote the regeneration of damaged tissue by stimulating cell proliferation and migration, promoting angiogenesis, and suppressing apoptosis and fibrosis [14]. The regenerative capacity of MSCs has been used to treat Alzheimer's disease, bone and cartilage diseases, diabetes, myocardial infarction, and osteoarthritis [22]. Another advantage of MSCs is that they do not form teratomas *in vivo*, which ensures an outstanding clinical safety profile. Human MSCs achieve senescence

The biological complexity and heterogeneity of MSCs hamper the translation of laboratory-scale experiments into industrial processes for cost-effective and reliable manufacturing. This can be addressed by developing MSC manufacturing processes that adhere to quality-by-design (QbD) principles [24]. QbD provides a rational framework and integrates scientific knowledge and risk analysis into process development. It is guided by a thorough understanding of the fundamental biology and engineering principles underlying an MSC product and its production process. QbD begins with a description of the desired product quality characteristics, known as the quality target product profile (QTPP). This is used to identify critical quality attributes (CQAs), which are physical, chemical, and biological attributes that define the quality of the product. The QTPP for MSCs describes properties such as identity, purity, and potency, which will be unique for each MSC product and

For MSCs, identity often means the cell phenotype, but as discussed above, there is no agreement on a single definition. Identity is often demonstrated by confirming a typical morphology and/or karyotype [25] and by detecting the presence or absence of surface markers. The minimal criteria for MSCs (see above) have led to a misconception that cells meeting these criteria are equivalent in identity and therapeutic functionality. In polyclonal MSC populations, the presence of multiple cell types can be a clinical benefit as stated above [26], and this should be reflected

The functionality and potency of MSCs are closely linked to their therapeutic efficacy and thus the clinical outcome, but potency is used to demonstrate manufacturing consistency for batch release so a measurable property is required. Viability can fulfill the role of a potency indicator because only living cells can act as a therapeutic

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

effective [16].

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

which in turn generate new tissues or exert positive immunomodulatory effects [1]. The MSC secretome comprises a pool of cytokines, chemokines, growth factors, and extracellular vesicles (carrying proteins, lipids, and various forms of RNA). This secretome differs widely among MSC isolates and subpopulations and can be used to functionally distinguish between several MSC types (e.g., type I, II, and III cells), revealing that the self-renewable type III cells are therapeutically the most effective [16].

The immunomodulatory properties of MSCs and their secretion of antiinflammatory molecules and extracellular vesicles are an important therapeutic functionality [14]. MSCs are therefore logical candidates for the treatment of immune disorders, including GvHD, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and diabetes [21]. MSCs also secrete peptides and factors that promote the regeneration of damaged tissue by stimulating cell proliferation and migration, promoting angiogenesis, and suppressing apoptosis and fibrosis [14]. The regenerative capacity of MSCs has been used to treat Alzheimer's disease, bone and cartilage diseases, diabetes, myocardial infarction, and osteoarthritis [22]. Another advantage of MSCs is that they do not form teratomas *in vivo*, which ensures an outstanding clinical safety profile. Human MSCs achieve senescence without evidence of transformation into tumor cells [23].
