**4. Harnessing cell-free therapeutics' variability to improve their safety and efficacy**

### **4.1. Age-related variability of MSC properties**

Since MSCs are used as a starting material for CM manufacturing, it is necessary to pay attention to the standardization of MSC. The effects of this biopharmaceutical are multifactorial, and even the minimal variability of its composition can strongly affect its activity. As properties of MSC influence CM composition crucially, the variability of MSC donor characteristics and manipulations during manufacturing should be taken into account.

more frequent in BM-MSC preparations from female donors. What is more important, no correlation of donor age to adipogenic, osteogenic, and chondrogenic differentiation in vitro as confirmed by an extended panel of lineage specific markers was found. In addition, the secretion rate of molecules important for tissue regeneration and immunomodulation was analyzed. The evidence of a critical role of factors such as VEGF, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), and angiopoietin-1 (Angpt-1) in contribution to male and female MSC variability was lacking [21]. As MSC-secreted factors are principal active components of MSC CM, donor sex might not influence MSC CM efficacy substantially. However, in vitro data concerning the impact of donor

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sex to variability of donor-derived MSC effects are controversial [22].

and in vivo if transferred in high amounts [7, 23].

**4.4. Variability determined by manufacture process**

expansion approach as well as the use of proper raw materials.

for certain pathology.

**4.3. Variability determined by the origin of starting material for MSC isolation**

MSC isolated from the most common sources can fit minimal characteristic criteria and share majority of biological properties. However, some studies demonstrated that MSCs from the popular sources such as adipose tissue and bone marrow have different transcription profiles. Among differentially expressing or expressing by only one cell type, one can find components involved in cellular communication and differentiation. Therefore, the secretory profile of different MSC types also varies. In addition, not only a spectrum of secreted paracrine factors differs but also nucleic acid, particularly microRNA composition, which might influence EV contents. The variability between EV of two types of MSC suggests that prior tissue-specific microenvironment might influence the exosomal sorting of micro-RNA. It is important as micro-RNA transferred via exosomes might be functional in repressing their targets in vitro

Taken together a plethora of donor-associated factors can influence MSC CM composition and effects. Thus to enhance the effect of this biopharmaceutical and harness lot-to-lot variability one can isolate MSC from selected donors that are optimal for MSC CM manufacturing

Manufacturing features can also substantially affect the safety and efficacy of MSC CM.Among them, one can name at least optimal cell isolation protocol, growth medium selection, and cell

Although isolated by the same way, MSCs often demonstrate different properties when cultured in different media. The choice of medium determines the potential of MSC for adhesion, growth, and also for maintaining a population of clonogenic cells: both a basal medium and a supplement influence these parameters. It is important to note that ready-to-use commercial media fit to MSC obtained from various sources differently. Media can cause negative effects up to early cell aging or termination of growth in the earliest passages for certain MSC type. In addition, some cell culture media can be positively selective for specific MSC subpopulations. This feature is important as the composition of MSC population might affect regenerative potential of MSC [24, 25]. The media can also influence MSC CM composition significantly.

One of the key factors affecting MSC CM composition and therapeutic effects is a donor's age. It is generally accepted that stem cell number and/or function decline with advanced age during the replacement and the turnover of damaged cells in compromised renewable tissues. Several studies showed that MSCs derived from old donors exhibit reduced proliferative capacity, differentiation potential, and, most importantly, impaired specific activity [15]. Older adipose-derived MSC showed impaired angiogenic properties in vitro and in vivo. The production of key pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and others by adipose-derived MSC, decreased with age [16]. It is important to note that factors involved in ECM remodeling like urokinase and its receptor, plasminogen activator inhibitor-1 (PAI-1), metalloproteinases 2 and 9 were overexpressed in adipose MSC from aged donors compared to younger ones, both in human and in mice [16, 17].

Furthermore, it was shown that age and age-associated conditions such as type 2 diabetes and atherosclerosis decrease the immunomodulatory capacity of human MSCs. MSCs from elderly subjects with atherosclerosis have impaired T-cell suppression compared to their nonelderly adult counterparts. Possibly, the coexistence of age and these age-related conditions could compromise MSC immunomodulatory function [18] and influence MSC CM functional properties.

Bioinformatic data also suggest that age-related changes in MSCs result in impaired therapeutic potential of aged progenitor cells [19]. In one of the studies among the most highly differentially expressed genes, transforming growth factor alpha (TGFα) overexpression in MSC from young donors was detected. TGFα has been shown to mediate the secretion of vascular endothelial growth factor A (VEGFA) by MSC, positively contributing to processes like wound healing and injury response [20]. Some other differentially expressed genes participate in MSC differentiation. As it is revealed that during early MSC differentiation, secretome of MSC can be changed, one should take into account age-related differentiation predisposition.

#### **4.2. Sex-related variation of MSC paracrine activity**

In an attempt to overcome MSC donor variation, one can explore donor sex variability. Analysis of human BM-MSC preparations to identify statistically robust influence of donor sex on MSC functional properties identified minor differences among MSC isolated from both the sexes. It was shown that high-clonogenic BM-MSCs divided more rapidly and were more frequent in BM-MSC preparations from female donors. What is more important, no correlation of donor age to adipogenic, osteogenic, and chondrogenic differentiation in vitro as confirmed by an extended panel of lineage specific markers was found. In addition, the secretion rate of molecules important for tissue regeneration and immunomodulation was analyzed. The evidence of a critical role of factors such as VEGF, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), and angiopoietin-1 (Angpt-1) in contribution to male and female MSC variability was lacking [21]. As MSC-secreted factors are principal active components of MSC CM, donor sex might not influence MSC CM efficacy substantially. However, in vitro data concerning the impact of donor sex to variability of donor-derived MSC effects are controversial [22].

#### **4.3. Variability determined by the origin of starting material for MSC isolation**

MSC isolated from the most common sources can fit minimal characteristic criteria and share majority of biological properties. However, some studies demonstrated that MSCs from the popular sources such as adipose tissue and bone marrow have different transcription profiles. Among differentially expressing or expressing by only one cell type, one can find components involved in cellular communication and differentiation. Therefore, the secretory profile of different MSC types also varies. In addition, not only a spectrum of secreted paracrine factors differs but also nucleic acid, particularly microRNA composition, which might influence EV contents. The variability between EV of two types of MSC suggests that prior tissue-specific microenvironment might influence the exosomal sorting of micro-RNA. It is important as micro-RNA transferred via exosomes might be functional in repressing their targets in vitro and in vivo if transferred in high amounts [7, 23].

Taken together a plethora of donor-associated factors can influence MSC CM composition and effects. Thus to enhance the effect of this biopharmaceutical and harness lot-to-lot variability one can isolate MSC from selected donors that are optimal for MSC CM manufacturing for certain pathology.

#### **4.4. Variability determined by manufacture process**

**4. Harnessing cell-free therapeutics' variability to improve their** 

and manipulations during manufacturing should be taken into account.

Since MSCs are used as a starting material for CM manufacturing, it is necessary to pay attention to the standardization of MSC. The effects of this biopharmaceutical are multifactorial, and even the minimal variability of its composition can strongly affect its activity. As properties of MSC influence CM composition crucially, the variability of MSC donor characteristics

One of the key factors affecting MSC CM composition and therapeutic effects is a donor's age. It is generally accepted that stem cell number and/or function decline with advanced age during the replacement and the turnover of damaged cells in compromised renewable tissues. Several studies showed that MSCs derived from old donors exhibit reduced proliferative capacity, differentiation potential, and, most importantly, impaired specific activity [15]. Older adipose-derived MSC showed impaired angiogenic properties in vitro and in vivo. The production of key pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and others by adipose-derived MSC, decreased with age [16]. It is important to note that factors involved in ECM remodeling like urokinase and its receptor, plasminogen activator inhibitor-1 (PAI-1), metalloproteinases 2 and 9 were overexpressed in adipose MSC from aged donors compared to younger ones, both in human and in mice [16, 17]. Furthermore, it was shown that age and age-associated conditions such as type 2 diabetes and atherosclerosis decrease the immunomodulatory capacity of human MSCs. MSCs from elderly subjects with atherosclerosis have impaired T-cell suppression compared to their nonelderly adult counterparts. Possibly, the coexistence of age and these age-related conditions could compromise MSC immunomodulatory function [18] and influence MSC CM functional

Bioinformatic data also suggest that age-related changes in MSCs result in impaired therapeutic potential of aged progenitor cells [19]. In one of the studies among the most highly differentially expressed genes, transforming growth factor alpha (TGFα) overexpression in MSC from young donors was detected. TGFα has been shown to mediate the secretion of vascular endothelial growth factor A (VEGFA) by MSC, positively contributing to processes like wound healing and injury response [20]. Some other differentially expressed genes participate in MSC differentiation. As it is revealed that during early MSC differentiation, secretome of MSC can be changed, one should take into account age-related differentiation predisposition.

In an attempt to overcome MSC donor variation, one can explore donor sex variability. Analysis of human BM-MSC preparations to identify statistically robust influence of donor sex on MSC functional properties identified minor differences among MSC isolated from both the sexes. It was shown that high-clonogenic BM-MSCs divided more rapidly and were

**safety and efficacy**

50 Biopharmaceuticals

properties.

**4.1. Age-related variability of MSC properties**

**4.2. Sex-related variation of MSC paracrine activity**

Manufacturing features can also substantially affect the safety and efficacy of MSC CM.Among them, one can name at least optimal cell isolation protocol, growth medium selection, and cell expansion approach as well as the use of proper raw materials.

Although isolated by the same way, MSCs often demonstrate different properties when cultured in different media. The choice of medium determines the potential of MSC for adhesion, growth, and also for maintaining a population of clonogenic cells: both a basal medium and a supplement influence these parameters. It is important to note that ready-to-use commercial media fit to MSC obtained from various sources differently. Media can cause negative effects up to early cell aging or termination of growth in the earliest passages for certain MSC type. In addition, some cell culture media can be positively selective for specific MSC subpopulations. This feature is important as the composition of MSC population might affect regenerative potential of MSC [24, 25]. The media can also influence MSC CM composition significantly. We observed a substantial difference between several growth factor concentrations in MSC CM manufactured with two different media. Importantly, the variability of factor concentrations between two MSC CM reflected on their potency in vitro [26]. The presence or absence of a certain biologically active component in the culture medium can also affect the function of MSC. The addition of FGF2 to bone marrow MSC culture medium influences the expression of some membrane proteins, which contributes to morphology and differentiation potential changes [27]. Another challenging feature is an impact of starting material or sample processing during CM preparation. Such a routine procedure as washing cells with PBS can change the secretion profile of cells dramatically. Thus, the attention to auxiliary component and adequate selection of them are necessary [28].

for determining the drug efficacy. At the registration stage, the presence of a clearly defined potency test is required by regulators. An established and valid potency test is required for drug manufacturing to check how scaling affects drug's therapeutic properties as well as the in-process testing for checking production lines as a factor that actually determines the lot release capability. The choice of a potency test should also be economically feasible. A potency test has to be quick and unambiguous in the context of drug efficacy determination

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It should be emphasized that the development of an appropriate technique for potency tests of biopharmaceuticals based on cells and their secreted products is complicated (**Table 1**). In particular, the recently released "Alofisel" product faced the greatest difficulties at registration stage due to justifying the choice of potency test. "Early in the procedure a major objection was raised in relation to the potency assay. During the procedure the applicant provided additional data to support the suitability of the potency assay and the major" [31]. However, the parties managed to come to a common opinion on this issue, and the very first recommendation from the EMA was "The Applicant will undertake to review the data generated for the potency assay from clinical experience after suitable experience has been generated and to follow any recommendation that is issued following the review of the data assessment report

Here, we will not dwell on the complexities of developing a potency test for cellular products, including those based on MSC; however, we note that FDA and EMA have at least a clear classification and regulatory framework for this category of therapeutics [32, 33]. At the same time, biopharmaceuticals based on MSC CM have not been classified by these agencies. Moreover, since the general rule for pharmaceutical certification is the presence of a defined, clearly characterized active substance, further progress in the development and registration of this category of therapeutics is bound to attention from regulatory agencies. According to many experts' opinions working in the field of MSC CM-based drug development, the most correct way is the approval of a new group of drugs called "cell-free therapeutics" with a less strict attitude to the issue of the multicomponent active substance [34]. The main obstacle for establishing the adequate potency tests for such biopharmaceuticals is an elusive nature of their mechanism of action (MOA) as well as the challenging choice of one active components between multiple cell-secreted factors. Nevertheless, the factors from CM composition might be isolated as single ones and classified as biological products. Therefore, it seems reasonable to apply the similar standards for characterization, safety, and dosage testing as well as potency evaluation for both the biologicals and CM, at least with any exceptions. Similar to biological drugs, potency test can be used for dose clarification from lot to lot. For complex biological medicinal products that cannot be fully characterized by physicochemical means, the established concept is to assign potency in units of biological activity based on the use of an international standard for biological activity. The units of biological activity are mostly traced back to an internationally adopted reference preparation (International Standard, IS). The quantitative composition and dosing recommendation of biological medicinal products for which an IS exists are expressed in international units (IUs) [35]. IS analogs could also be

**5.2. Development of potency tests for CM-based biopharmaceuticals**

at the earliest stages of lot release.

objection is considered to be resolved."

Apart from materials used for MSC expansion and isolation, we might note the selection of cell culture approaches as a substantial factor affecting the variability of MSC functional properties. The use of bioreactors has been suggested as a promising alternative to conventional static culture flasks for MSC expansion. The advantage of 3D cultivation is more complete modeling of the natural microenvironment of MSC, which allows to retain the proliferation and differentiation potential of MSC for longer time. Besides, selected cell culture method can affect the secretion profile of MSC directly. Three-dimensional growth of bone marrow MSC culture influenced the expression of such factors as pigment epithelium-derived factor (PEDF), Galectin-1, brain-derived neurotrophic factor (BDNF), VEGF, nerve growth factor (NGF), insulin-like growth factor 1 (IGF-1), and miR-16, which are considered to be important regulators/modulators of the neurogenic and neural differentiation processes. Using CM from 3D cultured MSC induced the differentiation of a significantly higher number of human neural progenitors into neurons at different stages of maturation compared with human MSC secretome collected under 2D conditions [29].
