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Cited by 37Viral Infectious Diseases and Gene Expression in InsectsGlycosylation and Glycoproteins ResearchProtein purification and stability

Glycoengineering Chinese hamster ovary cells: a short history

Open access
Donini, Roberto, Haslam, Stuart M., Kontoravdi, Cleo·2021·Biochemical Society Transactions
DOI 10.1042/bst20200840PMC PMC8106501
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Abstract

Biotherapeutic glycoproteins have revolutionised the field of pharmaceuticals, with new discoveries and continuous improvements underpinning the rapid growth of this industry. N-glycosylation is a critical quality attribute of biotherapeutic glycoproteins that influences the efficacy, half-life and immunogenicity of these drugs. This review will focus on the advances and future directions of remodelling N-glycosylation in Chinese hamster ovary (CHO) cells, which are the workhorse of recombinant biotherapeutic production, with particular emphasis on antibody products, using strategies such as cell line and protein backbone engineering.

AI snapshot

Written from the paper’s full text.

TL;DR

This review traces how Chinese hamster ovary (CHO) cell glycoengineering has advanced from early glycan structure–activity understanding to precise genome and protein backbone editing for biotherapeutic production. It emphasizes N-glycosylation as a critical quality attribute and highlights CRISPR-based cell line engineering, antibody Fc glycan control, and mass spectrometry-based analytics.

Key findings

  • N-glycosylation is a critical quality attribute influencing efficacy, half-life, and immunogenicity; between 2014 and 2018, 84% of newly approved antibody therapeutics were produced in CHO cells.
  • Fc core fucose weakens high-affinity binding to glycosylated FcγRIIIa and is undesirable for ADCC; afucosylation strategies include GlycoMab® obinutuzumab, approved in 2013 with 10–100-fold improved ADCC, and mogamulizumab, the first glycoengineered mAb approved in 2012, produced in FUT8 knockout CHO cells (Potelligent®). FUT8 siRNA knockdown gave 100-fold improved ADCC for highly afucosylated antibodies.
  • CHO glycoengineering has moved to targeted nucleases—ZFNs, TALENs, and CRISPR—with CRISPR now dominant; examples include triple knockout of FUT8/BAX/BAK, KO/KI screens of glycosylation enzymes, and CRISPRa to increase MGAT3 and ST6GAL1 expression. Protein backbone mutations such as F241A/F243A/V262E/V264E increased glycan processing, darbepoetin alfa was approved in 2001 with two additional N-oligosaccharides, and atezolizumab has an Fc engineered to lack glycosylation (2016 US, 2017 EU).
  • Glycosylation analysis relies heavily on MS-based glycomics and glycoproteomics, with MALDI and ESI dominating; HILIC and capillary electrophoresis with fluorescence detection can quantify glycomic profiles but are hindered in assigning glycan structures to specific signals.

Methods

  • Narrative review; no primary experimental data or sample. It synthesizes published literature on CHO glycoengineering, N-glycan structure–activity relationships, cell line and protein backbone engineering, and glycosylation analytics.
  • Main analysis is thematic synthesis, supported by Box 1, Figures 1–3, and Table 1 summarizing N-glycosylation, engineering timelines, analytical workflows, and chromatographic/MS techniques.

Limitations

  • The paper states that effects of glycoengineering on cell physiology and N-glycosylation in contexts other than IgG-Fc and EPO need further investigation, including host cell proteins, glycan scaffolds, glycosylated Fab domains, and other recombinant products.
  • The paper states that its protein backbone engineering overview is not meant as a comprehensive discussion of all biotherapeutics and cell types.
  • Evident from the text: as a narrative review, it does not use a systematic search or quantitative synthesis, and its focus on CHO cells and mAbs may limit generalizability to other production systems and products.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1042/bst20200840

Cites

158 of 158 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

41 of 78 cited sources checked · 14 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The non-templated biosynthesis of glycans produces heterogeneous glycomic profiles due to enzymatic competition for a common substrate, substrate availability and enzyme-substrate specificity.

    “the non-templated biosynthesis of glycans is a major hurdle that produces heterogeneous glycomic profiles as a result of enzymatic competition for a common substrate, substrate availability and enzyme-substrate specificity [6,7].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] 1986, Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides, Biochem. Cell Biol. source
    • cites [7] 2017, Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab, Eur. J. Hosp. Pharm. source

      The Introduction (page not supplied) describes consistent N-glycosylation profiles as difficult to obtain. The discussion (section labeled “Some,” page not supplied) attributes differing glycosylation patterns in the studied therapeutic proteins to the involvement of hundreds of enzymes. It does not state that glycan biosynthesis is non-templated or identify competition for a common substrate, substrate availability, or enzyme–substrate specificity as the causes. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    In CHO cells, knock down, knock out and overexpression of glycosylation enzymes and NSD transporters have been used in biotherapeutic functional studies and for development of biobetters.

    “In CHO cells, the knock down (KD), knock out (KO) and overexpression (OE) of glycosylation enzymes and NSD transporters have been used in biotherapeutic functional studies and for the development of biobetters, as summarised in Figure 2 [73,74].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [73] 2019, Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms, Biotechnol. Bioeng. source

      Section 5.1.1 and Table 2 describe knockdown, knockout and overexpression of glycosylation-related enzymes in CHO cells to alter biopharmaceutical glycosylation and product properties. The passages do not establish the claimed use of NSD transporter engineering in CHO cells; Table 2 lists overexpression of SLC35A2 in yeast instead. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [74] 2018, Glycoengineering in CHO cells: advances in systems biology, Biotechnol. J. source
  • Premisecitation

    Combining insertion of a third N-glycosylation site at the N-terminus with further mutation of Cys residues generated a range of monomers and multimers with tailored interactions with sialic acid-dependent receptors.

    “The insertion of a third N-glycosylation site at the N-terminus generated highly branched and hypersialylated glycans, which are mostly absent at the Asn297 site and when combined with further mutation of Cys residues, was able to generate a range of monomers and multimers that have tailored interactions with sialic acid-dependent receptors [120].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [120] 2017, Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors, J. Biol. Chem. source
  • Premisecitation

    Glycosylation sites can vary in accessibility and the surrounding amino acid environment on the surface of a protein.

    “Glycosylation sites can vary both in accessibility and the surrounding amino acid environment on the surface of a protein [109].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [109] 2015, Analysis of site-specific N-glycan remodelling in the endoplasmic reticulum and the Golgi, Glycobiology source
  • Premisecitation

    The availability of genomic sequences has enabled CHO cell engineering with unprecedented precision using engineered nucleases including ZFNs, TALENs and CRISPR.

    “The availability of genomic sequences has enabled CHO cell engineering with unprecedented precision using engineered nucleases, including zinc finger nucleases (ZFNs) and transcription activator like nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR) system [71–73,75,76].”
    Quote in the paperNot a substantial claim3 of 5 sources checked
    • cites [71] 2011, The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line, Nat. Biotechnol. source

      The Discussion (page unavailable) says the CHO-K1 sequence will facilitate targeted genetic manipulation and cell-line engineering. It presents this as an anticipated benefit, however, and does not describe engineering with ZFNs, TALENs or CRISPR, or establish that the sequence has already enabled it with unprecedented precision. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [72] 2018, A reference genome of the Chinese hamster based on a hybrid assembly strategy, Biotechnol. Bioeng. source

      The Introduction says published CHO genomes have enhanced genetic engineering, and the Discussion says precise genome-editing tools depend on a well-assembled genome and discusses CRISPR-based approaches. The paper does not identify ZFNs or TALENs as examples of those tools, so it does not support the full list in the citing sentence. Page numbers were unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [73] 2019, Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms, Biotechnol. Bioeng. source
    • cites [75] 2015, CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives, Biotechnol. J. source
    • cites [76] 2015, Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN, J. Control. Release source
  • May combine two claims. 23
  • Classified as probably not a substantial claim. 9

  • May combine two claims.

    Premisecitation

    Murine cell lines NS0 and Sp2/0 produce glycan structures similar to humans but also produce immunogenic epitopes such as Galα(1-3)Gal and have high NeuGc sialic acid, which is why they are less commonly used for biotherapeutic production.

    “while murine cell lines such as NS0 and Sp2/0 also produce glycan structures similar to humans, they also produce immunogenic epitopes such as the Galα(1–3)Gal epitope and they have a high content of NeuGc sialic acid, which is why they are less commonly used for biotherapeutic production [1,73].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [1] 2009, Glycosylation as a strategy to improve antibody-based therapeutics, Nat. Rev. Drug Discov. source
    • cites [73] 2019, Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms, Biotechnol. Bioeng. source

      The Accepted Article passage (page unavailable) says biopharmaceuticals from mouse cell lines have a higher proportion of Neu5Gc than those from CHO cells, and identifies this as a major reason mouse lines stopped being used for human antibody production. The passages do not establish the specific claims about NS0 and Sp2/0, their human-like glycans, or their production of the Galα(1–3)Gal epitope; the source also speaks specifically about human antibody production rather than biotherapeutics generally. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Enzymatic hypergalactosylation of mAbs enhances ADCC activity, albeit afucosylation remains the primary influence.

    “In contrast with early studies, enzymatic hypergalactosylation of mAbs was shown to enhance ADCC activity, albeit afucosylation remained the primary influence [22].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] 2016, Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies, Mol. Immunol. source

  • May combine two claims.

    Premisecitation

    CHO cells do not express alpha-1,3-galactosyltransferases (although they have the gene), and expression of CMP-N-acetylneuraminic acid hydroxylase is virtually absent.

    “CHO cells do not express alpha-1,3-galactosyltransferases, which produce the immunogenic alpha-galactosyl epitope (although they have the gene), and the expression of CMP-N-acetylneuraminic acid hydroxylase, which produces CMP-NeuGc, is virtually absent [27,71,72].”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [27] 2010, Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity, J. Biol. Chem. source

      The Results and Discussion section discusses fucosyltransferase genes in CHO cells, but not the α-1,3-galactosyltransferase gene or its expression. It also mentions glycans with terminal NeuGc without addressing CMP-N-acetylneuraminic acid hydroxylase expression. Searches for the named enzymes, the α-galactosyl epitope, and NeuGc did not locate support for either attributed expression claim. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [71] 2011, The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line, Nat. Biotechnol. source

      In Results (“Genes involved in immunogenic responses”), the paper reports that a Ggta1 homolog is present in the CHO-K1 genome but was not expressed, and that CMAH expression was not detected in its CHO-K1 analysis. The claim extends those expression findings to CHO cells generally; the same section also notes very small CHO subpopulations capable of producing α-Gal epitopes. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [72] 2018, A reference genome of the Chinese hamster based on a hybrid assembly strategy, Biotechnol. Bioeng. source

      The Results section on filled sequence gaps discusses an Xylt2 mutation affecting glycosaminoglycan biosynthesis, not expression of the enzymes in the claim. Searches for alpha-1,3-galactosyltransferase, CMP-N-acetylneuraminic acid hydroxylase, and NeuGc found no passage in our copy addressing their presence or expression in CHO cells; page numbers were unavailable. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Galactose is required for sialylation with NeuAc, which can improve solubility, anti-inflammatory activity, thermal stability and serum half-life of mAbs.

    “On the other hand, galactose is required for sialylation with N-acetylneuraminic acid (NeuAc), which in turn can improve the solubility, anti-inflammatory activity, thermal stability and serum half-life of mAbs [8,23–26].”
    Quote in the paperNot a substantial claim3 of 5 sources checked
    • cites [8] 2015, Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins, J. Pharm. Sci. source
    • cites [23] 1993, Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura, Lancet source
    • cites [24] 2008, Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc, Science source

      The section labeled Abstract (page not provided) describes adding galactose before sialylating IgG Fc glycans and reports anti-inflammatory activity for the 2,6-sialylated Fc. It does not report improved solubility, thermal stability, or serum half-life of mAbs. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [25] 2015, Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity, Proc. Natl Acad. Sci. U.S.A. source

      The Results describe galactose-containing reagents in Fc sialylation, and the paper reports enhanced anti-inflammatory activity for sialylated IVIg in animal models. It does not establish improved solubility or thermal stability of mAbs. The Results also report no significant pharmacokinetic difference between sialylated and parent IVIg, so they do not support an improved serum half-life for this product. Page numbers were not provided in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [26] 2016, Sialylated intravenous immunoglobulin suppress anti-ganglioside antibody mediated nerve injury, Exp. Neurol. source

      The Discussion and Results report that sialylated IVIg had greater anti-inflammatory efficacy than sialic-acid-depleted IVIg in a mouse nerve-injury model (page not provided). The paper does not establish that galactose is required for NeuAc sialylation or that sialylation improves the solubility, thermal stability, or serum half-life of mAbs; its efficacy finding concerns IVIg, not therapeutic mAbs. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Humans lack the ability to produce the Galα1-3Galβ1-4GlcNAc-R epitope, which is common in murine cells and has been shown to induce anaphylaxis.

    “Humans also lack the ability to produce the Galα1–3Galβ1–4GlcNAc–R epitope, which is common in murine cells, and this has been shown to induce anaphylaxis [8,33].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [8] 2015, Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins, J. Pharm. Sci. source
    • cites [33] 2008, Cetuximab-induced anaphylaxis and IgE specific for galactose-α-1,3-galactose, N. Engl. J. Med. source

      The Discussion (page unavailable) links severe cetuximab reactions to preexisting IgE against galactose-α-1,3-galactose and says cetuximab is produced in a mouse cell line expressing α-1,3-galactosyltransferase. The Characterization section (page unavailable) describes the epitope on proteins of most nonprimate mammals. The copy does not directly establish that humans cannot produce the epitope, and the anaphylaxis finding concerns susceptible patients exposed to cetuximab, not exposure to the epitope in general. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    α2,6-linked NeuAc (main sialic acid linkage in human IgG) is solely required for the anti-inflammatory activity of IgG mediated by non-canonical IgG-Fc receptors.

    “it is the α2,6-linked NeuAc (main sialic acid linkage in human IgG) that is solely required for the anti-inflammatory activity of IgG mediated by non-canonical IgG-Fc receptors [24].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [24] 2008, Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc, Science source

  • May combine two claims.

    Premisecitation

    Some hypersialylated mutants bind influenza viruses with high affinity and inhibit influenza-mediated hemagglutination.

    “Some of these hypersialylated mutants were also found to bind influenza viruses with high-affinity and inhibit influenza-mediated hemagglutination [121,122].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [121] 2019, Insertion of N-terminal hinge glycosylation enhances interactions of the Fc region of human IgG1 monomers with glycan-dependent receptors and blocks hemagglutination by the influenza virus, J. Immunol. source
    • cites [122] 2020, Choice of host cell line is essential for the functional glycosylation of the Fc region of human IgG1 inhibitors of influenza B viruses, J. Immunol. source

  • May combine two claims.

    Premisecitation

    Oligomannose glycans (Man5-9GlcNAc2) increase FcγRIIIa binding and ADCC of mAbs compared with fucosylated complex glycans, but decrease CDC activity and binding affinity to FcγRIIa/b.

    “While oligomannose glycans (Man5–9GlcNAc2) have been shown to increase the FcγRIIIa binding and ADCC of mAbs compared with fucosylated complex glycans, the CDC activity was decreased together with the binding affinity to FcγRIIa/b [30].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] 2012, Production, characterization, and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans, mAbs source

  • May combine two claims.

    Premisecitation

    Darbepoetin alfa, an EPO molecule approved in 2001 with two additional N-oligosaccharides, increased in vivo potency and half-life.

    “An early example worth mentioning is the development of darbepoetin alfa, an EPO molecule approved in 2001 with two additional N-oligosaccharides that increased in vivo potency and half-life [117].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [117] 2003, Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin, Exp. Hematol. source

      The Results section says darbepoetin alfa has two additional N-linked carbohydrate chains, and the Abstract reports a longer circulating half-life and greater in vivo potency than rHuEPO. Our copy does not establish the claimed 2001 approval date (searched for approval and 2001). (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    GlycoDelete reduces glycan heterogeneity by expressing a Golgi-targeted endo-β-N-acetylglucosaminidase in HEK 293SGnTI(−) cells to produce GlcNAc N-glycan stumps modified by galactosyltransferases and sialyltransferases to yield small sialylated trisaccharides.

    “GlycoDelete is an elegant glycoengineering strategy worth mentioning, which reduces glycan heterogeneity by expressing a Golgi-targeted endo-β-N-acetylglucosaminidase in HEK 293SGnTI(−) cells to produce GlcNAc N-glycan ‘stumps’ modified by galactosyltransferases and sialyltransferases to yield small sialylated trisaccharides [108].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [108] 2014, Glycodelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins, Nat. Biotechnol. source

  • May combine two claims.

    Premisecitation

    High-mannose structures are cleared more rapidly from circulation in humans and mice, likely due to uptake by endogenous receptors such as the mannose receptor.

    “Pharmacokinetic studies in both humans and mice revealed that high-mannose structures were cleared more rapidly from circulation, which is likely to be a result of uptake by endogenous receptors such as the mannose receptor [29,30].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [29] 2011, High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans, Glycobiology source
    • cites [30] 2012, Production, characterization, and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans, mAbs source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mass spectrometry-based techniques have dominated the glycomics and glycoproteomics fields for decades because of their high sensitivity and ability to characterise complex heterogeneous mixtures, especially when combined with upstream separation techniques such as liquid chromatography.

    “Mass spectrometry (MS)-based techniques, the experimental workflow for which is outlined in Figure 3, have dominated the glycomics and glycoproteomics fields for decades because of its high sensitivity and ability to characterise complex heterogeneous mixtures, especially when combined with upstream separation techniques such as liquid chromatography [142].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [142] 2018, Mass spectrometry approaches to glycomic and glycoproteomic analyses, Chem. Rev. source

      The Introduction (page unavailable) says MS has been central to glycomic and glycoproteomic analysis and describes its use with HPLC and UPLC. The instrumentation section (page unavailable) supports the sensitivity claim, while the separation-methods section explains why LC helps analyze heterogeneous glycan samples. The copy does not establish that MS dominated both fields for decades. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    NeuGc can be immunogenic in humans, who express a non-functional CMP-N-acetylneuraminic acid hydroxylase and have high levels of circulating anti-NeuGc antibodies.

    “N-glycolylneuraminic (sialic) acid (NeuGc), can also be immunogenic in humans, who express a non-functional CMP-N-acetylneuraminic acid hydroxylase and as a result have high-levels of circulating anti-NeuGc antibodies [31,32].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [31] 2003, Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid, Proc. Natl. Acad. Sci. U.S.A. source
    • cites [32] 2010, Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins, Nat. Biotechnol. source

      The copy states that the CMAH gene is irreversibly mutated in all humans and that humans have circulating anti-Neu5Gc antibodies, supporting the immunogenicity point. But the Abstract describes antibody levels as variable and only sometimes high; it does not support the claim that humans generally have high levels as a result of the CMAH defect. The supplied passages have no page numbers. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    MALDI and ESI are soft ionisation techniques that now dominate in the MS field.

    “Matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) are ‘soft ionisation' techniques, which now dominate in the MS field [142,145].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [142] 2018, Mass spectrometry approaches to glycomic and glycoproteomic analyses, Chem. Rev. source

      The “Ionization Considerations” section describes soft ionization techniques as normally used for glycan and glycopeptide analyses and says MALDI and ESI are the most widely used ionization methods in that context. It does not establish that they dominate the entire MS field, as the citing sentence claims. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [145] 2006, Mass spectrometric analysis of N- and O-glycosylation of tissues and cells, Curr. Opin. Struc. Biol. source

  • May combine two claims.

    Premisecitation

    IgG modulates cellular immune responses through FcγR-binding and direct receptor- or ligand-binding.

    “They modulate cellular immune responses through Fcγ receptor (FcγR)-binding as well as through direct receptor- or ligand-binding [9,10].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] 1998, Fc receptors are required in passive and active immunity to melanoma, Proc. Natl Acad. Sci. U.S.A. source
    • cites [10] 2000, Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets, Nat. Med. source

  • Classified as probably not a substantial claim.

    Premisenone given

    CHO cells are the workhorse of recombinant biotherapeutic production.

    “Chinese hamster ovary (CHO) cells, which are the workhorse of recombinant biotherapeutic production”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The conserved Fc site Asn297 in IgG1 is N-glycosylated and essential to the anti- or proinflammatory role of IgGs.

    “The conserved Fc site Asn297 in IgG1 is N-glycosylated, which is essential to the anti- or proinflammatory role of IgGs (Figure 1) [2].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] 2014, Emerging principles for the therapeutic exploitation of glycosylation, Science source

  • May combine two claims.

    Premisecitation

    Without N-glycosylation, the Fc portion cannot adopt the functional conformation required to bind Fc-dependent receptors (type 1), glycan dependent receptors (type 2) or initiate the complement cascade.

    “without N-glycosylation the Fc portion cannot adopt the functional conformation required to bind Fc-dependent receptors (type 1), glycan dependent receptors (type 2) or initiate the complement cascade [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] 2015, Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins, J. Pharm. Sci. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Between 2014 and 2018, 84% of newly approved antibody therapeutics were produced in CHO cells.

    “between 2014 and 2018 the vast majority (84%) of newly approved antibody therapeutics were produced in CHO cells [5].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] 2018, Biopharmaceutical benchmarks 2018, Nat. Biotechnol. source

  • May combine two claims.

    Premisenone given

    α1,6 core-linked L-fucose is a primary determinant of IgG inflammatory activity and is added by FUT8.

    “α1,6 core-linked L-fucose (Fuc) is a primary determinant of IgG inflammatory activity and is added onto N-glycans by an α1,6-fucosyltransferase (FUT8).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The risks of immunogenic N-glycoforms require a production system that closely mimics human glycosylation, which is one reason CHO cells are most often used for industrial biotherapeutic production.

    “The risks of immunogenic N-glycoforms require a production system that closely mimics human glycosylation, which is one of the reasons why CHO cells are most often used for industrial biotherapeutic production.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    MALDI has risen to prominence for its exquisite sensitivity, tolerance to impurities, high-throughput capacity, ease of data analysis (given that most ions are singly charged) and simplicity of sample preparation.

    “MALDI in particular has risen to prominence for its exquisite sensitivity, tolerance to impurities, high-throughput capacity, ease of data analysis (given that most ions are singly charged) and the simplicity of sample preparation [146].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [146] 2018, Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2013–2014, Mass Spectrom. Rev. source

  • May combine two claims.

    Premisecitation

    Atezolizumab is a clinically approved (2016-US, 2017-EU) glycoengineered anti-PD-L1 mAb whose Fc has been engineered to lack glycosylation.

    “Coming back to Ig-based biotherapeutics, atezolizumab, an oncolytic anti-PD-L1 mAb, is another clinically approved (2016-US, 2017-EU) glycoengineered product, except the Fc has been engineered to lack glycosylation [5].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] 2018, Biopharmaceutical benchmarks 2018, Nat. Biotechnol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Over the last two decades, increasingly effective genetic engineering strategies have enabled cell line development and protein backbone engineering with unprecedented precision and speed.

    “Over the last two decades, increasingly effective genetic engineering strategies have enabled cell line development and protein backbone engineering with unprecedented precision and speed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.

    Interpretationnone given

    A remarkable and growing list of studies is revolutionising understanding of the fundamentals of protein glycosylation, and advances in genome editing have made glycoengineering increasingly accessible and efficient for the development of biobetters with improved half-life, safety and efficacy.

    “A remarkable and growing list of studies is revolutionising our understanding of the fundamentals of protein glycosylation and advances in genome editing have made glycoengineering increasingly accessible and efficient for the development of biobetters with improved half-life, safety and efficacy.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In 2012, mogamulizumab was the first-ever glycoengineered mAb to gain approval and is produced in FUT8 knockout CHO cells (Potelligent®) for CCR4-positive T cell leukaemia or lymphoma.

    “In 2012, mogamulizumab was the first-ever glycoengineered mAb to gain approval and is produced in FUT8 knockout CHO cells (Potelligent® technology) for the treatment of CCR4-positive T cell leukaemia or lymphoma [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] 2015, Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins, J. Pharm. Sci. source

  • May combine two claims.

    Interpretationreasoning

    Engineering N-glycosylation in CHO cells is complex and specific to the target biotherapeutic, and advances in genetic manipulation strategies are enabling increasingly homogeneous and bespoke glycosylation with unprecedented precision and efficiency.

    “While it is evident from these examples that engineering N-glycosylation in CHO cells is complex and specific to the target biotherapeutic, advances in genetic manipulation strategies are enabling increasingly homogeneous and bespoke glycosylation with unprecedented precision and efficiency.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    Control of the glycosylation pathway is made difficult due to the non-template-based synthesis of glycans.

    “However, control of this pathway is made difficult due to the non-template-based synthesis of glycans.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    GlycoMab technology produces low-fucose biotherapeutics such as obinutuzumab, approved in 2013 for follicular lymphoma and chronic lymphocytic leukaemia, with 10-100-fold improved ADCC.

    “This principle is exploited in the GlycoMab® technology of GlycArt (acquired by Roche) that produces low-fucose biotherapeutics such as the anti-CD20 obinutuzumab, approved in 2013 for treatment of follicular lymphoma and chronic lymphocytic leukaemia, with 10–100-fold improved ADCC [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] 2015, Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins, J. Pharm. Sci. source
29 of 29

  • The paper relies on this without support.

    Most therapeutic mAbs are IgG, the dominant Ig type in human serum.

    “Most therapeutic mAbs are Immunoglobulin G (IgG) which is also the dominant Ig type in human serum.”

  • The paper relies on this without support.

    Glycosylation is a critical quality attribute of glycoprotein therapeutics that influences the efficacy, half-life and safety of recombinant biotherapeutics.

    “Secondly, glycosylation is a critical quality attribute of glycoprotein therapeutics.”
    “Glycosylation is a critical quality attribute that influences the efficacy, half-life and safety of recombinant biotherapeutics.”

  • The paper relies on this without support.

    Glycosylation analysis is necessary for elucidating the impact of cell line engineering strategies and for quality control in manufacturing and clinical phases.

    “Firstly, glycosylation analysis is necessary for elucidating the impact of cell line engineering strategies at both the cellular and recombinant product level, and secondly, highly sensitive and reliable analytics are required for strict quality control in the later manufacturing and clinical phases.”
    “Deconstructing the role of glycans and analysing changes in the glycomic profile of a biotherapeutic is necessary at the developmental stage and essential for quality control purposes and that is why the analytical methods used in support of glycoengineering will be discussed below.”

  • The paper relies on this without support.

    One-dimensional MS mode cannot distinguish structural isomers with varied branching patterns or epitopes.

    “While the 1-dimensional MS mode can typically determine monosaccharide composition unambiguously, structural isomers with varied branching patterns or epitopes cannot be distinguished.”
4 of 4

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Authors
Donini, R., Haslam, S. M., & Kontoravdi, C.
Affiliations
Imperial College London (GB)
Corresponding author
Kontoravdi, Cleo
ORCID
Venue
Biochemical Society Transactions
Publisher
Portland Press Ltd.
Published
2021-03-11
Volume
49
Issue
2
Pages
915-931
ISSN
  • 0300-5127 (ISSN-L)
  • 1470-8752
DOI
10.1042/bst20200840
PMC
PMC8106501
OpenAlex
W3133686863
Semantic Scholar
4b5ec85de93aeb13bd34763fc91b81366320cd94
Cited by
37
References
157
Citations by year
2026: 8 · 2025: 7 · 2024: 9 · 2023: 6 · 2022: 5 · 2021: 2
FWCI
0.58
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemical Society Transactions (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Spiral (Imperial College London) (submittedVersion, cc-by)
Funders
  • Directorate for Biological Sciences
  • Biotechnology and Biological Sciences Research Council (2283566)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
glycoengineering, CHO cells, N-glycosylation, antibody engineering, recombinant protein expression, cell line engineering, immunogenicity, half-life
MeSH terms
Animals, Cricetulus, Glycoproteins, Glycosylation, Cricetinae, Polysaccharides, Recombinant Proteins, Protein Engineering, CHO Cells, Proteomics, Glycomics, Metabolic Engineering

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-28.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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