Good Manufacturing Practice Guidelines Research Articles

Production of [211At]NaAt solution under GMP compliance for investigator-initiated clinical trial.

The alpha emitter astatine-211 (211At) is garnering attention as a novel targeted alpha therapy for patients with refractory thyroid cancer resistant to conventional therapy using beta emitter radioiodine (131I). Herein, we aimed to establish a robust method for the manufacturing and quality control of [211At]NaAt solution for intravenous administration under the good manufacturing practice guidelines for investigational products to conduct an investigator-initiated clinical trial. 211At was separated and purified via dry distillation using irradiated Bi plates containing 211At obtained by the nuclear reaction of 209Bi(4He, 2n)211At. After purification, the 211At trapped in the cold trap was collected in a reaction vessel using 15 mL recovery solution (1% ascorbic acid and 2.3% sodium hydrogen carbonate). After stirring the 211At solution for 1h inside a closed system, the reaction solution was passed through a sterile 0.22μm filter placed in a Grade A controlled area and collected in a product vial to prepare the [211At]NaAt solution. According to the 3-lot tests, decay collected radioactivity and radiochemical yield of [211At]NaAt were 78.8 ± 6.0 MBq and 40 ± 3%, respectively. The radiochemical purity of [211At]At- obtained via ion-pair chromatography at the end of synthesis (EOS) was 97 ± 1%, and remained > 96% 6h after EOS; it was detected at a retention time (RT) 3.2-3.3min + RT of I-. LC-MS analysis indicated that this principal peak corresponded with an astatide ion (m/z = 210.988046). In gamma-ray spectrometry, the 211At-related peaks were identified (X-ray: 76.9, 79.3, 89.3, 89.8, and 92.3keV; γ-ray: 569.7 and 687.0keV), whereas the peak at 245.31keV derived from 210At was not detected during the 22h continuous measurement. The target material, Bi, was below the 9 ng/mL detection limit in all lots of the finished product. The pH of the [211At]NaAt solution was 7.9-8.6; the concentration of ascorbic acid was 9-10mg/mL. Other quality control tests, including endotoxin and sterility tests, confirmed that the [211At]NaAt solution met all quality standards. We successfully established a stable method of [211At]NaAt solution that can be administered to humans intravenously as an investigational product.

Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products

BackgroundMesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. MethodsTo circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. Results and ConclusionsOur findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.

ANALYSIS OF CHEMICAL CONTAMINANTS IN SEVVIYADHI CHOORANAM

Aim: The aim of the study was to analyze the chemical contaminants such as heavy metals, aflatoxins and pesticide residues in Siddha polyherbal formulation Sevviyadhi Chooranam, in powder form is indicated for the treatment and management of Sinusitis. Materials and Methods: Sevviyadhi Chooranam, the Siddha polyherbal formulation was prepared as per GMP (Good Manufacturing Practices) guidelines. The formulation was analyzed for heavy metals such as mercury, arsenic, lead and cadmium, aflatoxins such as aflatoxin B1, B2, G1, G2 and pesticide residues such as organochlorine pesticides, organophosphorus pesticides, organo carbamates and pyrethroids at Noble Research Solutions, Kolathur, Chennai. Results: Heavy Metal analysis of SC showed that the presence of Lead about 0.26PPM, Arsenic about 0.61 PPM, Mercury and Cadmium in below detection level. Aflatoxin assay of SC resulted absence of Aflatoxin B1, B2, G1 and G2. Pesticide residue analysis revealed the absence of organochloride pesticides, organophosphorus pesticides, organo carbamates and pyrethroids. Conclusion: The findings of this study revealed the presence of heavy metals such as lead and arsenic in limited amount and absence of mercury and cadmium, and absence of aflatoxins and pesticide residue in the Siddha polyherbal formulation Sevviyadhi chooranam. The study ensured that Sevviyadhi chooranam was free from chemical contaminants such as heavy metals, aflatoxins and pesticide residue and validated the safety of SC for therapeutic usage in treatment of sinusitis.

Radionuclide Cisternography with [64Cu]Cu-DOTA.

Radionuclide cisternography (RNC) is a method for conducting imaging of the cerebrospinal system and can be used to identify cerebrospinal fluid leaks. So far, RNC has commonly employed radiopharmaceutical agents suitable only for single-photon emission tomography techniques, which are thus lacking in terms of image resolution and can potentially lead to false-negative results. Therefore, [64Cu]Cu-DOTA was investigated as an alternative radiopharmaceutical for RNC, employing positron emission tomography (PET) instead of single-photon emission tomography. A formulation of [64Cu]Cu-DOTA was produced according to the guidelines for good manufacturing practice. The product met the requirements of agents suitable for intrathecal application. [64Cu]Cu-DOTA was administered to a patient and compared to the approved scintigraphic RNC agent, [111In]In-DTPA. While no cerebrospinal fluid leak was detected with [111In]In-DTPA, [64Cu]Cu-DOTA RNC exhibited a posterolateral leak between the vertebral bodies C1 and C2. Thus, in this patient, PET RNC with [64Cu]Cu-DOTA was superior to RNC with [111In]In-DTPA. Since radiopharmaceuticals have a very good safety profile regarding the occurrence of adverse events, PET RNC with [64Cu]Cu-DOTA may become an attractive alternative to scintigraphic methods, and also to computed tomography or magnetic resonance imaging, which often require contrast agents, causing adverse events to occur much more frequently.

The effect of serum origin on cytokines induced killer cell expansion and function

BackgroundCytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function.MethodsPeripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture.ResultsCell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression.ConclusionhPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.

Phytochemical, Physicochemical and HPTLC Analysis of Siddha Herbal Formulation Muppirandai Chooranam

Aim: The aim of the study was to evaluate the Phytochemical, Physicochemical, HPTLC analysis of Siddha herbal formulation Muppirandai Chooranam (MRC), indicated for various ailments including menorrhagia.
 Study design
 Place of study: The Phytochemical and Physicochemical analysis were conducted at the Tamilnadu Dr. MGR Medical University, located at no.69, Anna salai, Guindy, Chennai-32. The high-performance thin layer chromatography (HPTLC) was carried out at Noble research solutions, Kolathur, Chennai -99.
 Methodology: Siddha formulation Muppirandai Chooranam was prepared as per good manufacturing practices (GMP) guidelines and the phytochemical analysis, physicochemical analysis were carried out at the Tamilnadu Dr. MGR Medical University, located at no.69, Anna salai, Guindy, Chennai-32. As per PLIM guidelines (Pharmacopeia Laboratory Of Indian Medicine) in accordance with the guidelines established by AYUSH (Ayurveda, Yoga, Unani, Siddha, Homoeopathy), the governing body for traditional health systems in India. The HPTLC analysis was conducted at Noble Research Solutions, Kolathur, Chennai -99.
 Results: The Phytochemical screening of MRC shown the presence of Alkaloids, Carbohydrates, Saponins, Flavanoids, Diterpenes, Gum and Mucilage. The physicochemical analysis of MRC revealed that it had a loss on drying of 0.42% at 105°C, a total ash content of 23.3%, an acid-insoluble ash content of 3.02%, a water-soluble ash content of 2.30%, and a water-soluble extract content of 17.67%, and alcohol soluble extract content of 5.23%.HPTLC finger printing analysis of the sample reveals the presence of five prominent peaks corresponds to the presence of five versatile phytocomponents present within it. Rf value of the peaks ranges from 0.02 to 0.37.
 Conclusion: From the findings, it is concluded that it shown the compendious understanding of presence of Phytochemical components, Physicochemical characteristics, and HPTLC analysis of MRC and it is competent to assess the quality profile of Muppirandai Chooranam as a reference standard for the development of the standardised pharmaceutical product.

Platelet lysate for expansion or osteogenic differentiation of bone marrow mesenchymal stem cells for 3D tissue constructs

BackgroundThe use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems. MethodsWe evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining. ResultsCell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT. ConclusionsPLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs.

Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues

BackgroundInduced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness.MethodsiPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods.ResultsMore attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective “whitish” outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type.ConclusionsThe xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.

Corneal Endothelial-like Cells Derived from Induced Pluripotent Stem Cells for Cell Therapy.

Corneal endothelial dysfunction is one of the leading causes of corneal blindness, and the current conventional treatment option is corneal transplantation using a cadaveric donor cornea. However, there is a global shortage of suitable donor graft material, necessitating the exploration of novel therapeutic approaches. A stem cell-based regenerative medicine approach using induced pluripotent stem cells (iPSCs) offers a promising solution, as they possess self-renewal capabilities, can be derived from adult somatic cells, and can be differentiated into all cell types including corneal endothelial cells (CECs). This review discusses the progress and challenges in developing protocols to induce iPSCs into CECs, focusing on the different media formulations used to differentiate iPSCs to neural crest cells (NCCs) and subsequently to CECs, as well as the characterization methods and markers that define iPSC-derived CECs. The hurdles and solutions for the clinical application of iPSC-derived cell therapy are also addressed, including the establishment of protocols that adhere to good manufacturing practice (GMP) guidelines. The potential risks of genetic mutations in iPSC-derived CECs associated with long-term in vitro culture and the danger of potential tumorigenicity following transplantation are evaluated. In all, this review provides insights into the advancement and obstacles of using iPSC in the treatment of corneal endothelial dysfunction.

Pharmacological Evaluation of Ovulation Inducing Potential of Siddha Herbomineral Formulation Arputha Mathirai on Estradiol Valerate Induced Poly Cystic Ovarian Syndrome Wistar Albino Rat Model

Aim: The aim of this study is to examine the potential of Arputha Mathirai, a tablet-based Siddha herbomineral formulation, in promoting ovulation in Wistar Albino rats with polycystic ovary syndrome (PCOS) induced by Estradiol Valerate (EV).
 Place of Study: The study took place at C.L. Baid Metha College of Pharmacy located in Thorapakkam, Chennai - 600 092, Tamil Nadu.
 Methodology: Arputha Mathirai, the Siddha herbomineral formulation, was prepared in accordance with Good Manufacturing Practices (GMP) guidelines. Prior to conducting the study, approval was obtained from the Institutional Animal Ethics Committee (IAEC). The research adhered to ethical principles and guidelines established by the committee responsible for overseeing and regulating animal experimentation, ensuring proper control and supervision.
 Female Wistar Albino rats were selected as the preferred rodent species for the study. Polycystic ovary syndrome (PCOS) was induced in the animals by administering subcutaneous injection of 100μg Estardiol valerate (EV). The reproductive cycles of the rats were synchronized to ensure consistency.
 The rats were divided into four groups, each containing six rats, as follows:
 
 Group I: Normal Control animals received 1 ml/kg of Sodium Carboxymethyl Cellulose (CMC) solution.
 Group II: Rats were orally administered Arputha Mathirai at a dosage of 100 mg/kg for 10 days.
 Group III: Rats were orally administered Arputha Mathirai at a dosage of 200 mg/kg for 10 days.
 Group IV: Received Clomiphene at a dosage of 10 mg/kg and served as the standard group.
 
 At the end of the study, blood samples were collected from the rats through retro-orbital and cardiac puncture. The levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, and progesterone were estimated using ELISA method. Ovaries from the experimental rats were dissected out for histopathological studies.
 Results: The results consistently showed that the higher dosage of the drug (group III) had a more pronounced effect in normalizing hormone levels compared to the lower dosage group. However, it is important to note that the lower dosage group also exhibited effectiveness. While the results in both groups did not closely match those of the standard group (group IV), the drug Arputha Mathirai demonstrated significant potential in the experimental groups, supporting its efficacy in inducing ovulation.
 Furthermore, the histopathological analysis confirmed the drug's potential in inducing ovulation, particularly in the higher dosage group (group III) compared to the lower dosage group (group II).
 Overall, the findings suggest that Arputha Mathirai has promising ovulation-inducing activity, with the higher dosage showing more pronounced effects.
 Conclusion: It is hypothesized that the bioactive phytocompounds present in Arputha Mathirai may exert their effects at various stages to restore hormone levels and reverse the pathological condition associated with PCOS. These natural compounds have the potential to regulate hormone levels and promote ovulation. However, it is important to acknowledge that there is a scarcity of published evidence regarding the specific ability of these phytochemicals to address the underlying causes of the condition. Further research is needed to explore the mechanisms by which these compounds act and their potential in addressing the root causes of PCOS.

Cryopreserved clinical-grade human embryonic stem cell-derived dopaminergic progenitors function in Parkinson's disease models

AimParkinson's Disease (PD) is a common age-related neurodegenerative disorder with a rising prevalence. Human pluripotent stem cells have emerged as the most promising source of cells for midbrain dopaminergic (mDA) neuron replacement in PD. This study aimed to generate transplantable mDA progenitors for treatment of PD. Materials and methodsHere, we optimized and fine-tuned a differentiation protocol using a combination of small molecules and growth factors to induce mDA progenitors to comply with good manufacturing practice (GMP) guidelines based on our clinical-grade human embryonic stem cell (hESC) line. Key findingsThe resulting mDA progenitors demonstrated robust differentiation and functional properties in vitro. Moreover, cryopreserved mDA progenitors were transplanted into 6-hydroxydopamine-lesioned rats, leading to functional recovery. SignificanceWe demonstrate that our optimized protocol using a clinical hESC line is suitable for generating clinical-grade mDA progenitors and provides the ground work for future translational applications.

An optimized cultivation method for future in vivo application of γδ T cells.

γδ T cells, with their properties of both the innate and acquired immune systems, are suitable candidates for cellular immunotherapy in cancer. Because of their non-major histocompatibility complex (MHC) binding T cell receptor, allogenic transfer is feasible without relevant graft versus host reactions. In recent years, much experience has been gained with ex vivo expansion and stimulation of γδ T cells using bisphosphonates and Interleukin 2. Unfortunately, many current stimulation protocols are based on the use of xenogenic materials and other potentially hazardous supplements, which conflicts with basic principles of Good Manufacturing Practice (GMP). Adherence to the concept and current guidelines of GMP is state of the art for production of Advanced Therapy Medicinal Products (ATMP) like cell therapeutics and a necessity for clinical use under a regulatory perspective. In this study, we developed a new stimulation protocol that induces a marked increase of γδ T cell counts and allows for an easier transition from research to clinical applications with minimized regulatory workload. It reliably leads to a cell product with a purity of more than 90% γδ T cells and improved in vitro anti-tumor activity compared to our previous standard procedure. Furthermore, by investigating correlations between properties of unstimulated γδ T cells and proliferation rate as well as degranulation ability of stimulated γδ T cells, we can draw conclusions about suitable donors. Finally, we examined if expansion can be improved by pulsing zoledronate and/or using Interleukin 15 with or without Interleukin 2. Significant improvements can be achieved with respect to intrinsic and antibody-dependent cell-mediated cytotoxicity. Our results demonstrate that the stimulation protocol presented here leads to an improved γδ T cell product for future clinical applications.

Optimization of 177Lu-labelling of DOTA-TOC, PSMA-I&T and FAPI-46 for clinical application

Background177Lu-radiopharmaceuticals are routinely used for the treatment of various tumor entities. The productions of radiopharmaceuticals follow strict good-manufacturing practice guidelines and synthesis optimizations thereof have a strong impact on e.g. the quality of the product, radiation safety and costs. The purpose of this study is to optimize the precursor load of three radiopharmaceuticals. For that, different precursor loads were evaluated and compared to previously reported findings.ResultsAll three radiopharmaceuticals were successfully synthesized in high radiochemical purities and yields on the ML Eazy. The precursor load was optimized for [177Lu]Lu-FAPI-46 from 27.0 to 9.7 µg/GBq, for [177Lu]Lu-DOTATOC from 11 to 10 µg/GBq and for [177Lu]Lu-PSMA-I&T from 16.3 to 11.6 µg/GBq.ConclusionsWe successfully reduced the precursor load for all three radiopharmaceuticals while maintaining their quality.

Quality of Co-trimoxazole Tablets Marketed in Nairobi County, Kenya

Background: Poor-quality medicines reduce the therapeutic efficacy of medicines, negatively impacting the treatment outcomes, prevention, and management of fatal infections. This cross-sectional study evaluated the quality of selected co-trimoxazole tablets marketed in Nairobi County, Kenya.
 Methodology: A total of 42 samples categorized into ten brands were evaluated for active pharmaceutical ingredient (API) content and uniformity of weight following United State Pharmacopeia (USP). Additionally, a visual inspection of the packaging and labelling was performed to confirm whether they adhered to World Health Organization's Good Manufacturing Practice (GMP) guidelines.
 Results: The majority of the samples were of local origin (70%). By 23rd October 2019, the retention status of one of the ten brands documented was not documented in the Pharmacy and Poisons Board retention register. Of the 42 samples analyzed, 97.6% and 69.01% complied with United States Pharmacopeia (USP) specifications for uniformity of weight and API, respectively, while all samples adhered to packaging and labelling requirements.
 Conclusion: This study has demonstrated that most co-trimoxazole tablets tested complied with USP requirements. Additionally, it has provided evidence of the presence of poorquality co-trimoxazole medicines that could compromise the treatment of infectious diseases. Therefore, regular surveillance and stringent penalties that ensure quality medicines are essential.

Identification of monoclonal antibody drug substances using non-destructive Raman spectroscopy

Monoclonal antibodies provide highly specific and effective therapies for the treatment of chronic diseases. These protein-based therapeutics, or drug substances, are transported in single used plastic packaging to fill finish sites. According to good manufacturing practice guidelines, each drug substance needs to be identified before manufacturing of the drug product. However, considering their complex structure, it is challenging to correctly identify therapeutic proteins in an efficient manner. Common analytical techniques for therapeutic protein identification are SDS-gel electrophoresis, enzyme linked immunosorbent assays, high performance liquid chromatography and mass spectrometry-based assays. Although effective in correctly identifying the protein therapeutic, most of these techniques need extensive sample preparation and removal of samples from their containers. This step not only risks contamination but the sample taken for the identification is destroyed and cannot be re-used. Moreover, these techniques are often time consuming, sometimes taking several days to process. Here, we address these challenges by developing a rapid and non-destructive identification technique for monoclonal antibody-based drug substances. Raman spectroscopy in combination with chemometrics were used to identify three monoclonal antibody drug substances. This study explored the impact of laser exposure, time out of refrigerator and multiple freeze thaw cycles on the stability of monoclonal antibodies. and demonstrated the potential of using Raman spectroscopy for the identification of protein-based drug substances in the biopharmaceutical industry.

67 Ga-PSMA I&T for Radioguided Surgery of Lymph Node Metastases in Patients With Biochemical Recurrence of Prostate Cancer.

Radioguided lymph node dissection in patients with prostate cancer, and suffering from biochemical recurrence has been described thoroughly during the past few years. Several prostate-specific membrane antigen (PSMA)-directed ligands labeled with 111 In, 99m Tc, and 68 Ga have been published; however, limitations regarding availability, short half-life, high costs, and unfavorable high energy might restrict frequent use. This study aims at introducing 67 Ga as a promising radionuclide for radioguided surgery. Retrospective analysis was performed on 6 patients with 7 PSMA-positive lymph node metastases. 67 Ga-PSMA I&T (imaging and therapy) was synthesized in-house and intravenously applied according to §13 2b of the German Medicinal Products Act. Radioguided surgery was performed 24 hours after injection of 67 Ga-PSMA I&T using a gamma probe. Patient urine samples were collected. Occupational and waste dosimetry was performed to describe hazards arising from radiation. 67 Ga-PSMA application was tolerated without adverse effects. Five of 7 lymph nodes were detected on 22-hour SPECT/CT in 4 of 6 patients. During surgery, all 7 lymph node metastases were identified by positive gamma probe signal. Relevant accumulation of 67 Ga was observed in lymph node metastases (32.1 ± 15.1 kBq). Histology analysis of near-field lymph node dissection revealed more lymph node metastases than PET/CT (and gamma probe measurements) identified. Waste produced during inpatient stay required decay time of up to 11 days before reaching exemption limits according to German regulations. Radioguided surgery using 67 Ga-PSMA I&T is a safe and feasible option for patients suffering from biochemical recurrence of prostate cancer. 67 Ga-PSMA I&T was successfully synthesized according to Good Manufacturing Practice guidelines. Radioguided surgery with 67 Ga-PSMA I&T does not lead to relevant radiation burden to urology surgeons and represents a novel interdisciplinary approach in nuclear medicine and urology.

Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNF-β), osteopontin, nerve growth factor beta (NGFβ), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD.

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Marketing Regulatory Oversight of Advanced Therapy Medicinal Products in Europe.

Advanced therapy medicinal products (ATMP) in the European Union (EU) are regulated by Regulation 1394/2007 and comprise gene and cell therapy and tissue-engineered products. Under this framework, ATMP are authorised by the centralised procedure, coordinated by the European Medicines Agency (EMA), whereas clinical trial authorisations remain at the remit of each National Competent Authority. The Committee for Advanced Therapies is responsible for the scientific evaluation of the marketing authorisation applications and for generating a draft opinion that goes to the Committee for Human Medicinal Products for a final opinion. For every application, data and information relating to manufacturing processes and quality control of the active substance and final product have to be submitted for assessment together with data from non-clinical and clinical safety and efficacy studies. Technical requirements for ATMP are defined in the legislation, and guidance for different products is available through several EMA/CAT guidelines.Due to the diverse and complex nature of ATMP, a need for some regulatory flexibility was recognised. Thus, a risk-based approach was introduced in Regulation 1394/2007 allowing adapted regulatory requirements. This has led, for instance, to the development of good manufacturing practice (GMP) guidelines specific for ATMP. This, together with enhanced regulatory support, has allowed an increasing number of successful marketing authorisation applications resulting in 25 licensed ATMP in the EU, mainly gene therapy medicinal products. The promise of messenger RNA and genome editing technologies as therapeutic tools make the future for these innovative medicinal products look even brighter.This chapter reviews the regulatory landscape together with some of the support initiatives developed for ATMP in the EU.

Articular Cartilage Repair After Implantation of Hyaline Cartilage Beads Engineered From Adult Dedifferentiated Chondrocytes: Cartibeads Preclinical Efficacy Study in a Large Animal Model

Background: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions. Hypothesis: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. Study Design: Controlled laboratory study. Methods: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. Results: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). Conclusion: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. Clinical Relevance: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.