A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells

WOS: 000419607700024PubMed ID: 28685239Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-alpha), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-alpha mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-alpha mAb production from the humanized anti-TNF-alpha mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-alpha production. The production of anti-TNF-alpha in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-alpha MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [2209-A, 1919B011402737]This study has been partly funded by TUBITAK through 2209-A project number 1919B011402737 under the supervision of Sultan GULCE IZ, PhD

Mediastinal lymph node removal ameliorates cytotoxic T-lymphocyte functions in patients with non-small cell lung cancer

Introduction: Mediastinal lymph node (MLN) removal by video-assisted mediastinoscopic lymphadenectomy (VAMLA) for preoperative cancer staging was reported to be associated with increased survival. The aim of this study was to evaluate the immunologic effects of complete MLN removal by VAMLA on cytotoxic T lymphocyte (CTL) phenotype and function. Methods: Seventeen patients with non-small cell lung cancer (NSCLC) (stage cT1-4N0-3M0-1A) and 20 healthy participants were included in this study. Blood samples were collected before and 4 weeks after the procedure. Lymphocytes were isolated from the removed MLNs. CTL phenotypes and functions were evaluated by flow cytometry. Plasma levels of soluble programmed cell death protein 1 (sPD-1), soluble programmed cell death protein 1 ligand, and soluble CTL antigen 4 (sCTLA-4) were measured with enzyme-linked immunosorbent assay. Results: The ratio of the immunosenescent CTLs (CD3(+)CD8(+)CD28(-)) was increased in peripheral blood and MLNs of the patients with NSCLC compared to controls (p = 0.037), and MLN removal did not change this ratio. PD-1 and CTL antigen 4 expressions were significantly reduced in peripheral blood CTLs after MLN removal by VAMLA (p = 0.01 and p = 0.01, respectively). Granzyme A expression was significantly reduced in the peripheral blood CTLs of the patients compared to controls (p = 0.006) and MLN removal by VAMLA significantly improved Granzyme A expression in CTLs (p = 0.003). Plasma concentrations of sPD-1 and sCTLA-4 remained unchanged after VAMLA. Conclusion: CTLs in the MLNs and peripheral blood of the patients with NSCLC had an immunosenescent phenotype, increased immune checkpoint receptor expression, and impaired cytotoxicity. MLN removal by VAMLA improved these phenotypic and functional characteristics of CTLs. These changes may explain the potential contribution of VAMLA to improved survival

Application Reasons Of Adult Patients Admitted To The Family Medicine Outpatient Clinic Of Duzce University And Related Factors

Objective: The aim of study is to determine the profile of patients who applied to primary health care services(PHCS) and who have applied to the emergency department(ED) in last six months, the purpose, quality of applications, whether the applications require urgency or not and the reasons why patients prefer ED instead of PHCS. Methods: This is a descriptive study. The study sample consisted of adult patients who applied to Family Medicine Policlinic of a university in March 2019 and who met the inclusion and exclusion criteria and applied to the ED in the last six months. Data collection form prepared by us was applied to patients by us. Results: 150 patients were included in study. 62% of the participants were female and 38% were male. In last 6 months, the mean admission number was 2.45. 67.3% of the patients applied in the evening and night hours, 92% came with their own means, 91.3% were discharged; 5.4% had been hospitalized. The most common reason for admission was UTI. When questioning the reasons of prefer ED instead of PHCS, most of patients stated that they prefer ED because of working hours and time is limited. Conclusions: As a conclusion, the reasons for patients to choose ED emerged as reasons increasing the number of inappropriate applications to the ED. In this case, the presence of appropriate triage systems in EDs may reduce the number of inappropriate applications. At this stage, the use of PHCS can reduce the burden on the EDs.Wo