Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells

2010 ◽  
Vol 107 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Donglin Guo ◽  
Albert Gao ◽  
David A. Michels ◽  
Lauren Feeney ◽  
Marian Eng ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster

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 Amino Acid Requirements of the Chinese Hamster Ovary Cell Metabolism during Recombinant Protein Production

2019 ◽  
Author(s):  
Bergthor Traustason
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Scale Model ◽  
Amino Acid Requirements

SummaryMajority of biopharmaceutical drugs today are produced by Chinese hamster ovary (CHO) cells, which have been the standard industry host for the past decades. To produce and secrete a substantial amount of the target recombinant proteins the CHO cells must be provided with suitable growth conditions and provided with the necessary nutrients. Amino acids play a key role in this as the building blocks of proteins, playing important roles in a large number of metabolic pathways and being important sources of nitrogen as well as carbon under certain conditions. In this study exploratory analysis of the amino acid requirements of CHO cells was carried out using metabolic modelling approaches. Flux balance analysis was employed to evaluate the optimal distribution of fluxes in a genome-scale model of CHO cells to gain information on the cells’ metabolic response in silico.The results showed that providing non-essential amino acids (NEAAs) has a positive effect on CHO cell biomass production and that cysteine as well as tyrosine play a fundamental role in this. This implies that extracellular provision of NEAAs limits the extent of energy loss in amino acid biosynthetic pathways and renders additional reducing power available for other biological processes. Detailed analysis of the possible secretion and uptake of D-serine in the CHO model was also performed and its influence on the rest of the metabolism mapped out, which revealed results matching various existing literature. This is interesting since no mention of D-serine in regard to CHO cells was found in current literature, as well as the fact that this opens up the possibility of using the model for better understanding of certain disorders in higher up organisms that have been implicated with D-serine, such as motor neuron and cognitive degeneration. Finally, outcome from the model optimisation of different recombinant proteins demonstrated clearly how the difference in protein structure and size can influence the production outcome. These results show that systematic and model-based approaches have great potential for broad de novo exploration as well as being able to handle the cellular burden associated with the production of different types of recombinant protein.

Increase in K+ and alpha-AIB active transport in CHO cells after low [K+] treatment

1982 ◽  
Vol 243 (3) ◽  
pp. C124-C132 ◽  
Author(s):  
J. S. Graves ◽  
D. D. Wheeler
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Transport Systems ◽  
Acid Transport ◽  
Transport Capacity ◽  
Prolonged Incubation ◽  
Apparent Increase ◽  
Low K

We have studied the effects of prolonged incubation in low [K+] medium (approximately 0.3 mM) on both K+ and amino acid transport in Chinese hamster ovary (CHO) cells. When incubated in low [K+] medium, CHO cells redressed partially the loss of intracellular K+ after 12 h. After 24 h of incubation, both the activity of Na+-K+-ATPase in crude homogenates, and the transport capacity (Vmax) for ouabain-sensitive (i.e., active) K+ influx approximately doubled. The magnitude of the ouabain-insensitive (i.e., passive) K+ influx decreased by 50%. Thus the regulatory response involves an apparent increase in Na+-K+ pump and a decrease in K+ leak. The transport capacity for the nonmetabolized amino acid, alpha-aminoisobutyric acid (alpha-AIB), also increased after 24 h in low [K+] medium. The Vmax for the Na+-dependent (i.e., active) alpha-AIB influx increased by about 150%, and the magnitude of the Na+-independent influx increased by 20-40%. These changes in alpha-AIB transport result in a twofold greater capacity to accumulate this amino acid. Thus the regulation of K+ and alpha-AIB transport systems appears to be linked and possible mechanisms of this linkage are discussed.

N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Lijun Xia ◽  
Vishwanath Ramachandran ◽  
J. Michael McDaniel ◽  
Kiem N. Nguyen ◽  
Richard D. Cummings ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Terminal Region ◽  
Wild Type ◽  
Ovary Cells ◽  
N Terminus

P-selectin binds to the N-terminal region of human P-selectin glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2O-glycosylation on a threonine. P-selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-selectin. The cells were assayed for binding to fluid-phase P-selectin and for tethering and rolling on P-selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 andO-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components andO-glycosylation to bind to P-selectin than does human PSGL-1.

scholarly journals Peroxisome proliferator-induced acyl-CoA thioesterase from rat liver cytosol: molecular cloning and functional expression in Chinese hamster ovary cells

1997 ◽  
Vol 323 (2) ◽  
pp. 525-531 ◽  
Author(s):  
Susanna T. ENGBERG ◽  
Toshifumi AOYAMA ◽  
Stefan E. H. ALEXSON ◽  
Takashi HASHIMOTO ◽  
L. Thomas SVENSSON
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rat Liver ◽  
Chinese Hamster Ovary ◽  
Specific Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Protein ◽  
Peroxisome Proliferator ◽  
Ovary Cells

We have isolated and cloned a cDNA that codes for one of the peroxisome proliferator-induced acyl-CoA thioesterases of rat liver. The deduced amino acid sequence corresponds to the major induced isoform in cytosol. Analysis and comparison of the deduced amino acid sequence with the established consensus sequences suggested that this enzyme represents a novel kind of esterase with an incomplete lipase serine active site motif. Analyses of mRNA and its expression indicated that the enzyme is significantly expressed in liver only after peroxisome proliferator treatment, but isoenzymes are constitutively expressed at high levels in testis and brain. The reported cDNA sequence is highly homologous to the recently cloned brain acyl-CoA thioesterase [Broustas, Larkins, Uhler and Hajra (1996) J. Biol. Chem. 271, 10470–10476], but subtle differences throughout the sequence, and distinct differences close to the resulting C-termini, suggest that they are different enzymes, regulated in different manners. A full-length cDNA clone was expressed in Chinese hamster ovary cells and the expressed enzyme was characterized. The palmitoyl-CoA hydrolysing activity (Vmax) was induced approx. 9-fold to 1 μmol/min per mg of cell protein, which was estimated to correspond to a specific activity of 250 μmol/min per mg of cDNA-expressed enzyme. Both the specific activity and the acyl-CoA chain length specificity were very similar to those of the purified rat liver enzyme.

Cloning of cDNA for goldfish activin βB subunit, and the expression of its mRNA in gonadal and non-gonadal tissues

1997 ◽  
Vol 19 (1) ◽  
pp. 37-45 ◽  
Author(s):  
W Ge ◽  
T Miura ◽  
H Kobayashi ◽  
R E Peter ◽  
Y Nagahama
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Culture Medium ◽  
Specific Activity ◽  
Chinese Hamster ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
Wide Range

ABSTRACT We have cloned a full length cDNA coding for activin βB subunit from the goldfish ovary. Sequence analysis of the goldfish activin βB shows that this peptide is extremely conserved across vertebrates. The mature region of goldfish activin βB has 93 and 98% amino acid identity with that of human and zebrafish βB subunit respectively. The identity of the cloned goldfish activin βB was further confirmed by expressing the protein in the Chinese hamster ovary (CHO) cells followed by detection of the specific activity of activin in the culture medium using F5-5 cell assay. mRNA of goldfish activin βB is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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