Quality by design strategy of human mesenchymal stem/stromal cell drug products for the treatment of knee osteoarthritis

Abstract

Knee osteoarthritis (KOA), characterized by heterogeneous arthritic manifestations and complex peripheral joint disorder, is one of the leading causes of disability worldwide, which has become a high burden due to the multifactorial nature and the deficiency of available disease-modifying treatments. The application of mesenchymal stem/stromal cells (MSCs) as therapeutic drugs has provided novel treatment options for diverse degenerative and chronic diseases including KOA. However, the complexity and specificity of the “live” cells have posed challenges for MSC-based drug development and the concomitant scale-up preparation from laboratory to industrialization. For instance, despite the considerable progress in ex vivo cell culture technology for fulfilling the robust development of drug conversion and clinical trials, yet significant challenges remain in obtaining regulatory approvals. Thus, there’s an urgent need for the research and development of MSC drugs for KOA. In this review, we provide alternative solution strategies for the preparation of MSC drugs on the basis of the principle of quality by design, including designing the cell production processes, quality control, and clinical applications. In detail, we mainly focus on the quality by design method for MSC manufacturing in standard cell-culturing factories for the treatment of KOA by using the Quality Target Product Profile as a starting point to determine potential critical quality attributes and to establish relationships between critical material attributes and critical process parameters. Collectively, this review aims to meet product performance and robust process design, and should help to reduce the gap between compliant products and the production of compliant good manufacturing practice.

Key Words: Mesenchymal stem/stromal cells; Knee osteoarthritis; Quality by design; Critical quality attributes; Critical material attributes

Core Tip: Knee osteoarthritis is one of the leading causes of disability worldwide and has become a high burden due to the multifactorial nature and the deficiency of available disease-modifying treatments. Herein, we review the state-of-the-art literatures of the quality by design strategy of mesenchymal stem/stromal cell drug products for knee osteoarthritis treatment, together with discuss the accompanied principles as well, which will collectively help to reduce the gap between compliant products and the production of compliant good manufacturing practice.

INTRODUCTION

Knee osteoarthritis (KOA) is the most common form of arthritis with high incidence, which affects approximately 650 million people worldwide, with a prevalence of 14%-38% in women and 4%-14% in men aged 40 and older[1]. The quality of life of KOA patients is significantly reduced due to the limitation of efficacy by current treatments, including lifestyle changes (e.g., exercise, weight loss, and smoking cessation) and injectable interventions [e.g., corticosteroids[2], hyaluronic acid (HA), and injections of platelet-rich plasma (PRP) with short-to medium-term analgesic effects[3]].

Human mesenchymal stem/stromal cells (MSCs) are heterogeneous cell populations with splendid immunosuppressive and hematopoietic-supporting properties, which have been extensively explored in both preclinical and clinical investigations for the illumination of pathogenesis and treatment of refractory and recurrent diseases[4,5]. For instance, our colleagues and we have demonstrated the feasibility of MSC-based regimens for a wide range of disease treatment such as osteoarthritis[6], acquired aplastic anemia[7,8], cerebral infarction[9], acute lung injury[10,11], Crohn’s-like enterocutaneous fistula[12], infected anal fistula[13], acute-on-chronic liver failure[14], acute graft-versus-host disease[4,15], aplastic anemia[7], critical limb ischemia[16], cerebral infarction[9], radiation-induced colorectal fibrosis[17], and even the coronavirus disease 2019 pneumonia-associated acute respiratory distress syndrome[18,19]. Unlike other stem cell subtypes that require matching, MSCs meet the demand of allogeneic transplantation and thus serve as the most rapidly developing stem cell type in clinical trials and play critical roles in bone cells as well[20,21].

State-of-the-art literatures have highlighted MSCs as an attractive candidate for the treatment of KOA attributes to the multi-modal mode of action. For instance, MSCs are adequate to suppress the pro-inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin-6) and immune cell responses (e.g., macrophages for cell clearance) in the knee-joint microenvironment via modulating diverse paracrine factors and extracellular vesicles[22,23]. To date, numerous clinical trials of MSCs-based regimens upon KOA have been registered and launched globally, and in particular, in South Korea and India[24]. However, no commercial MSC product expect for Mesoblast’s Ryoncil (remestemcel-L-rknd) have been approved in the United States, which further indicates the bottlenecks in the production of high-quality MSCs for industrial manufacturing and clinical application. Therefore, the introduction of new tools and methods including the quality by design (QbD) method will provide an important basis for the development of MSC products for KOA treatment.

DEFINING A QBD APPROACH FOR COMPLIANT GOOD MANUFACTURING PRACTICE MANUFACTURING

QbD is a scientific and risk-based framework for process design based on relating product and process attributes to product quality. As a systematic development approach that begins with predefined objectives, QbD places an emphasis on understanding the process control of products, which is grounded in scientific principles and quality risk management. The emergence of QbD has enhanced the assurance of providing consumers with safe and effective drug supplies, while also promising a significant improvement in manufacturing quality performance[25]. For instance, Sawada et al[26] highlighted the increasing robustness of in vitro analyses for immuno-suppressive effect of MSCs. Hoang et al[27] put forward the importance of QbD principle for advanced cell-based product generation. The output products by cell therapy products (CTP) are healthy and functional living cells, which makes cell products highly sensitive to environmental factors in the manufacturing process. Therefore, it is the biological complexity of cells that makes the production and preparation process based on laboratory operation habits seriously affect the quality and efficacy of cell products. The biological complexity of cells affects the end product of CTP, and the way to solve this challenge is to cell manufacturing process design according to the principle of QbD.

The QbD manufactured by CTP is composed of four functional modules, including cell culture equipment module, cell proliferation module, scale and control module. QbD is a product development and life cycle management framework promoted by regulatory agencies, which plays a very important role in the traditional pharmaceutical field. Considering that QbD is a common management framework, it can be applied to the field of cell therapy in combination with new specific technical solutions. Connecting measurable molecular and cellular characteristics of cell populations with final product quality through QbD is a key step to achieve large-scale cell products. To be more effective, cell therapy bioprocess design and optimization need to include several basic criteria: (1) Evaluation of relevant cell properties; (2) Determination and control of key parameters; (3) Robust prediction strategies for interrogating and evaluating many parameters that may affect cell culture output; and (4) Methods to test many different parameters that may affect cell output in a high-throughput and large-scale relevant manner[28]. In brief, QbD first describes the Quality Target Product Profile (QTPP), identifies the critical quality attributes (CQAs) that directly affect the safety and effectiveness of products, verifies the critical process parameters (CPPs) that affect these attributes, and develops a design space to quantify the impact of parameter variability on quality attributes. Subsequently, a control strategy is developed to maintain the process parameters to ensure that the product quality is within a certain range, and the process is verified on a scale. Once CTP manufacturing is implemented, QbD allows the production process to be monitored and repeatedly optimized with the increase of process knowledge (Figure 1)[29].

Figure 1 Summary of quality by design process for the production of mesenchymal stem/stromal cells. CQA: Critical quality attributes; QTPP: Quality Target Product Profile; CMA: Critical material attributes; CPP: Critical process parameters; PDT: Population doubling time.

IDENTIFYING QTPP

QTPP describes the use, safety and efficacy of the target product and is the starting point of QbD. Determining strict and easy to measure criteria is essential for establishing QTPP. The purpose of MSC products is to treat diseases (clinical-grade cell products), and its safety depends on: (1) Cell safety, that is, the genomic stability of MSC during in vitro culture and expansion; and (2) MSC preparation safety, that is, the cell injection cannot be contaminated by microorganisms, and the excipients meet the requirements of the Pharmacopoeia. Cell cultures are susceptible to many types of contamination, especially microbial (e.g., bacteria, fungi, and mycoplasma), endotoxin, and cell line cross contamination. In addition, non-cellular particles (including plastic debris, residual microcarriers and fibers) produced by manufacturing equipment and materials must be supervised, which might impact the expansion performances and differentiation potential of MSCs[30-32].

The production of CTPs usually requires the mixing of bioactive chemicals, including cytokines, small molecules, serum and carriers, etc. These auxiliary substances must be fully removed to avoid being regarded as drugs themselves. Therefore, QTPP must describe the maximum residue level of these auxiliary materials in the final MSC product to ensure the safety, and additional treatments that may affect the final cell yield. In additional, the potency of MSCs includes strength and effectiveness, that is, the effect achieved at the same dose (cell concentration)[33-35]. Therefore, the discussion of the quality or biological efficacy of MSCs is inseparable from the specific indications[36]. For example, MSCs are adequate for simultaneously immunosuppression and vascular regeneration acceleration, which thus respectively need the corresponding biological efficacy indicators for the two different kinds of diseases. For MSC manufacturers and clinicians with the application of new stem cell drugs, it’s of great importance to develop biological indicators at the cellular and molecular levels that are close enough to the effect of MSC treatment according to the indications[37].

IDENTIFYING CQAS

The definition of CQAs 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. CQAs are generally linked to drug substances, excipients, intermediates (in-process materials), and final drug products. CQAs directly determine product quality, while CPP and material attributes indirectly affect product quality by influencing CQAs[38-40]. Therefore, a main goal of the manufacturing process is to ensure that the CQAs of the final cell product (identity, purity, efficacy, and safety) are maintained at every step of the entire process until entering the market, and can be evaluated with the aid of specific feature tools and analytical methods[41-43].

The CQAs of MSC products should include cellular characteristics, such as dosage, cell quantity, cell viability, therapeutic potency, in vitro differentiation capacity, expected in vivo effect, product quality, genetic stability, and cell purity (Table 1). Commonly, MSCs involved in clinical applications should satisfy four types of quality attributes, including basic biological attributes, microbiological safety, biological safety, and biological efficacy. Among them, the biological efficacy of MSCs refers to the biological functions that correspond to or can be used to predict the clinical efficacy of MSCs during the preclinical stage[44], which serves as an important quality attribute that determines the clinical outcomes of MSCs at present and in future.

Table 1 The definition of critical quality attributes for mesenchymal stem/stromal cell-based therapy according to the defined Quality Target Product Profile.

QTPP	CQA	Quality control	Acceptable standards	Ref.
Cell characteristics	Morphology	Microscopic visual inspection	Shuttle shaped in wall attached state	[90]
	Cell viability	Cell count and viability detection	Cell viability ≥ 90% before product distribution	[91]
	MSC identity	Specific positive antigen expression ≥ 95%	Flow cytometry: CD105+, CD73+, CD90+	[92]
		Specific negative antigen expression ≤ 2%	Flow cytometry: CD45-, CD34-, CD14- or CD11b-, CD79α- or CD19-, HLA-DR-	[92]
	PDT	PDT calculation formula	8 hours < PDT < 48 hours	[93]
	Cell differentiation	Osteogenic differentiation	Color reaction with Alizarin Red S	[94]
		Adipogenic differentiation	Color reaction with Oil Red O	[95]
		Chondrogenic differentiation	Color reaction with Alcian Blue	[96]
	Cell cycle	PI staining	G0/G1% > 70%
Product quality	Purity	Sterility testing for each lot	No detectable	[97]
		Mycoplasma testing for each lot	No detectable	[98]
		Endotoxin testing for each lot	≤ 0.5 CFU/mL	[98]
	Genetic stability	Karyotype	23 pairs of chromosomes	[99]
		STR	Each cell passage is from the same source
	Biological safety	Human telomerase activity	No or weak expression	[100]
		MSC soft agar cloning	No detectable	[101]
Therapeutic potency	In vivo effect	Co-culture with T cells	Inhibition of T cell growth	[102]
	Paracrine action	The secretion of bioactive molecules	PGE-2, IL-10	[103]

QTPP: Quality Target Product Profile; CQA: Critical quality attribute; MSC: Mesenchymal stem/stromal cell; PDT: Population doubling time; STR: Short tandem repeat; CFU: Colony forming unit; PGE-2: Prostaglandin E2; IL-10: Interleukin-10.

IDENTIFYING CPP

Ensuring high-quality standards in production facilities necessitates a thorough understanding of process parameter interactions and their potential impact on CQAs. The culture parameters that affect the cell yield can be divided into two categories: (1) The “traditional” physicochemical bioprocess engineering parameters that are important for the optimization of bio-pharmaceutical process[45,46]; and (2) The “cell therapy related” parameters that are important for the control of cell fate and product quality[47,48].

Cell extraction

Since their first identification in bone marrow in the 1960s, MSCs of diverse origins have been consecutively isolated from adult tissues (e.g., adipose tissue, bone marrow, muscle tendon, uterine blood, dental pulp and sac) and perinatal tissues (e.g., umbilical cord, amniotic fluid and membrane, placenta), and even derived from pluripotent stem cells including induced pluripotent stem cells and embryonic stem cells[4-6,12,49]. Considering the variations in the inherent attributes of MSCs derived from the aforementioned sources, it’s of great importance to select suitable cell sources for MSC preparation for KOA treatment[4,6].

Compared to the invasive collection of MSCs from bone marrow and adipose tissue, umbilical cord-derived MSCs are more suitable for large-scale preparation due to their non-invasive collection, cost-effectiveness, and accessibility[50]. To ensure the sustainability of industrial production and the stability of quality, the large-scale cell bank construction based on standardized processes is the core steps for “seed cells” separation and the subsequent ex vivo amplification, together with the cryopreservation of MSC products[14,15,51]. Of note, current updates have verified that the traditional cryopreservation technology has minimal impact on CQAs of MSCs therapy products compared to the fresh MSC products[52].

Overall, the most important issue at this stage is the considerable donor-to-donor and intra-population heterogeneity[53-55]. Due to the limitations in the detection of diverse infection or functional abnormalities, each donor carries a “one to many” related risk for MSC-based products upon KOA treatment. For example, the age of the donor is an important factor affecting cell quality[56], including the number of primary isolated cells[57], cell differentiation ability[58], cell doubling level[59] and increasing genetic instability[60].

Cell amplification

For cell amplification, the traditional methods are usually based on two-dimensional cell culture flasks, and the introduction of personnel operation greatly increases the risk of contamination. Currently, common strategies to mitigate these risks include limiting the number of cell containers[61] or utilizing fully enclosed production system[62]. In most clinical trials, MSCs have been prepared by utilizing the basal culture medium containing L-glutamine, non-essential amino acid and specific ions, together with 5%-20% fetal bovine serum to ensure cell expansion[63]. It’s of note that the addition of ingredients will directly affect the functional performance of MSCs[64-66]. Relevant process parameters are usually measured by dynamic detection, such as cell number and viability, micronutrient concentration and physicochemical variables[67]. The advantage of physicochemical biological process is that most engineering parameters can be measured and controlled in real time, which helps guide cell output of the MSC products. It is worth noting that oxygen has been considered to play an important role in the maintenance and differentiation of MSCs, together with the regulation of cell fate in culture expansion system[68].

Cell senescence

In the process of subculture, telomere length will gradually decrease with the increase of passages, resulting in gradual senescence of cells[69]. Aging is irreversible once it occurs, which will not only lead to the loss of pluripotency[70], but also increase the risk of tumorigenicity[71] and reduce the ability of immunosuppression[72]. However, according to karyotype analysis, the proportion of stem cells with abnormal karyotype is high in the two generations of cells just after primary isolation[73]. Therefore, a better solution for in vitro culture as a clinical application is selecting seed cells at passages ranging from 4 to 8 for the subsequent large-scale generations of MSCs.

DESIGNING MSC GRUGS FOR TREATING KOA THROUGH THE QBD

The CQAs and clinical mechanisms of MSC products

Currently, evidence on the clinical mechanism of MSC treatment remains limited, yet it is crucial for the large-scale application of MSCs in disease intervention, including KOA. For instance, the Food and Drug Administration’s previous rejection of Mesoblast’s allogeneic bone marrow MSC (M) product as an allogeneic product for the treatment of steroid-refractory acute graft-versus-host disease, is partly due to the insufficient CQAs used by sponsors[74,75]. In detail, the investigators didn’t demonstrate a clear relationship between the product’s CQAs and the concomitant clinical efficacy[42]. Therefore, a better understanding of the underlying mechanism of action will be beneficial in the following aspects: (1) This will aid the design of novel and improved MSC therapies to realize the anticipated effects of KOA; (2) This will assist in the selection of CQAs for MSC products, which are associated with the mechanism of action and can serve as release criteria for these products; (3) This will facilitate the identification of patients who are more likely to respond to MSCs intervention based on the use of precision medicine methods; and (4) This will enhance the communication of expected clinical outcomes based on MSCs intervention.

Clinical trials show that MSCs can directly exert cartilage repair and/or cartilage protection effects on KOA by releasing cartilage protective factors or growth factors, and indirectly by regulating the joint microenvironment[76]. At the same time, more joint tissues should be taken into consideration, including the infrapatellar fat pad, subchondral bone, and ligaments. Meanwhile, it’s important to carefully consider the donor factors, process parameters, and delivery strategies of MSCs, which collectively contribute to the mode of action and the overall therapeutic efficacy (Figure 2).

Figure 2 The critical quality attributes-related clinical mechanism of mesenchymal stem/stromal cell-based therapy for knee osteoarthritis. CQA: Critical quality attributes; VEGF: Vascular endothelial growth factor; IL-6: Interleukin-6; IDO: Indoleamine 2,3-dioxygenase; TSG6: Tumor necrosis factor-stimulated gene 6; MSC: Mesenchymal stem/stromal cell; BMP-2: Bone morphogenetic protein 2; TNF: Tumor necrosis factor; TGF-β: Transforming growth factor beta; PGE2: Prostaglandin E2; Tregs: Regulatory T cells.

Preparation form and delivery strategies

The dose of MSCs in the treatment of KOA is a key issue that needs to be focused on. Clinical data indicate that low-dose MSCs do not show significant effects on pain relief and cartilage repair in KOA patients. Meta-analysis results suggest that a dose of (40-60) × 10⁶ MSCs is effective for treating osteoarthritis, which can significantly improve the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and function subscale scores[24]. However, it should be noted that high doses of MSCs (> 100 × 10⁶) may not provide better outcomes, but rather have a more pronounced protective effect on cartilage tissue[77]. As shown in a published randomized controlled trial (RCT) by Matas et al[78], repeat umbilical cord-derived MSCs intraarticular injections revealed superiority in ameliorating KOA over the single MSC dose. According to the WOMAC pain and function subscale scores in a first-in-human study, Anil et al[79] reported that KOA patients with repeat injection of infrapatellar fat pad-derived MSCs showed significant improvement compared to the control group. However, those with single MSC injection showed sustained improvement up to 9 months, but the WOMAC score returned to control group level at the 12-month follow-up visit. Therefore, longer follow-up visit would have afforded a better evaluation of the synergistic or additive effects of repeat MSC injections.

Biological effectiveness of MSC product

The main biological function of MSCs is immune regulation, which is also an important mechanism in alleviating KOA. To confirm the immunoregulatory mechanism of MSCs in KOA, Matas et al[78] investigated the relationship between the baseline immune regulatory fitness of MSCs in vitro and its clinical efficacy. They measured the interferon gamma induced immune regulatory gene expression [including prostaglandin E2, interleukin-10, CD274, indoleamine 2,3-dioxygenase (IDO), hepatocyte growth factor] and tumor necrosis factor-α induced tumor necrosis factor-stimulated gene 6 protein expression by MSCs in vitro, and found correlations with changes in WOMAC and knee injury and osteoarthritis outcome score. These evidences suggest that MSCs with higher baseline immunoregulatory fitness levels in vitro can bring better treatment experience and more significant efficacy to KOA patients[78].

Furthermore, Chen et al[80] found that the expression level of intracellular IDO protein in stimulated MSCs was significantly correlated with the inhibition of T cell proliferation in vitro and the improvement of knee injury and osteoarthritis outcome score sports, visual analogue scale, and international knee documentation committee scores in KOA patients after intraarticular injection of cells. Interestingly, Leijs et al[81] turned to enzyme-linked immunosorbent assay and relative methods to detect IDO protein expression by interferon-γ-stimulated MSCs, which could effectively evaluate the immunomodulatory effects of MSCs triggered by pro-inflammatory cytokines in arthritic synovial fluids. Therefore, it would be of great help to complete a satisfactory method validation and then incorporated into the CQAs for MSCs in the treatment of KOA. As a differentiation test for MSCs, the multi-lineage differentiation potential of MSCs under the conditioned medium also provides an in vitro source of drug quality control for the treatment of KOA with MSCs.

Comparison of clinical effects

To better verify the therapeutic potential of MSCs in treating KOA, numerous literatures have compared with other conventional treatment methods, including HA injection, PRP injection, arthroscopic debridement, high tibial osteotomy, and conservative treatment. For instance, Naja et al[82] conducted a meta-analysis based on recent RCTs to investigate the efficacy of commonly used non-surgical interventions in patients with mild to moderate KOA, and a 19 RCTs with diverse treatment options were included (e.g., bone marrow MSCs, PRP, HA, corticosteroids, non-steroidal anti-inflammatory drugs). They found the total WOMAC score of the MSC and PRP intervention groups significantly decreased from baseline to 12 months, and MSC treatment showed the greatest improvement in pain reduction and functional enhancement according to the meta-analysis. A systematic review further demonstrated the dynamics of MSC therapy upon KOA patients with significant improvement in every subsequent follow-up visit[83]. Compared to 18 months, the WOMAC score slightly decreased at 24 months, highlighting that the first 18-24 months post-treatment were expected to yield the most significant clinical benefits. In recent years, an increasing body of clinical evidence points to the superior effectiveness of MSC therapy for KOA. With the advancement of the cell therapy industry, cell therapy-derived entities such as exosomes are demonstrating their potential in alleviating KOA symptoms by modulating macrophage polarization, promoting angiogenesis, enhancing osteogenesis, and inhibiting tunnels osteolysis.

CONCLUSION

Unlike most small molecule drugs or biopharmaceuticals, stem cell-based therapies are typically produced in unique manufacturing environments, including equipment and processes for cultivating, testing, and packaging. The collected raw materials are processed in a good manufacturing practice environment and then applied in clinical practice. A key distinction between stem cell-based therapies and traditional pharmaceuticals is the complexity and quantity of raw materials used in manufacturing. For manufacturers, determining which components are most critical to quality is crucial. When adding raw materials (such as cytokines and growth factors) during the manufacturing process of cellular products, they can affect the growth, differentiation, or function of cells[7]. Some of stem cell-based therapy products face manufacturing process challenges after amplification (culture amplification), resulting in high cost of goods and high variability in production preparation processes[7,84,85]. Use planar techniques such as T-flasks or multi-layer cell engineering, or perform cell expansion via a single use of 3D microcarrier-based culture in a bioreactor. After enzyme treatment (e.g., trypsin) releases adherent cells, the downstream process stage includes removal of microcarriers and volume reduction for cell concentration and washing. The next step is to place the cells in a low-temperature preservation buffer for low-temperature preservation. Therefore, the understanding of how cells interact with their environment is currently deepening, and bioreactor systems that can control the cellular environment are collecting data that is increasingly focused on molecular and cellular information. At the same time, deepen the understanding of the molecular basis of stem cell state, including adhesion dependence, metabolic network state, clonality, and proliferation control, which is important for KOA efficacy but different from traditional medicine (e.g., icariin) to a large extent[86-89]. The QbD principle is in the ascendant and highly valued in the pharmaceutical industry. The development of the stem cell therapy industry is bound to combine the QbD principle to develop its own production preparation process and management system.

ACKNOWLEDGEMENTS

The co-authors thank the members in Faculty of Life Sciences and Medicine, Kunming University of Science and Technology, The Fourth People’s Hospital of Jinan Affiliated to Shandong Second Medical University, Gansu Provincial Hospital, and Nankai University School of Medicine. We also thank the support from Shandong Provincial Key Medical and Health Laboratory of Blood Ecology and Biointelligence, Jinan Key Laboratory of Medical Cell Bioengineering, and Shandong Health Youth Science and Technology Innovation Team.