**5. Developing approaches to assess the potency of cell-free therapeutics**

#### **5.1. Legal aspects of potency tests development**

The efficacy of a drug means a biological response caused by this drug in a certain dose. As part of a drug development, clinical research, and certified manufacturing, there is a need to develop and apply a standardized rapid method for assessing the potential efficacy of the drug. This technique is called a potency test. By definition of International Conference on Harmonization (ICH) [30], potency is the quantitative measure of biological activity based on the attribute of the product, which is linked to the relevant biological properties. The assay demonstrating the biological activity should be based on the intended biological effect which should ideally be related to the clinical response. This definition has been implemented by Food and Drug Administration (FDA) and European Medicines Agency (EMA).

At the step of research and development, the availability of an approved potency test ensures consistency among the results obtained by different scientific groups. While conducting clinical trials, postulating a presumptive potency test is necessary as one of the evaluated factors 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 at the earliest stages of lot release.

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

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

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

adequate selection of them are necessary [28].

secretome collected under 2D conditions [29].

**5.1. Legal aspects of potency tests development**

**therapeutics**

52 Biopharmaceuticals

**5. Developing approaches to assess the potency of cell-free** 

Food and Drug Administration (FDA) and European Medicines Agency (EMA).

The efficacy of a drug means a biological response caused by this drug in a certain dose. As part of a drug development, clinical research, and certified manufacturing, there is a need to develop and apply a standardized rapid method for assessing the potential efficacy of the drug. This technique is called a potency test. By definition of International Conference on Harmonization (ICH) [30], potency is the quantitative measure of biological activity based on the attribute of the product, which is linked to the relevant biological properties. The assay demonstrating the biological activity should be based on the intended biological effect which should ideally be related to the clinical response. This definition has been implemented by

At the step of research and development, the availability of an approved potency test ensures consistency among the results obtained by different scientific groups. While conducting clinical trials, postulating a presumptive potency test is necessary as one of the evaluated factors 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 objection is considered to be resolved."

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


activity and also could be used as a potency test for MSC CM-based biopharmaceutical with

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Thus, the new category of drugs to which MSC CM will belong should be based on its definitions at the junction of two existing regulating categories: biomedical drugs and CGT/ATMP.

Another complexity of creating and validating the potency test for MSC CM is the heterogeneity of the product from batch to batch. Many factors such as heterogeneity of donors, in vitro cell population during cultivation, and soluble factors that MSC can secrete under different conditions influence this phenomenon. In addition, one might develop rules for this biopharmaceutical by partial borrowing of available legal documents for preparations based on blood plasma [36] as these drugs are also highly heterogeneous, have uncertain active components, and their potency is difficult to be assessed. However, the most important hurdle in a potency test development is a variety of biological activities of factors secreted by MSC. Hence, there is a diversity of MOA, where extent and type depends on the area of application of MSC CM-based biopharmaceutical. For example, the angiogenic properties of MSC secretome are mediated by well-known pro-angiogenic factors; however, some of them may have another action. In addition, it is necessary to achieve the pleiotropic action of MSC CM, since many

It is also worth considering that in some cases, specific effects of MSC CM are contraindicated. For example, angiogenic effects necessary for the restoration of ischemic tissues may promote tumor development. Thus, the creation of a universal method for developing a potency test of MSC CM is questionable. The selection of a method for testing this substance should be

One can suggest the following mechanism for selection, testing, and validating the potency test for MSC CM (**Figure 1**). First, based on pathology nature, the most potentially effective MOA should be chosen. It is important to note that potency tests are available for the majority of MOA, and it might be possible to apply them for CM potency testing with minimal modifications.

Since there is no "gold standard" or sufficiently defined regulations in this field, developers of MSC CM-based therapeutics use a variety of approaches for determining the potency. To evaluate the immunomodulatory effect of MSC-secreted EV, an in vitro test based on a dose-dependent inhibition by vesicles of the proliferation of phytohemagglutinin-activated T lymphocytes was successfully used [38]. For the surrogate analysis of the immunomodulatory activity of MSC CM in the model of inflammatory organ failure, the effect of MSC CM on bacterial LPS-activated PBMC was tested. The degree of modulation correlated well with the level of IL-10 secreted by PBMC in this experimental model [39]. The subsidiary company of SteMedica, StemProtein, has successfully used two potency tests for its unique product, human fibroblast secretome dried by proprietary technology "Preservation by Vaporization." Because tissue regeneration critically depends on adequate blood supply, they used in vitro angiogenesis assays to evaluate the therapeutic potency of stem cell factors. They routinely monitored the concentrations of VEGF, HGF, interleukin 6 (IL-6), chemokine C-C motif ligand 2 (CCL2), C-X-C motif chemokine 5 (CXCL5),

different mechanisms are involved in the regeneration of injuries.

**5.4. Overcoming existing challenges in potency testing**

similar indications.

disease-focused [37].

Abbreviations: PT, potency test; MOA, mode of action; CM, conditioned medium; R&D, research and development; cGMP, current good manufacturing practice; GTP, [current] good tissue practice; cGLP, current good laboratory practice; EMA, European Medicines Agency (European Union); FDA, Food and Drug Administration (USA); CGT, cellular and gene therapy; ATMP, advanced therapy medicinal product.

**Table 1.** Key challenges in the development of valid PT for CM-based cell-free therapeutics and possible ways to overcome them.

developed for the biopharmaceuticals derived from MSC CM. An important advantage of this approach is applicability of the direct rapid potency tests as well as the use of surrogate analytical and instrumental assays.

#### **5.3. Challenges in the development of potency tests for CM-based products**

However, MSC CM-based biopharmaceuticals represent not only a mixture of defined bioactive factors but a complex of multiple components produced by a specific type of cells. MSC CM mimics the beneficial effects of MSC cell therapy, and potency tests applied for corresponding cell and gene therapy products/advanced therapy medicinal products (CGT/ ATMP) could also be relevant. For example, the measure of a defined cytokine like IL-10 in MSC secretome may serve as a potency test for a cell-based product with immunosuppressive activity and also could be used as a potency test for MSC CM-based biopharmaceutical with similar indications.

Thus, the new category of drugs to which MSC CM will belong should be based on its definitions at the junction of two existing regulating categories: biomedical drugs and CGT/ATMP.

Another complexity of creating and validating the potency test for MSC CM is the heterogeneity of the product from batch to batch. Many factors such as heterogeneity of donors, in vitro cell population during cultivation, and soluble factors that MSC can secrete under different conditions influence this phenomenon. In addition, one might develop rules for this biopharmaceutical by partial borrowing of available legal documents for preparations based on blood plasma [36] as these drugs are also highly heterogeneous, have uncertain active components, and their potency is difficult to be assessed. However, the most important hurdle in a potency test development is a variety of biological activities of factors secreted by MSC. Hence, there is a diversity of MOA, where extent and type depends on the area of application of MSC CM-based biopharmaceutical. For example, the angiogenic properties of MSC secretome are mediated by well-known pro-angiogenic factors; however, some of them may have another action. In addition, it is necessary to achieve the pleiotropic action of MSC CM, since many different mechanisms are involved in the regeneration of injuries.

It is also worth considering that in some cases, specific effects of MSC CM are contraindicated. For example, angiogenic effects necessary for the restoration of ischemic tissues may promote tumor development. Thus, the creation of a universal method for developing a potency test of MSC CM is questionable. The selection of a method for testing this substance should be disease-focused [37].

One can suggest the following mechanism for selection, testing, and validating the potency test for MSC CM (**Figure 1**). First, based on pathology nature, the most potentially effective MOA should be chosen. It is important to note that potency tests are available for the majority of MOA, and it might be possible to apply them for CM potency testing with minimal modifications.

#### **5.4. Overcoming existing challenges in potency testing**

developed for the biopharmaceuticals derived from MSC CM. An important advantage of this approach is applicability of the direct rapid potency tests as well as the use of surrogate

**Table 1.** Key challenges in the development of valid PT for CM-based cell-free therapeutics and possible ways to

Abbreviations: PT, potency test; MOA, mode of action; CM, conditioned medium; R&D, research and development; cGMP, current good manufacturing practice; GTP, [current] good tissue practice; cGLP, current good laboratory practice; EMA, European Medicines Agency (European Union); FDA, Food and Drug Administration (USA); CGT, cellular and

**Possible strategies to overcome**

Pleiotropic mode of action 1. One should choose the main mode of action (MOA) depending on pathological process.

preferred MOA as accurate as possible. Complex active substance 1. CM composition and its batch-to-batch variability should be carefully

"Batch-to-batch variability" 1. Complex of the most ubiquitous and crucial components should be defined at R&D phase to mitigate donor-to-donor variability.

account existing classes of innovative products:

• Biopharmaceuticals (in aspect of actual composition)

2. Compliance to cGMP/GTP and cGLP. Time consumption and high cost Replacement of *in vivo* PT by *in vitro* PT or analysis of crucial components concentration (surrogate PT).

account.

controlled.

2. PT must be disease-relevant and reflect preferred MOA.

3. Other MOAs specific to particular CM-based therapeutic must be taken into

2. Depending on preferred MOA, the most crucial components can be enriched.

4. Reproducible *in vitro* PT should be preferred. This model must reflect

Development of specific approach for regulation of multi-MOA and multicomponent CM-based therapeutics. Regulatory approach may take into

• CGTs/ATMPs (in aspect of usage of stem cells in manufacturing)

• Blood plasma-derived therapeutics (in aspect of high heterogeneity and

However, MSC CM-based biopharmaceuticals represent not only a mixture of defined bioactive factors but a complex of multiple components produced by a specific type of cells. MSC CM mimics the beneficial effects of MSC cell therapy, and potency tests applied for corresponding cell and gene therapy products/advanced therapy medicinal products (CGT/ ATMP) could also be relevant. For example, the measure of a defined cytokine like IL-10 in MSC secretome may serve as a potency test for a cell-based product with immunosuppressive

**5.3. Challenges in the development of potency tests for CM-based products**

variability)

analytical and instrumental assays.

gene therapy; ATMP, advanced therapy medicinal product.

**Challenges in development and** 

Lack of corresponding specific category in existing regulations

(EMA, FDA, etc.)

overcome them.

**validation of PT**

54 Biopharmaceuticals

Since there is no "gold standard" or sufficiently defined regulations in this field, developers of MSC CM-based therapeutics use a variety of approaches for determining the potency. To evaluate the immunomodulatory effect of MSC-secreted EV, an in vitro test based on a dose-dependent inhibition by vesicles of the proliferation of phytohemagglutinin-activated T lymphocytes was successfully used [38]. For the surrogate analysis of the immunomodulatory activity of MSC CM in the model of inflammatory organ failure, the effect of MSC CM on bacterial LPS-activated PBMC was tested. The degree of modulation correlated well with the level of IL-10 secreted by PBMC in this experimental model [39]. The subsidiary company of SteMedica, StemProtein, has successfully used two potency tests for its unique product, human fibroblast secretome dried by proprietary technology "Preservation by Vaporization." Because tissue regeneration critically depends on adequate blood supply, they used in vitro angiogenesis assays to evaluate the therapeutic potency of stem cell factors. They routinely monitored the concentrations of VEGF, HGF, interleukin 6 (IL-6), chemokine C-C motif ligand 2 (CCL2), C-X-C motif chemokine 5 (CXCL5),

As CM action is mediated by the soluble factors, the development of a surrogate test based on the correlations between defined factor concentration (e.g., measured by immunoassay) and potency seems to be reasonable. It is necessary to analyze secretome profiles of cells from a large number of donors in order to detect the most robust and relevant active factors in CM concerning current MOA. The molecules selected for this analysis should be the most significant for maintenance of the selected MOA, and less significant for another MOA. In **Figure 1**, if someone selects MOA-II as pivotal one, he should firstly consider factor-2 as the candidate for the surrogate test. Factor-12 and factor-23, which apart from MOA-II are involved in MOA-I and MOA-III, respectively, should be chosen only if their levels are crucial

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This chapter is dedicated to a novel class of biopharmaceuticals based on secretory components of MSC as products for regenerative medicine. CM-based cell-free therapeutics comprise multicomponent mixture with multiple targets and pleiotropic effects. These biopharmaceuticals reproduce many benefits of the rapidly developing cell therapy products. However, the use of cells might be complicated due to ectopic transplantation, tumorigenesis, and immune system reactions. MSC secretome is devoid of cell therapy side effects and has substantial advantages in manufacture, storage, and standardization making it a promising

We paid attention to safe and effective MSC CM-based cell-free therapeutics manufacturing conception. We have discussed several challenges concerning donor-associated variability, cell isolation procedure, optimal protocols for manufacturing and quality control, and lack of key regulatory decisions that must be overcome prior to the wide-scale clinical translation of such therapeutics. To reach the best safety and efficacy marks, developers could apply a system-wide approach to disease model analysis considering the principal mechanisms of tissue reparation and regeneration processes. In addition, it is rational to develop appropriate donor selection criteria that can help to involve only a suitable starting material to manufacturing process. Preventing the lot-to-lot variability together with improved efficacy could be facilitated by a relevant potency test development and validation required for the drug-quality control. The use of rational approach to the choice of MOA might help to relieve development and conduction of a potency test by transition to more simple surrogate approach. In addition, implementing of these approaches into practice would help to develop novel legal potency test guidelines for cell-free therapeutics with unambiguous rules and examples.

The reported study was funded by RFBR according to the research project No. 18-315-00403

and carried out within the state assignment of Lomonosov MSU.

for the MOA-II potential.

type of biopharmaceuticals.

**Funding information**

**6. Conclusion**

**Figure 1.** Schematic reflecting multicomponent multi-MOA nature of MSC and effects of factors secreted by them. A detailed description is provided in the subsequent text. Abbreviations: MOA, mode of action; MSC, mesenchymal stromal cells.

and interleukin 8 (CXCL8) in the product, as published literature has identified the important biological activity of these growth factors, cytokines, and chemokines [40, 41]. MultiStem also successfully used an analogous in vitro angiogenic test of biological activity, and also showed that three factors—VEGF, CXCL8, and CXCL5—are the crucial factors for the angiogenic activity of the secretome. The depletion of any of them drops the angiogenic effect of the secretome. Concentrations of each of the factors can be used as a threshold for the lot release [42]. Based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for human adipose-derived MSC CM developed for tissue reparation and regeneration on "Specific activity." As the most important type of cells involved in tissue repair and regeneration after injury are fibroblasts, one of the models for MSC CM potency measurement was the assessment of human skin fibroblasts migration in the model of the scratch assay. In this regard that angiogenesis is also an indispensable process for the successful regeneration of tissues, we additionally used a model of human endothelial cells direct migration upon MSC CM gradient [26].

Despite direct in vitro or in vivo biological activity tests seem to be more informative, they demonstrate several substantial disadvantages. The main challenge is to render some injuryspecific conditions in model objects. Many in vivo studies in the fields of oncology and adaptive immunity conducted on rodents have faced this problem. In accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and Directive 2010/63/EU on protection of animals used for scientific purposes, the 3R principles (replacement, reduction, and refinement) should be applied to production and control testing of medicinal products. [43]. Last but not the least, it is rational to take into account a high cost of these types of potency test.

As CM action is mediated by the soluble factors, the development of a surrogate test based on the correlations between defined factor concentration (e.g., measured by immunoassay) and potency seems to be reasonable. It is necessary to analyze secretome profiles of cells from a large number of donors in order to detect the most robust and relevant active factors in CM concerning current MOA. The molecules selected for this analysis should be the most significant for maintenance of the selected MOA, and less significant for another MOA. In **Figure 1**, if someone selects MOA-II as pivotal one, he should firstly consider factor-2 as the candidate for the surrogate test. Factor-12 and factor-23, which apart from MOA-II are involved in MOA-I and MOA-III, respectively, should be chosen only if their levels are crucial for the MOA-II potential.
