Good Manufacturing Practice Compliant Production Research Articles

O-31 - Introduction of terbium-161 into the clinics through its automated purification and the Good Manufacturing Practice compliant production of [161Tb]Tb-DOTA-LM3

O-31 - Introduction of terbium-161 into the clinics through its automated purification and the Good Manufacturing Practice compliant production of [161Tb]Tb-DOTA-LM3

Development of a fully automated low-pressure [11 C]CO carbonylation apparatus.

[11C]carbon monoxide ([11C]CO) is a versatile synthon for radiolabeling of drug‐like molecules for imaging studies with positron emission tomography (PET). We here report the development of a novel, user‐friendly, fully automated, and good manufacturing practice (GMP) compliant low‐pressure synthesis module for 11C‐carbonylation reactions using [11C]CO. In this synthesis module, [11C]CO was reliably prepared from cyclotron‐produced [11C]carbon dioxide ([11C]CO2) by reduction over heated molybdenum and delivered to the reaction vessel within 7 min after end of bombardment, with an overall radiochemical yield (RCY) of 71%. [11C]AZ13198083, a histamine type‐3 receptor ligand, was used as a model compound to assess the functionality of the radiochemistry module. At full batch production conditions (55 μA, 30 min), our newly developed low‐pressure 11C‐carbonylation apparatus enabled us to prepare [11C]AZ13198083 in an isolated radioactivity of 8540 ± 1400 MBq (n = 3). The radiochemical purity of each of the final formulated batches exceeded 99%, and all other quality control tests results conformed with specifications typically set for carbon‐11 labeled radiopharmaceuticals. In conclusion, this novel radiochemistry system offers a convenient GMP compliant production drugs and radioligands for imaging studies in human subjects.

GMP production of [68Ga]Ga-BOT5035 for imaging of liver fibrosis in microdosing phase 0 study

IntroductionEarly detection of liver fibrosis and monitoring response to treatment crucial for the management of patients are currently not feasible in clinical practice. Platelet derived growth factor receptor β (PDGFR-β) expression is regarded as a potential biomarker to determine the stages of fibrotic diseases including liver fibrosis. [68Ga]Ga-BOT5035 comprising a bicyclic peptide was developed for specific targeting of PDGFR-β overexpressed in pathological fibrosis. The realization of microdosing phase 0 study using [68Ga]Ga-BOT5035 positron emission tomography required automated good manufacturing practice (GMP) compliant production of [68Ga]Ga-BOT5035 presented herein. Moreover, the investigation of radiation dosimetry was conducted to ensure possibility of multiple annual examinations for disease monitoring in clinical setup. MethodsThe active pharmaceutical ingredient starting material BOT5035 (GMP grade) was provided by BiOrion Technologies BV. The 68Ga-labelling process was developed and automated using synthesis platform (Modular-Lab PharmTrace, Eckert & Ziegler), disposable cassettes for 68Ga-labelling, and pharmaceutical grade 68Ge/68Ga generator (GalliaPharm®) purchased from Eckert & Ziegler. Radiolysis sensitive BOT5035 required development and systematic optimization of the labelling synthesis parameters such as time, temperature, precursor concentration, radical scavenger, buffer concentration and pH. The validation process was conducted with regard to the product quality and quantity, as well as production reproducibility. Human organ equivalent doses and total body effective doses were calculated using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1), based on ex vivo organ distribution in Sprague-Dawley rats. ResultsThe GMP compliant automated production of [68Ga]Ga-BOT5035 with on-line documentation demonstrated high reproducibility. The time for the labelling synthesis and quality control was approximately 60 min. The non-decay corrected radiochemical yield and radiochemical purity of the radiopharmaceutical were 43.7 ± 7.6% (n = 3, process validation) and 97.7 ± 0.4% (n = 3, process validation), respectively. Predefined acceptance criteria were met for the sterility, endotoxins level, radionuclidic purity and residual solvent content. The stability at ambient temperature was controlled for 120 min with approved results. Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. The dose-limiting organs were kidney and bone marrow. The total effective dose as limiting parameter would allow for up to 3–4 PET scans per annum. ConclusionThe fully automated and GMP compliant production of [68Ga]Ga-BOT5035 was developed and thoroughly validated. The radiopharmaceutical was approved by Swedish Medicinal Products Agency and the Ethical Review Authority for the Phase 0 clinical study of the quantitative imaging of liver fibrosis. Human dosimetry calculations extrapolated from animal experiment indicated possibility of 3–4 PET examinations per year.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

BackgroundThe production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor.MethodsFive hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions.ResultsThree cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process.ConclusionA streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

Standard Operating Procedure for the Good Manufacturing Practice-Compliant Production of Human Endometrial Stem Cells for Multiple Sclerosis.

Multiple sclerosis (MS) is the most common cause of neurological diseases. Although, there are some effective medications with regulatory approval for treating MS, they are only partially effective and cannot promote repairing of tissue damage directly which occurs in the central nervous system. Therefore, there is an essential need to develop novel therapeutic approaches for neuroprotection or repairing damaged tissue in MS. Accordingly, cell-based therapies as a novel therapeutic strategy have opened a new horizon in treatment of MS. Each setting in cell therapy has potential benefits. Human endometrial stem cells as an invaluable source for cell therapy have introduced treatment for MS. In this respect, good manufacturing practice (GMP) has a pivotal role in clinical production of stem cells. This chapter tries to describe the protocol of GMP-grade endometrial stem cells for treatment of MS.

Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors.

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

Single-Stage Preparation of Human Cartilage Grafts Generated from Bone Marrow-Derived CD271+ Mononuclear Cells.

Due to the limited self-healing capacity of articular cartilage, innovative, regenerative approaches are of particular interest. The use of two-stage procedures utilizing in vitro-expanded mesenchymal stromal cells (MSCs) from various cell sources requires good manufacturing practice-compliant production, a process with high demands on time, staffing, and financial resources. In contrast, one- stage procedures are directly available, but need a safe enrichment of potent MSCs. CD271 is a surface marker known to marking the majority of native MSCs in bone marrow (BM). In this study, the feasibility of generating a single-stage cartilage graft of enriched CD271+ BM-derived mononuclear cells (MNCs) without in vitro monolayer expansion from eight healthy donors was investigated. Cartilage grafts were generated by magnetic-activated cell sorting and separated cells were directly transferred into collagen type I hydrogels, followed by 3D proliferation and differentiation period of CD271+, CD271-, or unseparated MNCs. CD271+ MNCs showed the highest proliferation rate, cell viability, sulfated glycosaminoglycan deposition, and cartilage marker expression compared to the CD271- or unseparated MNC fractions in 3D culture. Analysis according to the minimal criteria of the International Society for Cellular Therapy highlighted a 66.8-fold enrichment of fibroblast colony-forming units in CD271+ MNCs and the only fulfillment of the MSC marker profile compared to unseparated MNCs. In summary, CD271+ MNCs are capable of generating adequate articular cartilage grafts presenting high cell viability and notable chondrogenic matrix deposition in a CE-marked collagen type I hydrogel, which can obviate the need for an initial monolayer expansion.

Good Manufacturing Practice-Compliant Production and Lot-Release of Ex Vivo Expanded Regulatory T Cells As Basis for Treatment of Patients with Autoimmune and Inflammatory Disorders.

In recent years, the exploration of regulatory T cell (Treg)-based cellular therapy has become an attractive strategy to ameliorate inflammation and autoimmunity in various clinical settings. The main obstacle to the clinical application of Treg in human is their low number circulating in peripheral blood. Therefore, ex vivo expansion is inevitable. Moreover, isolation of Treg bears the risk of concurrent isolation of unwanted effector cells, which may trigger or deteriorate inflammation upon adoptive Treg transfer. Here, we present a protocol for the GMP-compliant production, lot-release and validation of ex vivo expanded Tregs for treatment of patients with autoimmune and inflammatory disorders. In the presented production protocol, large numbers of Treg, previously enriched from a leukapheresis product by using the CliniMACS® system, are ex vivo expanded in the presence of anti-CD3/anti-CD28 expander beads, exogenous IL-2 and rapamycin during 21 days. The expanded Treg drug product passed predefined lot-release criteria. These criteria include (i) sterility testing, (ii) assessment of Treg phenotype, (iii) assessment of non-Treg cellular impurities, (iv) confirmation of successful anti-CD3/anti-CD28 expander bead removal after expansion, and (v) confirmation of the biological function of the Treg product. Furthermore, the Treg drug product was shown to retain its stability and suppressive function for at least 1 year after freezing and thawing. Also, dilution of the Treg drug product in 0.9% physiological saline did not affect Treg phenotype and Treg function for up to 90 min. These data indicate that these cells are ready to use in a clinical setting in which a cell infusion time of up to 90 min can be expected. The presented production process has recently undergone on site GMP-conform evaluation and received GMP certification from the Bavarian authorities in Germany. This protocol can now be used for Treg-based therapy of various inflammatory and autoimmune disorders.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production.

BackgroundClinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL).MethodsIn this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors.ResultsWe characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells.ConclusionsOur study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria.Clinical trialsUse of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007. Registered August 26, 2013.MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540. Registered April 23, 2013.Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027. Registered October 31, 2014.Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145. Registered August 17, 2006.A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283. Registered May 18, 2012.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0370-8) contains supplementary material, which is available to authorized users.

Standard operating procedure for the good manufacturing practice-compliant production of human bone marrow mesenchymal stem cells.

According to the European Regulation (EC 1394/2007), Mesenchymal Stem Cells expanded in culture for clinical use are considered as Advanced Therapy Medicinal Products. As a consequence, they must be produced in compliance with Good Manufacturing Practice in order to ensure safety, reproducibility, and efficacy. Here, we report a Standard Operating Procedure describing the Good Manufacturing Practice-compliant production of Bone Marrow-derived Mesenchymal Stem Cells suitable for autologous implantation in humans. This procedure can be considered as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval. Possible clinical applications concern local uses in the regeneration of bone tissue in nonunion fractures or in orthopedic and maxillofacial diseases characterized by a bone loss.