scholarly journals Physicochemical and Biological Characterization of rhC1INH Expressed in CHO Cells

2021 ◽  
Vol 14 (11) ◽  
pp. 1180
Author(s):  
Ekaterina Zubareva ◽  
Maksim Degterev ◽  
Alexander Kazarov ◽  
Maria Zhiliaeva ◽  
Ksenia Ulyanova ◽  
...  
Keyword(s):  
Cho Cells ◽  
Specific Activity ◽  
Therapeutic Option ◽  
Chinese Hamster ◽  
Biological Characterization ◽  
Acquired Angioedema ◽  
C1 Esterase Inhibitor ◽  
Rabbit Milk ◽  
Esterase Inhibitor

The disfunction or deficiency of the C1 esterase inhibitor (C1INH) is associated with hereditary or acquired angioedema (HAE/AAE), a rare life-threatening condition characterized by swelling in the skin, respiratory and gastrointestinal tracts. The current treatment options may carry the risks of either viral infection (plasma-derived Berinert®) or immune reaction (human recombinant C1INH from rabbit milk, Ruconest®). This study describes the physicochemical and biological characterization of a novel recombinant human C1 esterase inhibitor (rhC1INH) from Chinese hamster ovary (CHO) cells for the treatment of hereditary angioedema compared to the marketed products Berinert® and Ruconest®. The mass spectrometry results of total deglycosylated rhC1INH revealed a protein with a molecular mass of 52,846 Da. Almost full sequence coverage (98.6%) by nanoLC-MS/MS peptide mapping was achieved. The purity and C1s inhibitory activity of rhC1INH from CHO cells are comparable with Ruconest®, although we found differences in charge isoforms distribution, intact mass values, and N-glycans profile. Comparison of the specific activity (IC50 value) of the rhC1INH with human C1 esterase inhibitor from blood serum showed similar inhibitory properties. These data allow us to conclude that the novel rhC1INH molecule could become a potential therapeutic option for patients with HAE/AAE.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

scholarly journals Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

1970 ◽  
Vol 12 (4) ◽  
Author(s):  
S.N.Z Zainul Abidin ◽  
And N. Anuar
Keyword(s):  
Recombinant Protein ◽  
Suspension Culture ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Shake Flask ◽  
Specific Activity ◽  
Chinese Hamster ◽  
Lactate Production ◽  
Spinner Flask ◽  
Lac Z

Chinese hamster ovary (CHO) cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO)) digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar dan kelalang goncang disesuaikan dengan sewajarnya. Eksperimen dijalankan menggunakan pendua dan sampel yang diambil untuk kiraan sel, penentuan penggunaan glukosa, penghasilan laktat dan aras protein dengan menggunakan cerakin biokimia. Keputusan menunjukkan tumbesaran sel di dalam kelalang putar lebih menggalakkan daripada dalam kelalang goncang. Kepekatan sel dalam kelalang putar adalah 58% lebih tinggi daripada dalam kelalang goncang. Sebaliknya, pada kelajuan agitasi yang sama, aktiviti tertentu β-galaktosidase adalah 25% lebih tinggi dalam kelalang putar dibandingkan dengan kelalang goncang.

scholarly journals Production by Human Lung Cells of Trypsin and Plasmin Inhibitor(s) Differing from a1-Antitrypsin, a2-Macroglobulin, C1-Esterase Inhibitor and Inter-a-Trypsin Inhibitor

1977 ◽  
Author(s):  
Maria B. Bernik
Keyword(s):  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Human Lung ◽  
Specific Activity ◽  
Cross Reaction ◽  
Lung Cells ◽  
C1 Esterase Inhibitor ◽  
Human Lung Cells ◽  
Wide Range ◽  
Esterase Inhibitor

Human lung cells in primary culture and serial subculture were used to study the production of inhibitory activity and to isolate and identify inhibitor(s) of trypsin and plasmin produced and released by the cells into the supernatant medium. Assays of inhibitory activity were performed on fibrin, and casein substrate and results expressed in BAEE units of trypsin inhibited on the former substrate. Inhibitory activity against plasmin and trypsin accumulated progressively in serum free supernates of cultures to concentrations of 80–150 BAEE units/ml and was isolated from the supernates by concentration with Amicon PM 30 membranes, gel filtration on Sephadex G—100 or G-200 columns and polyacrilamide gel electrophoresis. On calibrated columns inhibitory activity eluted in the range of 75,000 mol wt substances. On immunodiffusion, performed using a wide range of concentrations of chromatographed inhibitor preparations with specific activity of about 1,300 BAEE units/mg protein there was no cross-reaction with antiserum to a1-antitrypsin, a2-macroglobulin, C1-esterase inhibitor or inter-a-trypsin inhibitor. There was also no cross-reaction with antiserum to antithrombin III or antichymotrypsin. Immunoelectrophoresis showed no immunoreactive material with the antisera. These observation indicate the production in lung of inhibitor(s) differing from the major protease inhibitors and derivatives or subunits of these inhibitors described to date.

Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis

2001 ◽  
Vol 281 (1) ◽  
pp. G293-G301 ◽  
Author(s):  
E. Hall ◽  
P. Hylemon ◽  
Z. Vlahcevic ◽  
D. Mallonee ◽  
K. Valerie ◽  
...  
Keyword(s):  
Bile Acid ◽  
Cho Cells ◽  
Specific Activity ◽  
Recombinant Adenovirus ◽  
Cholesterol Acyltransferase ◽  
Chinese Hamster ◽  
Cholesterol Homeostasis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
G2 Cells

In the liver, sterol 27-hydroxylase (CYP27) participates in the classic and alternative pathways of bile acid biosynthesis from cholesterol (Chol). In extrahepatic tissues, CYP27 converts intracellular Chol to 27-hydroxycholesterol (27OH-Chol), which may regulate the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA-R). This study attempts to better define the role of CYP27 in the maintenance of Chol homeostasis in hepatic and extrahepatic cells by overexpressing CYP27 in Hep G2 cells and Chinese hamster ovary (CHO) cells through infection with a replication-defective recombinant adenovirus encoding for CMV-CYP27. After infection, CYP27 mRNA and protein levels increased dramatically. CYP27 specific activity also increased two- to fourfold in infected cells ( P ≤ 0.02), with a marked increase in conversion of [14C]Chol to [14C]27OH-Chol (∼150%; P ≤ 0.01). Accumulation of 27OH-Chol in CHO cells was associated with a 50% decrease in HMG-CoA-R specific activity ( P ≤ 0.02). In infected Hep G2 cells, the significant increase in bile acid synthesis (46%; P ≤ 0.006), which prevented the accumulation of intracellular 27OH-Chol, resulted in increased HMG-CoA-R activity (183%; P ≤ 0.02). Overexpression of CYP27 in Hep G2 cells also increased acyl CoA-cholesterol acyltransferase (71%, P ≤ 0.02) and decreased cholesteryl ester hydrolase (55%, P ≤ 0.02). In conclusion, CYP27 generates different physiological responses depending on cell type and presence or absence of bile acid biosynthetic pathways.

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen.

1980 ◽  
Vol 87 (3) ◽  
pp. 755-763 ◽  
Author(s):  
P A Harper ◽  
R L Juliano
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Divalent Cations ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Human Diploid Fibroblasts ◽  
Isolation And Characterization ◽  
A Cell ◽  
Cold Insoluble Globulin

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.

Anti-μ-opioid-receptor IgG antibodies are commonly present in serum from healthy blood donors: evidence for a role in apoptotic immune cell death

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3261-3268 ◽  
Author(s):  
Gaëtane Macé ◽  
Martial Jaume ◽  
Catherine Blanpied ◽  
Lionel Stephan ◽  
Jérôme D. Coudert ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Cho Cells ◽  
Blood Donors ◽  
Immune Cell ◽  
Specific Activity ◽  
Chinese Hamster ◽  
Murine Splenocytes ◽  
Healthy Humans ◽  
Extracellular Loops ◽  
Μ Opioid Receptor

Abstract We previously observed the presence of anti-human μ-opioid-receptor (anti-hMOR) autoantibodies in IgG pools prepared from several thousand healthy blood donors. These autoantibodies behaved agonistically because of their ability to bind to the first and third extracellular loops of the receptor. In this study, we found that each healthy donor's serum contained anti-hMOR IgG autoantibodies with a specific activity against both the first and the third extracellular loops of the receptor. Because of the inability of IgG to cross the blood-brain barrier, we investigated the effects of the expression of anti-hMOR autoantibodies on immune cells. In analogy to studies of the effects of morphine, we investigated the ability of antibodies to sensitize splenocytes to Fas (CD95)-mediated apoptosis. We took advantage of the high sequence homology between murine MOR and hMOR extracellular loops to estimate the effect on murine splenocytes of anti-hMOR antibodies raised by immunizing mice. Splenocytes from mice injected with Chinese hamster ovary (CHO) cells expressing MOR were sensitized to Fas-mediated apoptosis, whereas those from mice injected with CHO cells or phosphate-buffered saline were not. Similar sensitization to Fas-mediated apoptosis was observed in splenocytes from mice undergoing passive transfer either with IgG from mice previously immunized against CHO cells expressing MOR or with IgG directed against the first and third extracellular loops of the receptor. Together, our data show that anti-MOR autoantibodies are commonly expressed in healthy humans and could participate in the control of lymphocyte homeostasis by promoting Fas-mediated apoptosis.

scholarly journals Development of a Microfluidic GHz Impedance Cytometer

2013 ◽  
Vol 80 (12) ◽  
Author(s):  
Niels Haandbæk ◽  
Sebastian C. Bürgel ◽  
Flavio Heer ◽  
Andreas Hierlemann
Keyword(s):  
Dielectric Properties ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Single Cells ◽  
Chinese Hamster ◽  
Frequency Range ◽  
Cell Nuclei ◽  
Dielectric Characterization ◽  
The Difference

AbstractThis article presents a novel microfluidic impedance cytometer enabling dielectric characterization of single cells at frequencies up to 500 MHz. The dielectric properties of cells at lower frequencies contain information about their size and membrane capacitance. The increased frequency range of the presented cytometer potentially allows for characterization of intracellular components, such as vacuoles or the cell nuclei. We demonstrate the overall capabilities of the cytometer through discrimination of polystyrene beads from Chinese hamster ovary (CHO) cells. The discrimination is based on the difference in dielectric properties at frequencies up to 500 MHz.

scholarly journals Refractory Abdominal Pain in a Patient with Chronic Lymphocytic Leukemia: Be Wary of Acquired Angioedema due to C1 Esterase Inhibitor Deficiency

2018 ◽  
Vol 2018 ◽  
pp. 1-5
Author(s):  
Abdullateef Abdulkareem ◽  
Ryan S. D’Souza ◽  
Joshua Mundorff ◽  
Pragya Shrestha ◽  
Oluwaseun Shogbesan ◽  
...  
Keyword(s):  
Abdominal Pain ◽  
Chronic Lymphocytic Leukemia ◽  
Upper Airway ◽  
Recurrent Abdominal Pain ◽  
Final Diagnosis ◽  
Lymphocytic Leukemia ◽  
C1 Inhibitor Deficiency ◽  
Acquired Angioedema ◽  
C1 Esterase Inhibitor ◽  
Esterase Inhibitor

Acquired angioedema due to C1 inhibitor deficiency (C1INH-AAE) is a rare and potentially fatal syndrome of bradykinin-mediated angioedema characterized by episodes of angioedema without urticaria. It typically manifests with nonpitting edema of the skin and edema in the gastrointestinal (GI) tract mucosa or upper airway. Edema of the upper airway and tongue may lead to life-threatening asphyxiation. C1INH-AAE is typically under-diagnosed because of its rarity and its propensity to mimic more common abdominal conditions and allergic reactions. In this article, we present the case of a 62-year-old male with a history of recently diagnosed chronic lymphocytic leukemia (CLL) who presented to our hospital with recurrent abdominal pain, initially suspected to haveClostridium difficilecolitis and diverticulitis. He received a final diagnosis of acquired angioedema due to C1 esterase inhibitor deficiency due to concomitant symptoms of lip swelling, cutaneous nonpitting edema of his lower extremities, and complement level deficiencies. He received acute treatment with C1 esterase replacement and icatibant and was maintained on C1 esterase infusions. He also underwent chemotherapy for his underlying CLL and did not experience further recurrence of his angioedema.

Pharmacokinetics, Clinical Efficacy and Safety of Plasma-Derived Nanofiltered C1 Inhibitor Concentrate for Treatment of Hereditary and Acquired Angioedema.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1988-1988
Author(s):  
J. J. Hofstra ◽  
E. van Twuyver ◽  
I. Kleine-Budde ◽  
C. W. Choi ◽  
M. Levi ◽  
...  
Keyword(s):  
Phase Ii Study ◽  
Phase Iii ◽  
Pharmacokinetic Parameters ◽  
Efficacy And Safety ◽  
C1 Inhibitor ◽  
Acquired Angioedema ◽  
C1 Esterase Inhibitor ◽  
Phase Iii Study ◽  
Mean Time ◽  
Esterase Inhibitor

Abstract RATIONALE: From the early 70’s, C1 inhibitor concentrate manufactured from pooled human plasma has been available to patients with hereditary and acquired angioedema (HAE and AAE) and in 1997 a highly purified C1 inhibitor (Cetor®) was introduced. Many precautions have been taken to minimize the potential risk of viral transmission (e.g. rigorously controlled whole blood collection systems, extensive screening of each individual donation for a variety of blood-borne viruses, pasteurisation). To further minimize the potential risk of viral transmission, a 15 nm filtration was implemented in the manufacturing process giving rise to C1-inhibitor-N (anofiltered). DESIGN: A randomised, double-blind, controlled cross-over phase II study was conducted in which our primary objective was to compare the pharmacokinetics of the newly developed concentrate with conventional C1-inhibitor concentrate in HAE patients without signs of an attack of angioedema. Secondly, an open-label phase III study in patients with an HAE attack was performed to investigate whether the introduction of the virus reducing 15nm filtration step in the manufacturing process of the concentrate did not affect the efficacy and safety of the product. RESULTS: Thirteen patients were enrolled in the phase II study. No differences between conventional C1-inhibitor concentrate and nanofiltered C1-inhibitor concentrate were detected with regard to the primary pharmacokinetic parameters clearance, volume of distribution, and the fraction of C1-inhibitor-N detected by the antigen assay relative to the functional assay. Therefore incremental recovery, mean residence time, half-life and the area under curve were equivalent for both products. In the phase III study, 8 HAE patients were enrolled. In these 8 patients, 14 attacks qualified as acute angioedema attack. The mean time-to-relief for attacks treated with new C1-inhibitor concentrate was 3.0 (SD 2.5) hours. Historical data showed that treatment with conventional C1-inhibitor concentrate resulted in time-to-relief of 3.9 (SD 6.2) hours, whereas for untreated attacks this was 24.7 (SD 19.9) hours. The attacks treated with new C1-esterase inhibitor concentrate had a mean time-to-resolve of 18.6 hours (SD 13.1) whereas medical history showed an time-to-resolve of 17.8 hours (SD 17.2) for conventional C1-inhibitor. Untreated attacks showed a mean time-to-resolve of 63.6 hours (SD 31.0). CONCLUSION: The newly developed nanofiltered C1-esterase inhibitor has equal pharmacokinetic properties compared to the conventional concentrate. The viral reduction step (15 nm filtration) in the production process of Cetor did not induce changes in the efficacy and safety in the treatment of acute angioedema attacks and in the pharmacokinetic parameters.

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