Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure

Suen, Ka Fai; Turner, Mary S.; Gao, Feng; Liu, Bo; Althage, Alana; Slavin, Anthony; Ou, Weijia; Zuo, Elizabeth; Eckart, Michael; Ogawa, Tomoya; Yamada, Masao; Tuntland, Tove; Harris, Jennifer L.; Trauger, John W.

doi:10.1016/j.pep.2009.12.015

Cited by 36 publications

(19 citation statements)

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“…They also showed the CHO cells are not well suited to deliver reasonable amounts of protein following transient transfection (11). Suen et al has also indicated that the protein expression yield is generally 2 -5 times higher for PEI-transfected HEK vs. CHO cells (7). Other studies has also indicated that using a CHO-based transient production of proteins was limited by poor transfection efficiencies, viabilities, and production of insufficient quantities of recombinant proteins so that transient production of recombinant scFv-Fc antibodies in HEK293 is efficient and robust (18).…”

Section: Discussionmentioning

confidence: 99%

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

Vatandoost

Dolatabadi

2017

Gene Cell Tissue

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Background: Selecting a host system for the expression of recombinant proteins should be carefully evaluated prior to the initiation of any bio therapeutic development programs. Since different hosts express proteins with various efficiencies and with different posttranslational modifications, changing hosts may impact the expected activity of the protein. The main expression systems have members of the mammalian cell family, however, there are remarkable differences between the species. The most generally used mammalian hosts for the production of recombinant proteins are CHO and HEK293 cells. Objectives: In order to compare the differences between HEK versus CHO cells, the human coagulation factor IX in a transient and stable expression was examined. Methods: After transfection of CHO and HEK cells with an hFIX-expressing mammalian plasmid, pcDNA-hFIX, transient expression was analyzed on the culture supernatant during 72 hours. The stable clones were also recovered after 10 weeks of geneticin selection. The expression and activity of the hFIX was evaluated by performing enzyme-linked immunosorbent assay (ELISA) as well as

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Section: Discussionmentioning

confidence: 99%

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

Vatandoost

Dolatabadi

2017

Gene Cell Tissue

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“…Both cell lines are well established and have been utilized to produce clinically relevant proteins [5]. While both CHO and HEK cells are capable of extensive glycosylations, it has recently become more and more evident that proteins produced in CHO cells have a different glycosylation pattern than the same proteins produced in HEK cells, which may affect their function [6–10]. For example, a fusion protein containing the extracellular domain of the human interleukin-23 receptor and a crystalizing fragment (Fc) region produced in HEK cells was less stable in mice than the same protein produced in CHO cells [6].…”

Section: Introductionmentioning

confidence: 99%

Lentiviral expression system for the purification of secreted proteins from human cell cultures

et al. 2016

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BackgroundRecombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media.ResultsUsing a protein-based HIV entry inhibitor, soluble CD4 (sCD4), we demonstrated that 293 T cells transduced with a lentiviral vector mediated over 10-fold higher secretion of sCD4 in comparison to 293 T cells transfected with the corresponding plasmid. Secretion of sCD4 increased with the dose of the lentiviral vector up to a multiplicity of infection of 50. Exchanging the native signal peptide of sCD4 with the signal peptide of human alpha-1 antitrypsin increased expression by 50 %. There was no difference in expression from a monocistronic or bicistronic lentiviral vector. Reduction of the serum concentration in the culture media had no significant effect on the secretion of sCD4. Small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4. Purified sCD4 bound to recombinant HIV envelope glycoprotein 120 (Env gp120) and inhibited HIV entry at concentrations comparable to published results.ConclusionThe procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. Furthermore, the method could be used to produce sCD4 fusion proteins or other His-tagged proteins.

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“…Glycosylation is specific to various organisms, tissues and cell lines (22,45,46), as such, production of viral proteins in different expression systems can result in substantially different glycosylation profiles (47)(48)(49)(50). Defining glycosylation of viral surface proteins produced in vivo or in vitro is therefore important for the elucidation of host-virus interactions and for the design of viral therapeutics.…”

Section: Viral Protein Glycosylationmentioning

confidence: 99%

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

Pegg¹

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The family Paramyxoviridae (paramyxovirus) contains several significant human and animal pathogens.Represented within this family are human respiratory syncytial virus (hRSV) and Newcastle disease virus (NDV). The former contributes significantly to severe respiratory tract disease in infants, children and immunocompromised individuals. At present, highly efficacious therapeutics or safe and effective vaccines are not available for hRSV. NDV is the causative agent of Newcastle disease, afflicting a wide range of avian species. The desire to study NDV is due not only to the significant economic impact it has on the poultry industry worldwide, but also its potential use as an oncolytic agent and vaccine vector in humans and animals. Additionally, findings on NDV may be translated to closely related viruses that cause disease in humans, such as parainfluenza viruses.As outlined in Chapter 1 of this thesis, the infectious processes of all members of this family are driven by two major membrane glycoproteins whose ectodomains project from the viral envelope: the fusion (F) and attachment glycoproteins. The F glycoprotein is responsible for viral entry by means of fusion with host cell membranes while the variable attachment glycoproteins, haemagglutinin, haemagglutininneuraminidase (HN) and major surface glycoprotein (G), are involved in viral attachment to host cells.Previous studies have shown that altering the glycosylation profile of these proteins can modulate the ability of the virus to infect host cells and stimulate the host immune system. As yet, site-specific glycan heterogeneity of hRSV and NDV surface glycoproteins has not been defined at a chemical level. As described in Chapter 2, reverse-phase liquid chromatography tandem mass spectrometry (MS) strategies were implemented to characterise intact glycopeptides from the attachment and F glycoproteins of hRSV and NDV. Site-occupancy and monosaccharide compositions at a given site were determined using collision-induced dissociation, higher-energy collision dissociation, electron-transfer dissociation and electron-transfer dissociation combined with collision dissociation. As described in Chapter 3, a spectral processing program was developed called OxoExtract to aid identification of intact glycopeptides that were fragmented with higher-energy collision dissociation.The F and HN proteins of NDV were derived from virions propagated in embryonic eggs. Analyses of HN in Chapter 4 revealed high mannose N-linked glycans and complex or hybrid N-linked glycans that were variably fucosylated, sialylated and sulfated or phosphorylated. In total 63, 58, and 37 glycans were identified at sites N341, N433 and N481, respectively. In addition, a previously undocumented Olinked glycosylation site was identified in the stalk domain of the protein. Observed glycans from NDV F described in Chapter 5 were mainly high mannose, containing variations of five to nine mannose ii residues across four sites, N85, N191, N366 and N471. There was also evidence of fucosylated c...

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Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure

Cited by 36 publications

References 20 publications

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

Lentiviral expression system for the purification of secreted proteins from human cell cultures

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

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