Small-scale end-to-end mAb platform with a continuous and integrated design

Fully continuous manufacturing of therapeutic proteins is an emerging trend in the biopharmaceutical industry. Integration of the upstream and downstream processes and conversion to continuous manufacturing leads to increased productivities, decreased equipment size, improved product quality, and overall reduced production costs. An integrated continuous bioprocess (ICB) from the early stages of process development can accelerate the transfer of a new drug candidate to commercial manufacturing. The process presented in this work is a proof-of-concept of an end-to-end monoclonal antibody (mAb) production platform at small scale. It was implemented by a 200 mL ATF perfusion bioreactor, integrated with a single lab-scale chromatography system, performing all purification steps for the mAb from the cell culture harvest in a continuous way. The downstream process consisted of a periodic twin-column capture with protein A resin, followed by a virus inactivation step, a cation exchange step in bind-elute mode, and an anion exchange step in flow-through mode. MAbs were produced for 17 days in a high cell density perfusion culture of CHO cells and purified continuously with a recovery yield of up to 60 % by the following downstream train. A 5-log reduction of host cell protein levels revealed that impurities were sufficiently removed. A consistent glycosylation pattern of the purified product was ensured by the steady-state operation of the process. With this proof-of-concept, we demonstrated the technical feasibility of a fully continuous end-to-end process with a compact design, integrating several unit operations in a single chromatography station and using small-size equipment for the upstream and downstream operations. The work presented here can become a useful development tool for the future of continuous bioprocesses

Clinical and biochemical outcomes of cinacalcet treatment of familial hypocalciuric hypercalcemia: a case series

<p>Abstract</p> <p>Introduction</p> <p>Familial hypocalciuric hypercalcemia is a rare benign autosomal-dominant genetic disease with high penetrance. In most cases, patients with familial hypocalciuric hypercalcemia experience unspecific physical discomfort or asymptomatic disease. These patients are typically characterized by mild to moderately increased blood ionized calcium and a normal to slightly elevated serum parathyroid hormone.</p> <p>Case presentation</p> <p>Four female patients with familial hypocalciuric hypercalcemia with inactivating mutations in the <it>CaSR </it>gene were included in the treatment study. Three patients were related: two were siblings and one was the daughter of one of these. The ages of the related patients were 51 years, 57 years and 35 years. All three patients were carriers of the same mutation. The fourth patient, unrelated to the others, was 53 years old, and a carrier of a novel and previously unknown mutation leading to familial hypocalciuric hypercalcemia. All four patients were Caucasians of Danish nationality. Biochemically, all patients had elevated blood ionized calcium, serum parathyroid hormone, serum magnesium and total serum calcium, except one, whose serum parathyroid hormone was within the normal range prior to treatment. All patients were treated with cinacalcet in a dosage of 30 mg to 60 mg per day.</p> <p>Conclusion</p> <p>Three months after the initiation of cinacalcet treatment, all our patients experiencing clinical signs of hypercalcemia had improved in self -reported well-being and in biochemical parameters. None of our patients suffered adverse events to cinacalcet treatment. Biochemical markers of calcium homeostasis were improved and remained stable during the observation period of 12 months (two patients), 24 and 36 months, in both the symptomatic and the asymptomatic patients.</p

The P2X7 Receptor: A Key Player in Immune-Mediated Bone Loss?

Inflammatory diseases are often multiorganic diseases with manifestations not related directly to the primary affected organ. They are often complicated by a generalized bone loss that subsequently leads to osteoporosis and bone fractures. The exact mechanism for the accompanying bone loss is not understood in full detail, but factors such as glucocorticoid treatment, immobilization, malnutrition, and insufficient intake of vitamin D play a role. However, it has become evident that the inflammatory process itself is involved and the resulting bone loss is termed immune-mediated bone loss. It stems from an increase in bone resorption and the pro-inflammatory cytokines tumor necrosis factor alpha and interleukin 1 beta and has been shown to not only mediate the inflammatory response but also to strongly stimulate bone degradation. The purinergic P2X7 receptor is central in the processing of these two cytokines and in the initiation of the inflammatory response, and it is a key molecule in the regulation of both bone formation and bone resorption. The aim of this review is therefore to provide evidence-based novel hypotheses of the role of ATP-mediated purinergic signalling via the P2X7 receptor in immune-mediated bone loss and -osteoporosis

The Evidence for Efficacy of Osteoporosis Treatment in Men with Primary Osteoporosis: A Systematic Review and Meta-Analysis of Antiresorptive and Anabolic Treatment in Men

Purpose. Fragility fractures in men constitute a major worldwide public health problem with a life-time risk of 13%. It cannot be directly inferred that antiosteoporotic drugs effective in women have the same effect in men. Our aim was to appraise the existing evidence for efficacy of osteoporosis treatment in men. Methods. This study was a systematic review of the published literature on the clinical efficacy of medical osteoporosis therapy in the reduction of fracture risk in men (age > 50 years). Studies included were randomised, placebo-controlled trials of men. Results. Five BMD studies of antiresorptive treatment were included. All studies showed an increase in BMD, but there was only a nonsignificant trend in the reduction of clinical fractures. Three BMD studies of anabolic treatment with teriparatide were also included. These showed a significant mean increase in spine BMD and for vertebral fractures a non-significant trend towards a reduction was seen. Conclusion. The evidence of medical osteoporosis treatment in men is scant and inconclusive due to the lack of prospective RCT studies with fracture prevention as primary end point. So far, all evidence is based on BMD increases in small RCT studies showing BMD increases comparable to those reported in postmenopausal women

A Complementary and Revised View on the N-Acylation of Chitosan with Hexanoyl Chloride

The modification of the biobased polymer chitosan is a broad and widely studied field. Herein, an insight into the hydrophobization of low-molecular-weight chitosan by substitution of amino functionalities with hexanoyl chloride is reported. Thereby, the influence of the pH of the reaction media was investigated. Further, methods for the determination of the degree of substitution based on 1H-NMR, FTIR, and potentiometric titration were compared and discussed regarding their accuracy and precision. 1H-NMR was the most accurate method, while FTIR and the potentiometric titration, though precise and reproducible, underlie the influence of complete protonation and solubility issues. Additionally, the impact of the pH variation during the synthesis on the properties of the samples was investigated by Cd2+ sorption experiments. The adjusted pH values during the synthesis and, therefore, the obtained degrees of substitution possessed a strong impact on the adsorption properties of the final material

Waterborne phenolic, triazine-based porous polymer particles for the removal of toxic metal ions

Highly functional and also highly porous materials are presenting great advantages for applications in energy storage, catalysis and separation processes, which is why a continuous development of new materials can be seen. To create a material combining the promising potential interactions of triazine groups with the electrostatic or hydrogen bonding interactions of phenolic groups, a completely new polymeric resin was synthesized. From an eco-friendly dispersion polymerization in water, a copolymer network was obtained, which includes nine hydroxyl groups and one s-triazine ring per repetition unit. The polymer forms highly porous particles with specific surface areas up to 531 ​m2/g and a negative streaming potential over a great pH range. The adsorption isotherms of Ni2+, Cd2+, and Pb2+ were studied in more detail achieving very good adsorption capacities (16 mg Ni2+/g, 24 mg Cd2+/g, and 90 mg Pb2+/g). Demonstrating excellent properties for adsorption applications. The adsorbent exhibited selectivity for the adsorption of Pb2+ over more commonly occurring but non-toxic metal ions such as Fe2+, Ca2+, Mg2+, and K+. Furthermore, reusability of the material was demonstrated by facile, quantitative desorption of adsorbed Pb2+ with a small amount of diluted HCl, circumventing organic chelators. Subsequently, adsorption was carried out without decrease in adsorption performance

The role of the P2X7 receptor on bone loss in a mouse model of inflammation-mediated osteoporosis

In inflammatory autoimmune diseases, bone loss is frequent. In most cases, secondary osteoporosis is caused by treatment with systemic glucocorticoid. However, the pathogenesis behind the bone loss is presumed multifactorial. We aimed to elucidate the role of the P2X7 receptor on bone mineral density (BMD), microarchitecture, and bone strength in a standardized mouse model of inflammation-mediated osteoporosis (IMO). In total 146 mice completed our protocol, 70 wild type (WT) mice and 76 P2X7−/− (knockout, KO). BMD at the femur and spine decreased significantly from baseline to day 20 in the WT IMO mice (p < 0.01). In the WT vehicle, KO vehicle and KO IMO, no significant BMD changes were found. Bone strength showed a lower mid-shaft max strength (p = 0.038) and also a non-significant trend towards lower strength at the femoral neck of the WT IMO group. Trabecular bone volume fraction (BV/TV) and connectivity density (CD) after 20 days were significantly decreased in the WT IMO group (p = 0.001). In contrast, the WT vehicle and KO vehicle, BV/TV and CD did no change at 20 days. Cortical bone revealed no significant microarchitectural changes after 20 days in the WT IMO group, whereas the total cortical area increased significantly in WT vehicle and KO IMO after 20 days (5.2% and 8.8%, respectively). In conclusion, the P2X7 receptor KO mice did not respond to inflammation with loss of BMD whereas the WT mice had a significant loss of BMD, bone strength and trabecular microarchitecture, demonstrating a role for the P2X7 receptor in inflammatory bone loss

Coatings of different carbon nanotubes on platinum electrodes for neuronal devices: Preparation, cytocompatibility and interaction with spiral ganglion cells

Cochlear and deep brain implants are prominent examples for neuronal prostheses with clinical relevance. Current research focuses on the improvement of the long-term functionality and the size reduction of neural interface electrodes. A promising approach is the application of carbon nanotubes (CNTs), either as pure electrodes but especially as coating material for electrodes. The interaction of CNTs with neuronal cells has shown promising results in various studies, but these appear to depend on the specific type of neurons as well as on the kind of nanotubes. To evaluate a potential application of carbon nanotube coatings for cochlear electrodes, it is necessary to investigate the cytocompatibility of carbon nanotube coatings on platinum for the specific type of neuron in the inner ear, namely spiral ganglion neurons. In this study we have combined the chemical processing of as-delivered CNTs, the fabrication of coatings on platinum, and the characterization of the electrical properties of the coatings as well as a general cytocompatibility testing and the first cell culture investigations of CNTs with spiral ganglion neurons. By applying a modification process to three different as-received CNTs via a reflux treatment with nitric acid, long-term stable aqueous CNT dispersions free of dispersing agents were obtained. These were used to coat platinum substrates by an automated spray-coating process. These coatings enhance the electrical properties of platinum electrodes, decreasing the impedance values and raising the capacitances. Cell culture investigations of the different CNT coatings on platinum with NIH3T3 fibroblasts attest an overall good cytocompatibility of these coatings. For spiral ganglion neurons, this can also be observed but a desired positive effect of the CNTs on the neurons is absent. Furthermore, we found that the well-established DAPI staining assay does not function on the coatings prepared from single-wall nanotubes. © 2016 Burblies et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.DFG/EXC 1077/1 “Hearing4all

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24 h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL–NK cell anti-melanoma therapies

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments