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.

Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial.

BACKGROUND: Articular cartilage injuries have poor repair capacity, leading to progressive joint damage, and cannot be restored predictably by either conventional treatments or advanced therapies based on implantation of articular chondrocytes. Compared with articular chondrocytes, chondrocytes derived from the nasal septum have superior and more reproducible capacity to generate hyaline-like cartilage tissues, with the plasticity to adapt to a joint environment. We aimed to assess whether engineered autologous nasal chondrocyte-based cartilage grafts allow safe and functional restoration of knee cartilage defects. METHODS: In a first-in-human trial, ten patients with symptomatic, post-traumatic, full-thickness cartilage lesions (2-6 cm2) on the femoral condyle or trochlea were treated at University Hospital Basel in Switzerland. Chondrocytes isolated from a 6 mm nasal septum biopsy specimen were expanded and cultured onto collagen membranes to engineer cartilage grafts (30 × 40 × 2 mm). The engineered tissues were implanted into the femoral defects via mini-arthrotomy and assessed up to 24 months after surgery. Primary outcomes were feasibility and safety of the procedure. Secondary outcomes included self-assessed clinical scores and MRI-based estimation of morphological and compositional quality of the repair tissue. This study is registered with ClinicalTrials.gov, number NCT01605201. The study is ongoing, with an approved extension to 25 patients. FINDINGS: For every patient, it was feasible to manufacture cartilaginous grafts with nasal chondrocytes embedded in an extracellular matrix rich in glycosaminoglycan and type II collagen. Engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of defect filling and development of repair tissue approaching the composition of native cartilage. INTERPRETATION: Hyaline-like cartilage tissues, engineered from autologous nasal chondrocytes, can be used clinically for repair of articular cartilage defects in the knee. Future studies are warranted to assess efficacy in large controlled trials and to investigate an extension of indications to early degenerative states or to other joints. FUNDING: Deutsche Arthrose-Hilfe.

Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial.

BACKGROUND: Autologous native cartilage from the nasal septum, ear, or rib is the standard material for surgical reconstruction of the nasal alar lobule after two-layer excision of non-melanoma skin cancer. We assessed whether engineered autologous cartilage grafts allow safe and functional alar lobule restoration. METHODS: In a first-in-human trial, we recruited five patients at the University Hospital Basel (Basel, Switzerland). To be eligible, patients had to be aged at least 18 years and have a two-layer defect (≥50% size of alar subunit) after excision of non-melanoma skin cancer on the alar lobule. Chondrocytes (isolated from a 6 mm cartilage biopsy sample from the nasal septum harvested under local anaesthesia during collection of tumour biopsy sample) were expanded, seeded, and cultured with autologous serum onto collagen type I and type III membranes in the course of 4 weeks. The resulting engineered cartilage grafts (25 mm × 25 mm × 2 mm) were shaped intra-operatively and implanted after tumour excision under paramedian forehead or nasolabial flaps, as in standard reconstruction with native cartilage. During flap refinement after 6 months, we took biopsy samples of repair tissues and histologically analysed them. The primary outcomes were safety and feasibility of the procedure, assessed 12 months after reconstruction. At least 1 year after implantation, when reconstruction is typically stabilised, we assessed patient satisfaction and functional outcomes (alar cutaneous sensibility, structural stability, and respiratory flow rate). FINDINGS: Between Dec 13, 2010, and Feb 6, 2012, we enrolled two women and three men aged 76-88 years. All engineered grafts contained a mixed hyaline and fibrous cartilage matrix. 6 months after implantation, reconstructed tissues displayed fibromuscular fatty structures typical of the alar lobule. After 1 year, all patients were satisfied with the aesthetic and functional outcomes and no adverse events had been recorded. Cutaneous sensibility and structural stability of the reconstructed area were clinically satisfactory, with adequate respiratory function. INTERPRETATION: Autologous nasal cartilage tissues can be engineered and clinically used for functional restoration of alar lobules. Engineered cartilage should now be assessed for other challenging facial reconstructions. FUNDING: Foundation of the Department of Surgery, University Hospital Basel; and Krebsliga beider Basel.

Engineered autologous nasal cartilage for repair of nasal septal perforations - a case series.

OBJECTIVE: This phase I clinical trial assessed the use of autologous nasal chondrocyte tissue-engineered cartilage (N-TEC) for functional repair of nasal septal perforations (NSP). BACKGROUND: The most widely used technique to treat NSP, namely interposition grafting with a polydioxanone (PDS) plate combined with a deep temporal fascia (DTF) graft, is still suboptimal towards patient satisfaction and revision rates. METHODS: Patients (n=5, all female, age range: 23-54 years) had a 0.5-2.0 cm diameter NSP. N-TEC was manufactured by expansion and 3D culture of autologous nasal septum chondrocytes into Chondro-Gide® collagen membranes. N-TEC was then shaped intraoperatively and enveloped in the harvested DTF before suturing it into the NSP. Safety (primary outcome) was assessed by the number of serious adverse reactions (SAR) until 12 months. Secondary outcomes included feasibility, assessed by surgical graft manipulation, and efficacy, assessed using subjective scoring (Nasal Obstruction Symptom Evaluation, NOSE, and Visual Analogue Scale, VAS, scores) and objective breathing function tests. Structural closure of NSP after 12 months was defined using endoscopy and computed tomography (CT) scans. RESULTS: NSP treatment by N-TEC implantation was safe and feasible, as no SAR and no challenge in graft manipulation was recorded for any of the patients. One year postoperative, subjective scoring improved in all patients, unless already optimal (average improvement of 23 and 28.6 points out of 100 respectively for NOSE and VAS scores). Objective respiratory function overall confirmed - with the exception of one case - the observations above (average improvement of 172 ml/s). NSP were closed and the mucosae completely healed in three patients. CONCLUSION: Autologous N-TEC is a valid treatment for NSP and warrants further clinical tests.

Toward clinical application of tissue-engineered cartilage.

Since the late 1960s, surgeons and scientists envisioned use of tissue engineering to provide an alternative treatment for tissue and organ damage by combining biological and synthetic components in such a way that a long-lasting repair was established. In addition to the treatment, the patient would also benefit from reduced donor site morbidity and operation time as compared with the standard procedures. Tremendous efforts in basic research have been done since the late 1960s to better understand chondrocyte biology and cartilage maturation and to fulfill the growing need for tissue-engineered cartilage in reconstructive, trauma, and orthopedic surgery. Starting from the first successful generation of engineered cartilaginous tissue, scientists strived to improve the properties of the cartilaginous constructs by characterizing different cell sources, modifying the environmental factors influencing cell expansion and differentiation and applying physical stimuli to modulate the mechanical properties of the construct. All these efforts have finally led to a clinical phase I trial to show the safety and feasibility of using tissue-engineered cartilage in reconstructive facial surgery. However, to bring tissue engineering into routine clinical applications and commercialize tissue-engineered grafts, further research is necessary to achieve a cost-effective, standardized, safe, and regulatory compliant process.

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.

Case Report: Reconstruction of a Large Maxillary Defect With an Engineered, Vascularized, Prefabricated Bone Graft.

The reconstruction of complex midface defects is a challenging clinical scenario considering the high anatomical, functional, and aesthetic requirements. In this study, we proposed a surgical treatment to achieve improved oral rehabilitation and anatomical and functional reconstruction of a complex defect of the maxilla with a vascularized, engineered composite graft. The patient was a 39-year-old female, postoperative after left hemimaxillectomy for ameloblastic carcinoma in 2010 and tumor-free at the 5-year oncological follow-up. The left hemimaxillary defect was restored in a two-step approach. First, a composite graft was ectopically engineered using autologous stromal vascular fraction (SVF) cells seeded on an allogenic devitalized bone matrix. The resulting construct was further loaded with bone morphogenic protein-2 (BMP-2), wrapped within the latissimus dorsi muscle, and pedicled with an arteriovenous (AV) bundle. Subsequently, the prefabricated graft was orthotopically transferred into the defect site and revascularized through microvascular surgical techniques. The prefabricated graft contained vascularized bone tissue embedded within muscular tissue. Despite unexpected resorption, its orthotopic transfer enabled restoration of the orbital floor, separation of the oral and nasal cavities, and midface symmetry and allowed the patient to return to normal diet as well as to restore normal speech and swallowing function. These results remained stable for the entire follow-up period of 2 years. This clinical case demonstrates the safety and the feasibility of composite graft engineering for the treatment of complex maxillary defects. As compared to the current gold standard of autologous tissue transfer, this patient's benefits included decreased donor site morbidity and improved oral rehabilitation. Bone resorption of the construct at the ectopic prefabrication site still needs to be further addressed to preserve the designed graft size and shape.

Sensing tissue engineered cartilage quality with Raman spectroscopy and statistical learning for the development of advanced characterization assays.

Nasal chondrocyte-derived engineered cartilage has been demonstrated to be safe and feasible for the treatment of focal cartilage lesions with promising preliminary evidences of efficacy. To ensure the quality of the products and processes, and to meet regulatory requirements, quality controls for identity, purity, and potency need to be developed. We investigated the use of Raman spectroscopy, a nondestructive analytical method that measures the chemical composition of samples, and statistical learning methods for the development of quality controls to quantitatively characterize the starting biopsy and final grafts. We provide a proof-of-concept to show how Raman spectroscopy can be used to identify the types of tissues found in a nasal septal biopsy, i.e., hyaline cartilage and perichondrium, for a novel tissue identity assay. The tissues could be classified with a sensitivity of 89% and specificity of 77%. We also show how clinically relevant and mature nasal chondrocyte-derived engineered cartilage can be assessed with Raman spectroscopy for the development of potency assays. The maturity of engineered grafts, based on the quantified ratio of glycosaminoglycans to DNA and histological score, could be accurately assessed (R2 = 0.78 and 0.89, respectively, between predicted and measured values). Our results demonstrate the potential of Raman spectroscopy for the development of characterization assays for regenerative therapies that could be integrated into a good manufacturing practice-compliant process.

Do we really need cartilage tissue engineering?

The in vitro engineering of functionally developed biological cartilage substitutes, based on cells and appropriate structural and soluble factors, is an attractive concept for the clinical treatment of cartilage injuries and degeneration. The field of cartilage tissue engineering has developed strongly in the last few years, bringing together the scientific, clinical and commercial interests of highly interdisciplinary communities. However, engineered grafts are still far from being the standard of care for cartilage repair. In this review we present some of the issues challenging the reproducible engineering of functional cartilage templates starting from human cells. We then discuss the need to identify the mode of action of cartilage tissue engineering approaches, which in turn is expected to define potency markers and quality controls for grafts capable of inducing durable cartilage regeneration. Finally, we propose the use of engineered cartilage tissues not only as implants to be implemented in the clinic, but also as models to understand mechanisms and processes related to cartilage development and repair. The knowledge generated using these models will be instrumental in moving to the next generation of cartilage repair approaches, namely those inducing regeneration in situ, based on the recruitment of resident cells.

Tissue engineering for paediatric patients.

The effects of oncological treatment, congenital anomalies, traumatic injuries and post-infection damage critically require sufficient amounts of tissue for structural and functional surgical reconstructions. The patient’s own body is typically the gold standard source of transplant material, but in children autologous tissue is available only in small quantities and with severe morbidity at donor sites. Engineering of tissue grafts starting from a small amount of autologous material, combined with suitable surgical manipulation of the recipient site, is expected to enhance child and adolescent health, and to offer functional restoration for long-term wellbeing. Moreover, engineered tissues based on patient-derived cells represent invaluable models to investigate mechanisms of disease and to develop/test novel therapeutic approaches. In view of these great opportunities, here we introduce the currently limited successful implementation of tissue engineering in paediatric settings and discuss the open challenges in the field. A particular focus is on the specific needs and envisioned strategies in the areas of bone and osteochondral regeneration in children.

Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies.

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.

Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling.

Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop. Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier. Arrays can be designed and produced within hours according to the user's requirements. They are processed in a fully automatic workflow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature.