NAD+ supplementation improves mAbs productivity in CHO cells via a glucose metabolic shift

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Karyotype-based analysis of cell line instability and clonality in CHO cells

Chinese hamster ovary (CHO) cell line instability that can result in unexpected changes in phenotypes such as cell growth, productivity, or product quality is challenging for the biomanufacturing of therapeutic proteins. In addition, understanding cell line instability and its relationship to clonality is critical. We hypothesize that chromosomal rearrangements resulting from genomic instability are associated with cell line instability. We developed cell line instability models using two cell lines: secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) and their host cells (CHO-DUK). We also developed a karyotype-based framework to quantify chromosomal rearrangements. In the absence of methotrexate (MTX), long-term cultured CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes when compared to the CHO-SEAP cells grown with MTX, demonstrating production instability. Fluorescence in situ hybridization and karyotyping analyses indicated a chromosomal loss of the SEAP gene-containing region, leading to the emergence of a non-producing subpopulation. These results support a mutation-and-selection mechanism of production instability wherein random chromosomal rearrangements can give rise to faster growing cells with low or non-producing phenotypes. Long-term cultured CHO-DUK cells exhibited an increased growth rate and an increase in the population ratio containing a particular chromosome. Growth rate and karyotyping analyses of limiting dilution CHO-DUK clones showed a correlation between the faster growing clones and the presence of that particular chromosome. Finally, karyotyping analysis indicated that CHO-DUK cells, as well as limiting dilution clones, were karyotypically heterogeneous, suggesting that chromosomal rearrangements occur spontaneously and can compromise the clonality of a cell line that has been developed from a single cell

Host cell protein control via CHO genome engineering

Chinese hamster ovary (CHO) cells, a major mammalian platform in biomanufacturing, produce and secret recombinant proteins along with host cell proteins (HCPs). Because residual HCPs in the final drug product can adversely affect (1) patients by causing immune responses, (2) drug efficacy, and (3) product stability, the effective removal of HCPs is necessary. Unfortunately, many studies have reported that many HCPs can be difficult to remove through downstream purification processes because they share similar biophysical properties to biopharmaceuticals. In this study we employed a genome engineering approach using clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) system-mediated knockout to address difficult-to-remove HCP problems. Three HCPs (Cathepsin D, Nidogen-1, and Prosaposin) that are known to be difficult to remove were selected, and respective knockout clones were isolated without using selective reagents or reporter genes. Clones for each HCP were characterized using various analysis methods. Taken together, we demonstrate the applicability of the CRISPR/Cas9 system to eliminate difficult-to-remove HCP expression in an industry-relevant setting

Comparison of Posterior Lumbar Interbody Fusion and Posterolateral Lumbar Fusion in Monosegmental Vacuum Phenomenon within an Intervertebral Disc

Study DesignRetrospective.PurposeTo compare the clinical and radiological outcomes of posterolateral lumbar interbody fusion (PLIF) and posterolateral lumbar fusion (PLF) in monosegmental vacuum phenomenon within an intervertebral disc.Overview of LiteratureThe vacuum phenomenon within an intervertebral disc is a serious form of degenerative disease that destabilizes the intervertebral body. Outcomes of PLIF and PLF in monosegmental vacuum phenomenon are unclear.MethodsMonosegmental instrumented PLIF and PLF was performed on 84 degenerative lumbar disease patients with monosegmental vacuum phenomenon (PLIF, n=38; PLF, n=46). Minimum follow-up was 24 months. Clinical outcomes of leg and back pain were assessed using visual analogue scales for leg pain (LVAS) and back pain (BVAS), and the Oswestry disability index (ODI). The radiographic outcome was the estimated bony union rate.ResultsLVAS, BVAS, and ODI improved in both groups. There was no significant difference in the degree of these improvements between PLIF and PLF patients (p>0.05). Radiological union rate was 91.1% in PLIF group and 89.4% in PLF group at postoperative 24 months (p>0.05).ConclusionsNo significant differences in clinical results and union rates were found between PLIF and PLF patients. Selection of the operation technique will reflect the surgeon's preferences and patient condition

Short range scattering effect of InAs quantum dots in the transport properties of two dimensional electron gas

Short range interaction between two dimensional electron gas (2DEG) and InAs quantum dots embedded in the GaAs/AlGaAs quantum well is investigated as a function of carrier density. At low carrier density the interaction is significantly characterized by a transport to quantum lifetime ratio of less than 5. However, with an increase in carrier density, quantum lifetime is observed to undergo a sharp transition from 0.17 to 0.25 ps. This is attributed to the screening of short range repulsive scattering due to InAs quantum dots by the 2DEG.open7

Physicochemical factors that affect electroporation of lung cancer and normal cell lines

Electroporation is used for cancer therapy to efficiently destroy cancer tissues by transferring anticancer drugs into cancer cells or by irreversible tumor ablation without resealing pores. There is growing interest in the electroporation method for the treatment of lung cancer, which has the highest mortality rate among cancers. Improving the cancer cell selectivity has the potential to expand its use. However, the factors that influence the cell selectivity of electroporation are debatable. We aimed to identify the important factors that influence the efficiency of electroporation in lung cells. The electropermeabilization of lung cancer cells (H460, A549, and HCC1588) and normal lung cells (MRCS, WI26 and L132) was evaluated by the transfer of fluorescence dyes. We found that membrane permeabilization increased as cell size, membrane stiffness, resting transmembrane potential, and lipid cholesterol ratio increased. Among them, lipid composition was found to be the most relevant factor in the electroporation of lung cells. Our results provide insight into the differences between lung cancer cells and normal lung cells and provide a basis for enhancing the sensitivity of lung cancers cells to electroporation. (C) 2019 Elsevier Inc. All rights reserved.N

Lack of Association between Hepatitis B Virus Infection and Polymorphism of Mannose-Binding Lectin Gene in Korean Population

Mannose-binding lectin (MBL) plays an important role in immune defense. This study was undertaken to investigate the association between hepatitis B virus infection and polymorphisms of MBL gene. We assessed the single nucleotide polymorphism at codon 54 in exon 1 of MBL in patients with hepatitis B virus infection and HBsAg negative controls in Korean population. A total of 498 enrolled subjects was classified into four groups. Group 1; Clearance, Group 2; Inactive healthy carrier, Group 3; Chronic hepatitis, Group 4; Liver cirrhosis. MBL gene polymorphisms at codon 54 led to three genotypes (G/G, G/A, A/A). When we divided subjects into clearance group (group 1) and persistence group (group 2-4), G/G genotype and A-allele carrier were observed in 55.6% and 44.4% in clearance group, 64.8% and 35.2% in persistence group (p=0.081), respectively. When hepatitis B virus persistent cases were divided into inactive healthy carrier (group 2) and disease progression group (group 3 and 4), MBL gene polymorphisms at codon 54 were not related to disease progression (p=0.166). MBL gene polymorphism at codon 54 was not associated with the clearance of hepatitis B virus infection nor progression of disease in chronic hepatitis B virus infection