Good Manufacturing Practice-compliant change of raw material in the manufacturing process of a clinically used advanced therapy medicinal product-a comparability study.

The development of medicinal products often continues throughout the different phases of a clinical study and may require challenging changes in raw and starting materials at later stages. Comparability between the product properties pre- and post-change thus needs to be ensured. Here, we describe and validate the regulatory compliant change of a raw material using the example of a nasal chondrocyte tissue-engineered cartilage (N-TEC) product, initially developed for treatment of confined knee cartilage lesions. Scaling up the size of N-TEC as required for the treatment of larger osteoarthritis defects required the substitution of autologous serum with a clinical-grade human platelet lysate (hPL) to achieve greater cell numbers necessary for the manufacturing of larger size grafts. A risk-based approach was performed to fulfill regulatory requirements and demonstrate comparability of the products manufactured with the standard process (autologous serum) already applied in clinical indications and the modified process (hPL). Critical attributes with regard to quality, purity, efficacy, safety and stability of the product as well as associated test methods and acceptance criteria were defined. Results showed that hPL added during the expansion phase of nasal chondrocytes enhances proliferation rate, population doublings and cell numbers at passage 2 without promoting the overgrowth of potentially contaminant perichondrial cells. N-TEC generated with the modified versus standard process contained similar content of DNA and cartilaginous matrix proteins with even greater expression levels of chondrogenic genes. The increased risk for tumorigenicity potentially associated with the use of hPL was assessed through karyotyping of chondrocytes at passage 4, revealing no chromosomal changes. Moreover, the shelf-life of N-TEC established for the standard process could be confirmed with the modified process. In conclusion, we demonstrated the introduction of hPL in the manufacturing process of a tissue engineered product, already used in a late-stage clinical trial. Based on this study, the national competent authorities in Switzerland and Germany accepted the modified process which is now applied for ongoing clinical tests of N-TEC. The described activities can thus be taken as a paradigm for successful and regulatory compliant demonstration of comparability in advanced therapy medicinal products manufacturing.

From Single Batch to Mass Production-Automated Platform Design Concept for a Phase II Clinical Trial Tissue Engineered Cartilage Product.

Advanced Therapy Medicinal Products (ATMP) provide promising treatment options particularly for unmet clinical needs, such as progressive and chronic diseases where currently no satisfying treatment exists. Especially from the ATMP subclass of Tissue Engineered Products (TEPs), only a few have yet been translated from an academic setting to clinic and beyond. A reason for low numbers of TEPs in current clinical trials and one main key hurdle for TEPs is the cost and labor-intensive manufacturing process. Manual production steps require experienced personnel, are challenging to standardize and to scale up. Automated manufacturing has the potential to overcome these challenges, toward an increasing cost-effectiveness. One major obstacle for automation is the control and risk prevention of cross contaminations, especially when handling parallel production lines of different patient material. These critical steps necessitate validated effective and efficient cleaning procedures in an automated system. In this perspective, possible technologies, concepts and solutions to existing ATMP manufacturing hurdles are discussed on the example of a late clinical phase II trial TEP. In compliance to Good Manufacturing Practice (GMP) guidelines, we propose a dual arm robot based isolator approach. Our novel concept enables complete process automation for adherent cell culture, and the translation of all manual process steps with standard laboratory equipment. Moreover, we discuss novel solutions for automated cleaning, without the need for human intervention. Consequently, our automation concept offers the unique chance to scale up production while becoming more cost-effective, which will ultimately increase TEP availability to a broader number of patients.

Ex vivo osteochondral test system with control over cartilage defect depth - A pilot study to investigate the effect of oxygen tension and chondrocyte based treatments in chondral and full thickness defects in an organ model.

Objective: Cartilage defect treatment strategies are dependent on the lesion size and severity. Osteochondral explant models are a platform to test cartilage repair strategies ex vivo. Current models lack in mimicking the variety of clinically relevant defect scenarios. In this controlled laboratory study, an automated device (artificial tissue cutter, ARTcut®) was implemented to reproducibly create cartilage defects with controlled depth. In a pilot study, the effect of cartilage defect depth and oxygen tension on cartilage repair was investigated. Design: Osteochondral explants were isolated from porcine condyles. 4 â€‹mm chondral and full thickness defects were treated with either porcine chondrocytes (CHON) or co-culture of 20% CHON and 80% MSCs (MIX) embedded in collagen hydrogel. Explants were cultured with tissue specific media (without TGF-ß) under normoxia (20% O2) and physiological hypoxia (2% O2). After 28 days, immune-histological stainings (collagen II and X, aggrecan) were scored (modified Bern score, 3 independent scorer) to quantitatively compare treatment outcome. Results: ARTcut® represents a software-controlled device for creation of uniform cartilage defects. Comparing the scoring results of the MIX and the CHON treatment, a positive relation between oxygen tension and defect depth was observed. Low oxygen tension stimulated cartilaginous matrix deposition in MIX group in chondral defects and CHON treatment in full thickness defects. Conclusion: ARTcut® has proved a powerful tool to create cartilage defects and thus opens a wide range of novel applications of the osteochondral model, including the relation between oxygen tension and defect depth on cartilage repair.

Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells.

The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 × 106 intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 × 103 to 1:1 × 106 human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies.

Validation of Microbiological Testing of Cellular Medicinal Products Containing Antibiotics.

BACKGROUND: The risk of microbial contamination of cellular products can be reduced when cultured in the presence of antibiotics. This however, may impact the sensitivity of microbiological tests. Given that the addition of antibiotics to cell/tissue products does not guarantee sterility but may just reduce the proliferation rate of microorganisms, microbiological testing of medicinal products remains obligatory. Thus, an appropriate method to test for microbial contamination of antibiotic-containing products has to be validated. OBJECTIVES: In the context of microbiological testing of a cellular advance therapy medicinal product, the method was validated and approved by German competent authorities for four different matrices with three matrices containing antibiotics. The paper shall provide help for establishing test methods for other investigational medicinal products which contain antibiotics. METHODS: Matrices were spiked individually with Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus pyogenes, Escherichia coli, Clostridium sporogenes, Propionibacterium acnes, Candida albicans, and Aspergillus brasiliensis. Samples were pretreated with penicillinase for 1 h before inoculation and incubation in BacT/ALERT iFA Plus and iFN Plus culture bottles using 3D BacT/ALERT automates. Microorganisms within positive BacT/ALERT bottles were specified. The procedure was performed in two different laboratories to prove robustness of test. RESULTS: All nine tested microorganisms were detected within 14 days of incubation in accordance with requirements of the European Pharmacopoiea in terms of sensitivity, specificity and robustness of the test. Penicillin and streptomycin did not have any influence on specifications defined within the investigational medicinal product dossier. CONCLUSIONS: Culturing cellular products in the presence of antibiotics can serve as an effective method to reduce contamination risk but only if the chosen antibiotics neither have any influence on specifications of the investigational medicinal product nor interfere with microbiological tests. Consequently, cells and tissues primarily contaminated with microorganisms, like placenta, may be considered as a source of cellular therapeutics when cultured for a sufficient time with antibiotics and tested with a validated method. The choice of microorganisms for the validation of the microbiological test should always consider all conceivable scenarios and should not be reduced to minimal criteria defined in European Pharmacopoiea, wrongfully believing to thus save time and effort.

Injection of Adipose-Derived Stromal Cells in the Knee of Patients with Severe Osteoarthritis has a Systemic Effect and Promotes an Anti-Inflammatory Phenotype of Circulating Immune Cells.

Rationale: Recent studies confirmed that osteoarthritis (OA) is associated with systemic inflammation. Adipose-derived stromal cells (ASCs) could become the most promising cell-based therapy in OA, based not only on their differentiation capacities and trophic and paracrine effects on the existing cartilage, but also on their immunomodulatory properties. Here, we wanted to determine the biological effect of autologous ASC intra-articular (IA) injection. Method: To this aim, we monitored the profile of immune cells in fresh peripheral blood after IA injection of autologous ASCs in the knee of 18 patients with severe OA (ADIPOA phase I study). Specifically, we used 8-color flow cytometry antibody panels to characterize the frequencies of innate and adaptive immune cell subsets (monocytes, dendritic cells, regulatory T cells and B cells) in blood samples at baseline (before injection) and one week, one month and three months after ASC injection. Results: We found that the percentage of CD4+CD25highCD127lowFOXP3+ regulatory T cells was significantly increased at 1 month after ASC injection, and this effect persisted for at least 3 months. Moreover, CD24highCD38high transitional B cells also were increased, whereas the percentage of classical CD14+ monocytes was decreased, at 3 months after ASC injection. These results suggest a global switch toward regulatory immune cells following IA injection of ASCs, underscoring the safety of ASC-based therapy. We did not find any correlation between the scores for the Visual Analogic Scale for pain, the Western Ontario and McMaster Universities Osteoarthritis Index (pain subscale and total score) at baseline and the immune cell profile changes, but this could be due to the small number of analyzed patients. Conclusion: ASCs may drive an immediate local response by releasing paracrine factors and cytokines, and our results suggest that ASCs could also initiate a cascade resulting in a long-lasting systemic immune modulation.

Strategies and First Advances in the Development of Prevascularized Bone Implants.

Despite the great regenerative potential of human bone, large bone defects are a serious condition. Commonly, large defects are caused by trauma, bone disease, malignant tumor removal, and infection or medication-related osteonecrosis. Large defects necessitate clinical treatment in the form of autologous bone transplantation or implantation of biomaterials as well as the application of other available methods that enhance bone defect repair. The development and application of prevascularized bone implants are closely related to the development animal models and require dedicated methods in order to reliably predict possible clinical outcomes and the efficacy of implants. Cell sheet engineering, 3D-printing, arteriovenous loops, and naturally derived decellularized scaffolds and their respective testings in animal models are presented as alternative to the autologous bone graft in this article.

Adipose Mesenchymal Stromal Cell-Based Therapy for Severe Osteoarthritis of the Knee: A Phase I Dose-Escalation Trial.

UNLABELLED: : Osteoarthritis (OA) is the most widespread musculoskeletal disorder in adults. It leads to cartilage damage associated with subchondral bone changes and synovial inflammation, causing pain and disability. The present study aimed at evaluating the safety of a dose-escalation protocol of intra-articular injected adipose-derived stromal cells (ASCs) in patients with knee OA, as well as clinical efficacy as secondary endpoint. A bicentric, uncontrolled, open phase I clinical trial was conducted in France and Germany with regulatory agency approval for ASC expansion procedure in both countries. From April 2012 to December 2013, 18 consecutive patients with symptomatic and severe knee OA were treated with a single intra-articular injection of autologous ASCs. The study design consisted of three consecutive cohorts (six patients each) with dose escalation: low dose (2 × 10(6) cells), medium dose (10 × 10(6)), and high dose (50 × 10(6)). The primary outcome parameter was safety evaluated by recording adverse events throughout the trial, and secondary parameters were pain and function subscales of the Western Ontario and McMaster Universities Arthritis Index. After 6 months of follow-up, the procedure was found to be safe, and no serious adverse events were reported. Four patients experienced transient knee joint pain and swelling after local injection. Interestingly, patients treated with low-dose ASCs experienced significant improvements in pain levels and function compared with baseline. Our data suggest that the intra-articular injection of ASCs is a safe therapeutic alternative to treat severe knee OA patients. A placebo-controlled double-blind phase IIb study is being initiated to assess clinical and structural efficacy. SIGNIFICANCE: Although this phase I study included a limited number of patients without a placebo arm, it showed that local injection of autologous adipose-derived stem cells was safe and well tolerated in patients with knee osteoarthritis. This study also provides encouraging preliminary evidence of efficacy. Larger and controlled long-term studies are now mandatory to confirm whether this new strategy of cell therapy can improve pain and induce structural benefit in osteoarthritis.

Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head.

Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN.

Identification of the receptor for advanced glycation end products in synovial tissue of patients with rheumatoid arthritis.

OBJECTIVES: Generation of advanced glycation end products (AGE) is an inevitable process in vivo and can be accelerated under pathologic conditions such as oxidative stress, e.g. in rheumatoid arthritis (RA). This process is mediated by the AGE-specific receptor (RAGE). In this study we analysed the presence of RAGE in RA and osteoarthritic (OA) synovial tissue using immunohistology. METHODS: Frozen synovial tissue samples from 11 RA patients and 12 OA patients were treated with goat anti-RAGE immunoglobulin G (IgG) and rabbit antigoat IgG. Immunostaining was visualised with streptavidin horse radish peroxidase (chromogen amino-ethyl-carbazole). Cell differentiation was performed with antibodies against CD68, CD45RO, and CD20. RESULTS: In 9/11 RA and 8/12 OA synovial specimens, RAGE was detected in synovial lining, sublining, and stroma. In RA, many T cells (CD45RO(+)) and some macrophages (CD68(+)) showed positive immunostaining for RAGE, whereas B cells were mostly negative. We found no difference in staining patterns between the RA and OA samples. CONCLUSIONS: We detected RAGE in RA and OA synovial tissue. The presence of RAGE on macrophages, T cells, and some B cells suggests its role in the pathogenesis of inflammatory joint disease.