**1.3 The critical quality attributes of MSCs**

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 dependent on the therapeutic indication.

## *1.3.1 Identity*

*New Advances on Fermentation Processes*

MSCs become heterogeneous during expansion [16].

[19]. A unique MSC surface marker has yet to be identified.

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

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

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,

*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*

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*factor 1; MMP1/2/9, matrix metalloproteinase-1/2/9.*

**Figure 1.**

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 in the identity attributes.

## *1.3.2 Potency*

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

entity. Potency can also be measured using *in vitro* functional assays that determine MSC activity directly or via an indirect metric that correlates to MSC activity *in vivo*. An assay that measures differentiation potential is only appropriate to describe MSC potency if the therapeutic aim involves engraftment of the cells or tissue formation (notwithstanding the controversy over the assumption that MSCs differentiate *in vivo*, as discussed above). The FDA mandates that potency is measured using quantitative biological assays [27], so the standard approach is to differentiate MSCs *in vitro* by cultivating them in differentiation medium and then testing them after 21 days [17]. Staining for differentiation markers is nonquantitative, so alternative methods such as postdifferentiation RNA or protein analysis [28, 29], or the online monitoring of differentiation by Raman spectroscopy [30], are more suitable.

If the therapeutic effect of MSCs is conferred by the secretome, then the differentiation potential may not be the primary determinant of potency. The profile of secreted factors would be a more appropriate measure, and this could be determined by multiplex enzyme-linked immunosorbent assays (ELISAs) or mass spectroscopy [31]. However, a clear link between the secretome profile and *in vivo* efficacy must be established, so that animal models or cell-based assays can be used to determine the limits of the relevant factors. This is a typical way to move from a complex and highly variable *in vivo* assay to a multiassay approach combining the quantification of viability, target-specific cytotoxicity or cytokine release, surrogate biomarkers (morphological phenotype or released factors that correlate with function), bioactivity (e.g., presentation of surface markers), cell-based assays, and genomic, transcriptomic, and proteomic profiles [32].

#### *1.3.3 Sterility and purity*

Impurities are unwanted components from within the process, whereas contaminants come from outside the process. Impurities during MSC manufacturing include unwanted cell types, particles (e.g., residual microcarriers, or plastics and fibers from manufacturing equipment and materials), or components of culture medium. Contaminants include bacteria, fungi, viruses, endotoxins, and mycoplasma. The heterogeneity of MSCs makes it difficult to detect unwanted cell types. MSC preparations should ideally be pure, but fibroblasts are often present as impurities. Cell-specific sorting based on the marker CD166 (which is expressed at higher levels on MSCs) and CD9 (which is expressed at higher levels on fibroblasts) may help to achieve sufficient purity [33]. In other cases, it may be sufficient if most of the cells in the final product (>98%) fulfill the ISCT minimal criteria based on MSC surface markers. All other impurities and contaminants must be measured and the maximum residual levels must be defined to ensure safety and efficacy. A final sterilization step is not possible when the product is living cells, so the entire MSC production process must be carried out under aseptic conditions.

From the QTPP list, CQAs must be identified, which directly influence the safety and efficacy of the MSC product. This means that a risk assessment is carried out to reduce the QTPP list to the most influential attributes based on impact and certainty. According to ICHQ8, a CQA is "*A physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.*" Therefore, every process parameter "*whose variability has an impact on a CQA"* is a critical process parameter (CPP) that "*should be monitored or controlled to ensure that the process produces the desired quality."* There is no precise delimitation of the degree of impact required to define a CPP, so the broad definition of a CPP is generally divided into parameters that have a substantial impact on the CQAs and those with minimal or zero impact. Each process step has multiple CPPs. For example, during the *in vitro* expansion

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*Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products*

Therapeutic applications of MSCs require at least 1 × 108

CPPs affecting MSC quality are discussed in more detail below.

step, CPPs can be directly associated with the MSCs (e.g., cell density and cell age) or raw material attributes (e.g., medium, serum, and growth factors) or operational features of the culture vessel/bioreactor system (e.g., pH, temperature, dissolved oxygen, and agitation). The effect of each CPP on the CQAs must be quantified in a design space. With an appropriate control strategy, the CPPs are kept in their normal operational range, which ensures the production of high-quality MSCs that meet all the required CQAs. Based on the heterogeneity and the complexity of MSCs, each MSC product can have unique CQAs and the corresponding CPPs must

many more than can be isolated by tissue aspiration. All MSC production processes must therefore include an *in vitro* expansion. Having generated or isolated the starting cell population, *in vitro* expansion is followed by harvest, concentration, purification, formulation, fill and finish, storage, and shipping. The manufacturing steps of MSCs are therefore similar to the production of recombinant proteins, but MSCs are more challenging due to the variability of the starting material, the complexity of living cells as a product, an incomplete understanding of their mechanism of action, and the inherent difficulties encountered during product characterization.

The properties of MSCs are strongly influenced by the environment because MSCs in nature interact with surrounding cells and tissues, with the extracellular matrix and with various bioactive molecules. Even in an artificial environment like a bioreactor or T-flask, MSCs are very sensitive to their environment, and the most influential factors give rise to CPPs. By identifying CPPs that affect MSC quality, the process can be designed to favor the recovery of MSCs with specific phenotypes of interest, in this case those with the greatest therapeutic efficacy [34, 35]. The

During MSC isolation, the seeding density is important because all sources contain different quantities of MSCs. For example, only 1 in 100,000 bone marrow cells is an MSC, whereas in adipose tissue, the ratio is nearer to 1 in 100 [36]. If plastic adherence is selected as a strategy for MSC isolation, the number of adherent cells therefore differs according to the source if a similar number of tissue cells are seeded. Standardization during this step can be achieved by isolating MSCs using a strategy of surface marker sorting, allowing a defined number of cells to be seeded into the culture vessel. The seeding density selected for the *in vitro* expansion step

is a CPP. MSCs can be seeded at a very low density (50–100 cells per cm2

proliferate until they achieve confluence. This corresponds to a high expansion factor, but the process takes a long time and requires more rounds of cell division for each seeded cell, so the cells experience significant aging [37]. The aging of MSCs during expansion is a problem, because older cells lose competence to behave as stem cells and have a tendency to enter senescence or even to undergo transformation. The manufacturing of Prochymal provided a clear example of this issue: 10,000 or more doses were manufactured from one donor, and the corresponding expansion stress led to replicative senescence, in which the cells retained a typical

cells per dose, which is

) and will

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

**2. Expansion of human MSCs** *in vitro*

**2.1 CPPs that affect MSC manufacturing**

*2.1.1 Cell density and age*

be identified case by case.

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

step, CPPs can be directly associated with the MSCs (e.g., cell density and cell age) or raw material attributes (e.g., medium, serum, and growth factors) or operational features of the culture vessel/bioreactor system (e.g., pH, temperature, dissolved oxygen, and agitation). The effect of each CPP on the CQAs must be quantified in a design space. With an appropriate control strategy, the CPPs are kept in their normal operational range, which ensures the production of high-quality MSCs that meet all the required CQAs. Based on the heterogeneity and the complexity of MSCs, each MSC product can have unique CQAs and the corresponding CPPs must be identified case by case.