Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Croset, Amelie; Delafosse, Laurence; Gaudry, Jean-Philippe; Arod, Christian; Glez, Loïc; Losberger, Christophe; Bégué, Damien; Krstanovic, Ana; Robert, Flavie; Vilbois, Francis; Chevalet, Laurent; Antonsson, Bruno

doi:10.1016/j.jbiotec.2012.06.038

Cited by 194 publications

(171 citation statements)

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“…In some cases, the scaling factor may represent the differences in transporter protein abundance between in vitro and in vivo, but it may also reflect the dissimilarities in activity per milligram of protein owing to several factors, including membrane environment or differences in posttranslational modifications (Straumann et al, 2006;Croset et al, 2012). Is important to note that in this study the V max and K m values were estimated from in vitro experiments performed at room temperature and not at 37°C.…”

Section: Discussionmentioning

confidence: 99%

Prediction of Renal Transporter Mediated Drug-Drug Interactions for Pemetrexed Using Physiologically Based Pharmacokinetic Modeling

et al. 2014

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Pemetrexed, an anionic anticancer drug with a narrow therapeutic index, is eliminated mainly by active renal tubular secretion. The in vitro to in vivo extrapolation approach used in this work was developed to predict possible drug-drug interactions (DDIs) that may occur after coadministration of pemetrexed and nonsteroidal anti-inflammatory drugs (NSAIDs), and it included in vitro assays, risk assessment models, and physiologically based pharmacokinetic (PBPK) models. The pemetrexed transport and its inhibition parameters by several NSAIDs were quantified using HEK-PEAK cells expressing organic anion transporter (OAT) 3 or OAT4. The NSAIDs were ranked according to their DDI index, calculated as the ratio of their maximum unbound concentration in plasma over the concentration inhibiting 50% (IC 50 ) of active pemetrexed transport. A PBPK model for ibuprofen, the NSAID with the highest DDI index, was built incorporating active renal secretion in Simcyp Simulator. The bottom-up model for pemetrexed underpredicted the clearance by 2-fold. The model we built using a scaling factor of 5.3 for the maximal uptake rate (V max ) of OAT3, which estimated using plasma concentration profiles from patients given a 10-minute infusion of 500 mg/m 2 of pemetrexed supplemented with folic acid and vitamin B 12 , recovered the clinical data adequately. The observed/predicted increases in C max and the area under the plasma-concentration time curve (AUC 0-inf ) of pemetrexed when ibuprofen was coadministered were 1.1 and 1.0, respectively. The coadministration of all other NSAIDs was predicted to have no significant impact on the AUC 0-inf based on their DDI indexes. The PBPK model reasonably reproduced pemetrexed concentration time profiles in cancer patients and its interaction with ibuprofen.

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Section: Discussionmentioning

confidence: 99%

Prediction of Renal Transporter Mediated Drug-Drug Interactions for Pemetrexed Using Physiologically Based Pharmacokinetic Modeling

et al. 2014

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“…The use of a eukaryotic host may provide a smoother transition of the selected scFv to large-scale protein production for therapeutic applications. However, further optimization of a particular protein may be needed to optimize expression and/or posttranslational modifications in a production cell line, as there can be significant differences even between mammalian cell lines (27). The RCAS vectors normally infect and replicate optimally in avian cells that express the appropriate receptors (19).…”

Section: Discussionmentioning

confidence: 99%

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

Changming¹,

Pike²,

Rinkoski³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.antibody engineering | avian leukosis virus | protein display technology | antibody binding affinity | avian leukosis virus polypeptide display

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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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Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Cited by 194 publications

References 39 publications

Prediction of Renal Transporter Mediated Drug-Drug Interactions for Pemetrexed Using Physiologically Based Pharmacokinetic Modeling

Prediction of Renal Transporter Mediated Drug-Drug Interactions for Pemetrexed Using Physiologically Based Pharmacokinetic Modeling

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

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

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