scholarly journals Techno-Economic Assessment of Cell-Free Synthesis of Monoclonal Antibodies Using CHO Cell Extracts

Processes ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 454
Author(s):  
Vaishali Thaore ◽  
Dimitrios Tsourapas ◽  
Nilay Shah ◽  
Cleo Kontoravdi
Keyword(s):  
Protein Synthesis ◽  
Monoclonal Antibodies ◽  
Cho Cells ◽  
Transient Gene Expression ◽  
Genetic Material ◽  
Chinese Hamster ◽  
Economic Assessment ◽  
Product Yield ◽  
Living Cells ◽  
Cell Extracts

Cell-free protein synthesis (CFPS) is an emerging tool for the rapid production of difficult-to-express proteins as well as for identifying protein synthesis bottlenecks. In CFPS, the biotic phase is substituted by extracts of living cells devoid of any of their own genetic material. The main advantage is that these systems delineate cell growth from recombinant protein production, enabling the expression of targets that would otherwise place too big a burden on living cells. We have conducted a techno-economic analysis of a CFPS system to produce monoclonal antibodies (mAbs) using extracts of Chinese hamster ovary (CHO) cells. We compare the performance of the CFPS system with two alternative production strategies: stable and transient gene expression in CHO cells. Our assessment shows that the viability of CFPS for mAb production requires a significant increase in the product yield and the recycling of high-cost components such as DNA. Nevertheless, CFPS shows significant promise for personalized medicine applications, providing a platform for on-demand production and simplified supply chains.

Chinese Hamster Cell Variants Resistant to the A Chain of Ricin Carry Altered Ribosome Function

1982 ◽  
Vol 2 (6) ◽  
pp. 599-606
Author(s):  
Mayumi Ono ◽  
Michihiko Kuwano ◽  
Kei-Ichi Watanabe ◽  
Gunki Funatsu
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Cho Cells ◽  
Ricinus Communis ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Cytotoxic Action ◽  
A Chain ◽  
Ricin A

Ricin, a toxic lectin from Ricinus communis , is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 μg/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [ 125 I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [ 125 I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

scholarly journals Hydrophobic glycosides of N-acetylglucosamine can act as primers for polylactosamine synthesis and can affect glycolipid synthesis in vivo

1995 ◽  
Vol 307 (3) ◽  
pp. 791-797 ◽  
Author(s):  
D C A Neville ◽  
R A Field ◽  
M A J Ferguson
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Culture Medium ◽  
Linear Array ◽  
Chinese Hamster ◽  
Living Cells ◽  
Anomeric Configuration ◽  
Glycolipid Synthesis ◽  
Synthetic Capacity

Several hydrophobic glycosides of N-acetylglucosamine (GlcNAc) served as primers for polylactosamine synthesis when added to Chinese hamster ovary (CHO) cells. The modified glycosides, containing one to six lactosamine repeats in linear array, were sialylated and secreted into the culture medium. The relative efficiencies of the glycosides to serve as primers were dependent on the nature of the aglycone and on the anomeric configuration of the GlcNAc residue. The same compounds were tested for their effects on glycolipid synthesis in CHO cells. All of the beta-glycosides significantly inhibited the synthesis of the lactoseries glycolipid GM3 whereas the alpha-glycoside was inactive. The compound GlcNAc alpha 1-O-benzyl- was the most efficient primer of polylactosamine synthesis and had no effect on glycolipid synthesis. This compound may have potential for the assay of the polylactosamine synthetic capacity of living cells.

Generation of monoclonal antibodies to mitotic and interphase cytosolic proteins of Chinese Hamster Ovary (CHO) cells

2009 ◽  
Vol 18 (4) ◽  
pp. 139-143
Author(s):  
Ravi Maddaly ◽  
Lakshmi Srinivasan ◽  
Shruti Balaji ◽  
Solomon F.D. Paul
Keyword(s):  
Monoclonal Antibodies ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Cytosolic Proteins

Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

2017 ◽  
Vol 33 (3) ◽  
pp. 786-794 ◽  
Author(s):  
Zhilan Hu ◽  
Danming Tang ◽  
Shahram Misaghi ◽  
Guoying Jiang ◽  
Christopher Yu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Product Quality ◽  
Heavy Chain ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster

scholarly journals Engineering of Chinese Hamster Ovary Cells With NDPK-A to Enhance DNA Nuclear Delivery Combined With EBNA1 Plasmid Maintenance Gives Improved Exogenous Transient Reporter, mAb and SARS-CoV-2 Spike Protein Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
James D. Budge ◽  
Robert J. Young ◽  
Christopher Mark Smales
Keyword(s):  
Recombinant Protein ◽  
Plasmid Dna ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Nuclear Antigen ◽  
Stable Cell Line ◽  
Nuclear Pore

Transient gene expression (TGE) in mammalian cells is a method of rapidly generating recombinant protein material for initial characterisation studies that does not require time-consuming processes associated with stable cell line construction. High TGE yields are heavily dependent on efficient delivery of plasmid DNA across both the plasma and nuclear membranes. Here, we harness the protein nucleoside diphosphate kinase (NDPK-A) that contains a nuclear localisation signal (NLS) to enhance DNA delivery into the nucleus of CHO cells. We show that co-expression of NDPK-A during transient expression results in improved transfection efficiency in CHO cells, presumably due to enhanced transportation of plasmid DNA into the nucleus via the nuclear pore complex. Furthermore, introduction of the Epstein Barr Nuclear Antigen-1 (EBNA-1), a protein that is capable of inducing extrachromosomal maintenance, when coupled with complementary oriP elements on a transient plasmid, was utilised to reduce the effect of plasmid dilution. Whilst there was attenuated growth upon introduction of the EBNA-1 system into CHO cells, when both NDPK-A nuclear import and EBNA-1 mediated technologies were employed together this resulted in enhanced transient recombinant protein yields superior to those generated using either approach independently, including when expressing the complex SARS-CoV-2 spike (S) glycoprotein.

scholarly journals Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells

2020 ◽  
Vol 26 (4) ◽  
pp. 393-398
Author(s):  
Ehsan Naghneh ◽  
Es'hagh Pourmaleki ◽  
Azam Rahimpour
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Chinese Hamster ◽  
Vascular Endothelial ◽  
Cell Clones ◽  
Fc Fusion Protein ◽  
Endothelial Growth Factor

Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.

scholarly journals Chinese Hamster Cell Variants Resistant to the A Chain of Ricin Carry Altered Ribosome Function

1982 ◽  
Vol 2 (6) ◽  
pp. 599-606 ◽  
Author(s):  
Mayumi Ono ◽  
Michihiko Kuwano ◽  
Kei-Ichi Watanabe ◽  
Gunki Funatsu
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Cho Cells ◽  
Ricinus Communis ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Cytotoxic Action ◽  
A Chain ◽  
Ricin A

Ricin, a toxic lectin fromRicinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 μg/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

scholarly journals Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin αIIbβ3

2008 ◽  
Vol 181 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
Naohide Watanabe ◽  
Laurent Bodin ◽  
Manjula Pandey ◽  
Matthias Krause ◽  
Shaun Coughlin ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Related Protein ◽  
Guanosine Triphosphate ◽  
Integrin Αiibβ3 ◽  
Adaptor Molecule ◽  
Thrombin Receptors

Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered αIIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates αIIbβ3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1–guanosine triphosphate–interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to αIIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and αIIbβ3 activation. However, one talin mutant (L325R) is recruited to αIIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated αIIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and αIIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to αIIbβ3 by RIAM mediates agonist-induced αIIbβ3 activation, with implications for hemostasis and thrombosis.

scholarly journals Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

1981 ◽  
Vol 1 (6) ◽  
pp. 552-559 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pseudomonas Aeruginosa Exotoxin

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.

Sign in / Sign up

Export Citation Format

Share Document