Therapeutic glycoprotein production in mammalian cells

In the paper

“Moreover, different gene amplification systems have been developed and used in CHO cells, which allow for high titer yields and good specific productivity (Durocher and Butler, 2009;Kim et al., 2012;Lai et al., 2013).”

What the sources say

• ?Not checked — no full text of the sourcecites Durocher and Butler, 2009 source
• ?Not checked — no full text of the sourcecites Kim et al., 2012 source
• Supported

  The Introduction describes the MTX amplification and GS systems in CHO cell-line development and says further gene amplification can increase cell protein productivity. It also attributes improved titers to the establishment of stable, high-producing clones, alongside culture-process optimization. The paper does not imply that amplification alone determines titer. (LLM, conf=0.88)

  cites Lai et al., 2013 source

In the paper

“Since CHO cells produce recombinant glycoproteins with human-like glycans, the generated products are to more likely be compatible and bioactive within human hosts (Kim et al., 2012;Lai et al., 2013).”

What the sources say

• ?Not checked — no full text of the sourcecites Kim et al., 2012 source
• Supported

  The Introduction states that CHO cells produce glycoproteins with more human-like glycosylation and allow post-translational modifications that make recombinant proteins compatible and bioactive in humans. This supports the citing sentence’s paraphrase. (LLM, conf=0.94)

  cites Lai et al., 2013 source

In the paper

“CHO cells also have limited ability to gamma-carboxylate recombinant proteins such as clotting factors (Kumar 2015), even though some improvements have been achieved through metabolic engineering work (Rehemtulla et al., 1993;Liu et al., 2014).”

What the sources say

• ?Not checked — no full text of the sourcecites Kumar 2015 source
• Partly supported

  The paper reports that expressing gamma-carboxylase in CHO cells increased in vitro enzyme activity, but did not improve the specific procoagulant activity of secreted factor IX. Expressing the propeptide-processing enzyme PACE improved factor IX activity threefold. Thus, the cited work supports an improvement through cell engineering, but not an improvement in gamma-carboxylation of the secreted clotting factor. (LLM, conf=0.84)

  cites Rehemtulla et al., 1993 sourceExperimental — accuracy not yet measured
• Supported

  Liu et al. is cited for improvements through metabolic engineering, not for the preceding claim about CHO cells’ limited ability, which is attributed to Kumar (2015). The Introduction describes engineering a CHO cell line with enzymes supporting γ-carboxylation and reports dramatically improved productivity of functional recombinant factor IX; the Discussion likewise attributes improved productivity to co-expression of those enzymes. (LLM, conf=0.93)

  cites Liu et al., 2014 source

In the paper

“Moreover, CHO cells produce glycans that do not occur in human cells, namely N-glycolylneuraminic acid (Neu5Gc) and galactose-α1,3-galactose (α-gal), even though these occurring at very low levels (e.g < 2% and < 0.2% respectively) (Bosques et al., 2010;Ghaderi et al., 2010;Dietmair et al., 2012b;Ghaderi et al., 2012).”

What the sources say

• Partly supported

  In “To the Editor” (page unavailable), the paper shows that CHO-produced proteins can contain α-Gal and reports an α-Gal–containing glycan in abatacept at approximately 0.2% of total N-linked glycans. That is a product-specific estimate, not a general level below 0.2%. Our copy does not address Neu5Gc or its claimed level below 2%, nor does it explicitly state that these glycans do not occur in human cells. (LLM, conf=0.87)

  cites Bosques et al., 2010 sourceExperimental — accuracy not yet measured
• Partly supported

  The paper states that humans cannot synthesize α-gal or Neu5Gc and that glycoproteins produced in CHO cells carry small amounts of Neu5Gc (body text, indexed under Abstract; page unavailable). It does not establish α-gal production specifically in CHO cells or give the claimed <2% and <0.2% levels. It also notes that human cells can incorporate Neu5Gc from animal-derived culture materials, so ‘do not occur in human cells’ is too absolute. (LLM, conf=0.88)

  cites Ghaderi et al., 2010 sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Dietmair et al., 2012b

In the paper

“For example, co-expression of furin was shown to allow the production of fully cleaved and active von Willebrand factor in an industrial-scale CHO perfusion system (Fischer et al., 1995)”

What the sources say

• Partly supported

  The Expression section describes furin-coexpressing CHO cells grown in industrial-scale perfusion bioreactors. The structural analysis (section labeled Preparation; page unavailable) reports that the resulting vWF was completely processed and composed of intact, mature subunits. It identifies multimers associated with hemostatic activity but does not demonstrate the produced vWF’s activity with a functional test. (LLM, conf=0.85)

  cites Fischer et al., 1995 sourceExperimental — accuracy not yet measured

In the paper

“It was shown to be effective for establishing CHO cell lines producing respectable titers of monoclonal antibody (Lindenbaum et al., 2004;Kennard et al., 2009;Kennard 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Lindenbaum et al., 2004 source
• Supported

  The Abstract reports that the ACE System generated stable CHO cell lines expressing a human monoclonal IgG1 antibody at over 1 g/L. The Generation of Double Load Single Cell Subclones section reports a maximum titer of 1,144 mg/L for a candidate line, supporting the description of respectable titers. (LLM, conf=0.94)

  cites Kennard et al., 2009 source
• ?Not checked — no full text of the sourcecites Kennard 2011 source

In the paper

“Murine myeloma cells (NS0 and Sp2/0), derived from tumor cells that no longer produce their original immunoglobulins, also are being used to produce some commercial monoclonal antibodies such as Cetuximab (ERBITUX ® ) and Palivizumab (SYNAGIS ® ) (Barnes et al., 2000;Dumont et al., 2015).”

What the sources say

• Partly supported

  The ‘Products generated using the GS-NS0 system’ section identifies Synagis (palivizumab) as a marketed monoclonal antibody produced using the GS-NS0 system. The ‘NS0 cell history’ section also describes NS0 as a non-secreting murine myeloma line. Our copy does not identify Sp2/0 or Cetuximab, so it does not support those parts of the attributed claim. (LLM, conf=0.89)

  cites Barnes et al., 2000 sourceExperimental — accuracy not yet measured
• Supported

  In the Introduction’s section on non-human expression systems, the paper says NS0 and Sp2/0 are murine myeloma cell lines used to produce monoclonal antibodies and describes their origin in tumor cells that no longer produce their original immunoglobulins. Its product table lists palivizumab under NS0 and cetuximab under Sp2/0, both as approved products. Page numbers are not provided in our copy. (LLM, conf=0.95)

  cites Dumont et al., 2015 source

In the paper

“Similarly, the DHFR selection uses the fact that CHO cell mutants have been selected to be deficient for DHFR (Urlaub and Chasin, 1980).”

What the sources say

• Supported

  The introduction says the authors developed a selective system and used it to isolate DHFR-deficient mutants in a Chinese hamster ovary (CHO) cell line. The Discussion confirms that the selected CHO variants lacked DHFR catalytic activity. (LLM, conf=0.94)

  cites Urlaub and Chasin, 1980 source

In the paper

“These nucleases induce DNA double-strand breaks at precise locations into the host genome, facilitating the integration of the cassette by non-homologous end-joining (NHEJ) or homology-directed repair (HDR) (Gaj et al., 2013).”

What the sources say

• Supported

  The “Genome editing with site-specific nucleases” section says that nuclease-induced double-strand breaks enable targeted integration of donor transgenes through HDR. It also reports that large transgenes have been introduced into endogenous loci through NHEJ-mediated ligation. This supports the claim’s description of cassette integration through either repair pathway. (LLM, conf=0.91)

  cites Gaj et al., 2013 source

In the paper

“Only two marketed recombinant glycoproteins are currently manufactured in these cells, Factor VIIa (NovoSeven ® ) and Factor VIII (Kogenate ® and Kovaltry ® ), which are other clotting factors (Durocher and Butler, 2009;Dumont et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Durocher and Butler, 2009 source
• ?Could not verify

  The non-human expression systems section lists NovoSeven and Kogenate among product references and defines BHK as baby hamster kidney, but the passages retrieved do not link either product to BHK manufacture or establish that only two marketed glycoproteins use those cells. The references to Kogenate and the comparison of factor VIII expression with BHK21 cells also do not establish the marketed-product claim. Searches for BHK, both factor names, and the product names did not settle it. (LLM, conf=0.68)

  cites Dumont et al., 2015 sourceExperimental — accuracy not yet measured

In the paper

“Insect cells produce N-glycan precursors that are trimmed, creating high mannose or paucimannose structures (Kost et al., 2005).”

What the sources say

• Supported

  The Main section states that insect cells assemble N-glycans and trim their precursors to produce high-mannose or paucimannose end products. This directly supports the citing sentence. (LLM, conf=0.95)

  cites Kost et al., 2005 source

In the paper

“Murine cells can also produce α-gal and Neu5Gc at considerably higher levels than hamster cells, increasing the risks of immunogenicity (Tangvoranuntakul et al., 2003;Chung et al., 2008;Macher and Galili, 2008;Padler-Karavani et al., 2008;Ghaderi et al., 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Tangvoranuntakul et al., 2003 source
• Partly supported

  Evaluation of Antigens says mouse-cell-produced cetuximab differs in glycosylation from its Chinese hamster ovary (CHO)-produced variant because CHO cells lack α-1,3-galactosyltransferase. Characterization of the Epitope on Cetuximab identifies both α-gal and N-glycolylneuraminic acid (Neu5Gc) on cetuximab, while the Discussion links α-gal-specific IgE to severe hypersensitivity reactions. The paper does not establish that murine cells produce both sugars at considerably higher levels than hamster cells or that Neu5Gc increases immunogenicity. (LLM, conf=0.88)

  cites Chung et al., 2008 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Macher and Galili, 2008 source

In the paper

“Likewise, signal recognition particle 14 (SRP14), which targets secreted proteins to the endoplasmic reticulum membrane, enhanced productivity of recombinant IgG in CHO cells when overexpressed (Lakkaraju et al., 2008;Le Fourn et al., 2014).”

What the sources say

• Partly supported

  The section ‘Elongation arrest-defective SRP impairs the accumulation of membrane and secreted proteins’ (page unavailable) shows that functional SRP14 is important for secretion: restoring it in depleted 293T cells restored normal SEAP secretion. The paper does not report SRP14 overexpression increasing recombinant IgG productivity in CHO cells; searches for CHO cells, IgG, and overexpression found no such result. (LLM, conf=0.87)

  cites Lakkaraju et al., 2008 sourceExperimental — accuracy not yet measured
• Supported

  Section 3.4 reports that CHO subclones overexpressing SRP14 had increased recombinant IgG secretion and specific productivity compared with controls. The reported gains include a sevenfold increase in the low-producer cells and a 30% increase in the high-producer cells. (LLM, conf=0.94)

  cites Le Fourn et al., 2014 source

In the paper

“Recently, the same effect was observed for human IgG4 (Gong et al., 2016).”

What the sources say

• Supported

  The section on production and receptor-binding measurements reports increased binding of afucosylated human IgG4 to FcγRIIIa, including a 3.8-fold increase for the F158 allotype and a greater than 15-fold increase for V158. It also explicitly compares this effect with that observed for IgG1 antibodies. (LLM, conf=0.94)

  cites Gong et al., 2016 source

In the paper

“Since most of these elements are composed of nucleosome depleted regions, this could also contribute to the inhibition of the propagation of an inactive chromatin state surrounding the transgene, serving as genetic boundaries (Arope et al., 2013;Harraghy et al., 2015).”

What the sources say

• Partly supported

  The Discussion (page not provided) proposes that nucleosome depletion over MARs can block heterochromatin propagation and protect adjacent transgenes from silencing. It does not establish that most epigenetic elements have nucleosome-depleted regions; its evidence and proposed mechanism concern MARs and promoters. (LLM, conf=0.84)

  cites Arope et al., 2013 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Harraghy et al., 2015 source

In the paper

“Successful apoptosis inhibition was also achieved by overexpression of other related anti-apoptotic proteins (Durocher and Butler, 2009;Bandaranayake and Almo, 2014).”

What the sources say

• ?Not checked — no full text of the sourcecites Durocher and Butler, 2009 source
• Supported

  In the ‘Transient gene expression (TGE)’ section, the review says that overexpressing anti-apoptotic proteins, including Bcl-2 family members and Bcl-x(L), increases cell viability. This supports the claim that overexpression of related proteins was used successfully to inhibit apoptosis; the passage does not provide a page number. (LLM, conf=0.88)

  cites Bandaranayake and Almo, 2014 source

In the paper

“This increased affinity allows better therapeutic activity of Mabs used in cancer therapy, by triggering the antibody-dependent-cell-mediated cytotoxicity (ADCC) pathway (Niwa et al., 2005;Iida et al., 2006;Chung et al., 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Niwa et al., 2005 source
• Partly supported

  The Introduction (page not available) links higher FcγRIIIa affinity with a superior clinical response to rituximab, and the Discussion reports that improved FcγRIIIa binding enhances ADCC. However, this paper’s test of nonfucosylated antibodies used a human ex vivo model; it supports improved ADCC and therapeutic potential, not established better therapeutic activity in cancer patients. (LLM, conf=0.82)

  cites Iida et al., 2006 sourceExperimental — accuracy not yet measured
• Partly supported

  The ADCC assay section reports that increased FcγRIIIa binding appeared to produce a proportional increase in ADCC activity. The Discussion also reports better responses to cancer antibodies in patients with a higher-affinity receptor variant. But the paper does not establish that increasing an antibody’s affinity itself improves clinical therapeutic outcomes; its direct antibody findings concern binding and ADCC assays. (LLM, conf=0.84)

  cites Chung et al., 2012 sourceExperimental — accuracy not yet measured

In the paper

“UCOEs were also found to be helpful for increasing the productivity of recombinant protein cell lines (Benton et al., 2002;She et al., 2009).”

What the sources say

• Supported

  In “Vector component selection” (page not provided), the paper reports that adding UCOEs dramatically increased antibody expression in CHO cells. Its Figure 3 caption also reports increasing antibody production with UCOE vectors, compared with little increase without them. (LLM, conf=0.93)

  cites Benton et al., 2002 source
• ?Could not verify

  The Results and Discussion section on ultraconserved elements discusses UCEs and their enrichment in housekeeping genes, not UCOEs. Searches for UCOEs and recombinant-protein cell-line productivity found no passage in our copy addressing the claimed benefit. (LLM, conf=0.78)

  cites She et al., 2009 sourceExperimental — accuracy not yet measured

In the paper

“Inclusion of these various epigenetic elements not only can help to increase the expression level of biotherapeutics, but can also increase the number of clones that have integrated the transgene with a more defined copy number of transgene per cell, thus accelerating the selection process (Grandjean et al., 2011;Harraghy et al., 2015).”

What the sources say

• Supported

  The Abstract reports that MAR-containing vectors, using an epigenetic regulatory sequence, increased transgene expression. The Introduction identifies expression of therapeutic proteins as a potential application, supporting the claim that including such an element can help increase biotherapeutic expression. (LLM, conf=0.90)

  cites Grandjean et al., 2011 source
• ?Not checked — no full text of the sourcecites Harraghy et al., 2015 source

In the paper

“Recently, both ZFN and a CRISPR/Cas9 approaches were also used for FUT8 gene-specific knockout (Malphettes et al., 2010;Ronda et al., 2014).”

What the sources say

• Supported

  The Introduction states that the authors created zinc-finger nucleases (ZFNs) that disrupt FUT8, and the Discussion describes ZFN reagents for knockout of the C. griseus FUT8 gene. In the sentence with two citations, Malphettes et al. supports the ZFN part; the CRISPR/Cas9 part is associated with the other citation. (LLM, conf=0.94)

  cites Malphettes et al., 2010 source
• Supported

  The Results section, “Homozygous Knockout of FUT8 in CHO Cells Generated by CRISPR,” reports Cas9-mediated functional knockout of FUT8 in CHO cells. The paper also identifies earlier ZFN targeting of FUT8 as Malphettes et al. (2010), the other citation in the citing sentence. (LLM, conf=0.93)

  cites Ronda et al., 2014 source

In the paper

“Its overexpression has shown positive results on productivity in the case of monoclonal antibodies in both CHO cells and NS0 cells (Smales et al., 2004;Mohan et al., 2007).”

What the sources say

• ?Not checked — no full text of the sourcecites Smales et al., 2004 source
• Supported

  The Results section (Cultures of Tet-Ab-PDI Clones With Regulated PDI Expression) reports that elevated PDI expression increased specific antibody productivity by 15–27% in recombinant CHO cells. This supports the CHO-cell finding attributed to Mohan et al.; the sentence also cites Smales et al. for the NS0-cell finding. (LLM, conf=0.91)

  cites Mohan et al., 2007 source

In the paper

“Inclusion of these various epigenetic elements not only can help to increase the expression level of biotherapeutics, but can also increase the number of clones that have integrated the transgene with a more defined copy number of transgene per cell, thus accelerating the selection process (Grandjean et al., 2011;Harraghy et al., 2015).”

What the sources say

• Partly supported

  The Results section, “MAR elements increase the copy number of genome-integrated transgenes” (page not available), reports increased integration and higher copy numbers in CHO cell pools and individual clones with MAR-containing plasmids. It does not establish that more clones have a more defined copy number per cell or that clone selection is accelerated. (LLM, conf=0.84)

  cites Grandjean et al., 2011 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Harraghy et al., 2015 source

In the paper

“The CDKL3 gene insertion also accelerates cell proliferation of 293 and CHO cells (Jaluria et al., 2007).”

What the sources say

• Supported

  Results §5 reports that HEK-293 cells expressing cdkl3 had a 19% higher maximum specific growth rate than controls, while CHO cells expressing cdkl3 had a 9% higher rate. Both differences were statistically significant, supporting the claim for both cell lines. (LLM, conf=0.94)

  cites Jaluria et al., 2007 source

In the paper

“Disruption of the fucosyltransferase in these cell lines completely abolished the fucosylation on the Fc domain of IgG.”

What the sources say

• Supported

  The Results report that antibodies produced by FUT8-knockout CHO cells completely lacked fucosylation. The Introduction identifies Fut8 as the enzyme that attaches fucose to the Fc core region. Page numbers are not provided in the supplied passages. (LLM, conf=0.94)

  cites Malphettes et al., 2010 source
• Partly supported

  The Results section (‘Homozygous Knockout of FUT8 in CHO Cells Generated by CRISPR’; page unavailable) identifies FUT8 as the enzyme that adds fucose to CHO-produced IgG1 and reports FUT8 disruption. But its measurements concern cell-surface fucose and FUT8 indels, not fucosylation of the IgG Fc domain; moreover, 98.6% of selected cells, not all cells, were lectin-negative. It does not establish complete abolition of IgG Fc fucosylation in these cells. (LLM, conf=0.86)

  cites Ronda et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“Furthermore, to improve N-glycan homogeneity on therapeutic proteins, a technique called “GlycoDelete” engineering was also recently developed in HEK293S cells (Meuris et al., 2014).”

What the sources say

• Supported

  In “GlycoDelete technology: simplifying mammalian cell N-glycosylation for recombinant protein expression” (page not provided), the authors introduce GlycoDelete as a strategy to reduce N-glycan heterogeneity and describe creating a 293S-derived GlycoDelete cell line. This supports the citing sentence’s attribution. (LLM, conf=0.92)

  cites Meuris et al., 2014 source

In the paper

“The publication of the CHO-K1 genome sequence in 2011, followed by the publication of two Chinese hamster and six CHO cell line genomes in 2013, bring new opportunities in developing and engineering CHO cells for improved glycoprotein production (Xu et al., 2011;Brinkrolf et al., 2013;Lewis et al., 2013).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Xu et al., 2011
• Partly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites Brinkrolf et al., 2013 sourceExperimental — accuracy not yet measured
• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites Lewis et al., 2013 source

In the paper

“Co-expression of ST6Gal1 and β4GalT1 in CHO cells also significantly enhanced the sialylation level of the mutated version of trastuzumab (Raymond et al., 2015).”

What the sources say

• Supported

  The Results (Impact of the F243A mutation) identify the mutated antibody as TZMm and report that its glycans were 55% sialylated when expressed alone, while co-expression with GT and ST6 produced mostly sialylated glycans. The Discussion reports more than 80% sialylated glycans in the co-expressed F243A mutant. The Introduction identifies GT and ST6 as the human galactosyltransferase and sialyltransferase used in CHO cells. (LLM, conf=0.92)

  cites Raymond et al., 2015 source

In the paper

“Transient expression of ST6Gal1 in CHO and HEK293 cells also increased the α2,6 sialylation of a trastuzumab F243A mutant antibody (Raymond et al., 2012).”

What the sources say

• Partly supported

  Section 4 describes transient coexpression of ST6Gal-I with trastuzumab in CHO and HEK293 cells, and the section on ST6Gal-I overexpression reports increased α2,6-sialylation of the mutant antibody. However, the trastuzumab mutant studied is F246A; the paper says it is equivalent to a previously described IgG3 F243A mutant, not that trastuzumab itself has the F243A mutation. (LLM, conf=0.90)

  cites Raymond et al., 2012 sourceExperimental — accuracy not yet measured

In the paper

“As genome-editing strategies, the ST6Gal1 gene of the Chinese hamster was also recently used in stable expression of different CHO cell lines producing recombinant IgGs (Lin et al., 2015).”

What the sources say

• Supported

  The Introduction states that Chinese hamster ST6GAL1 was overexpressed in three recombinant CHO lines producing humanized or human IgG. The Results section reports that the three IgG-producing lines were stably transfected with the St6gal1 expression vector. (LLM, conf=0.93)

  cites Lin et al., 2015 source

In the paper

“For example, a Pichia pastoris yeast strain was engineered to generate human-like glycans but devoid of core-fucose (Choi et al., 2003;Hamilton et al., 2006;Hamilton and Gerngross 2007).”

What the sources say

• Supported

  The Results report an engineered P. pastoris strain whose secreted protein was almost uniformly modified with GlcNAc-(Man)5-(GlcNAc)2, which the paper identifies as a human glycosylation intermediate. The reported glycan structure contains no fucose, supporting the claim that it lacks core fucose. The paper describes a human-like hybrid glycan, not a fully complex human glycan. (LLM, conf=0.89)

  cites Choi et al., 2003 source
• ?Not checked — no full text of the sourcecites Hamilton et al., 2006 source
• ?Not checked — no full text of the sourcecites Hamilton and Gerngross 2007 source

In the paper

“This multidimensional study also resolved the codon frequency in these cells, which largely contributed to sequence optimization of genes encoding recombinant proteins for this expression system.”

What the sources say

• Partly supported

  The Results (Codon Frequency) report CHO codon frequencies and compare them with human codon frequencies, then suggest replacing suboptimal codons when producing recombinant proteins. The Discussion likewise describes codon optimization as relevant to improving expression, but neither section establishes that this study largely contributed to actual gene-sequence optimization. (LLM, conf=0.85)

  cites Baycin-Hizal et al., 2012 sourceExperimental — accuracy not yet measured

In the paper

“This decreased susceptibility could be attributed to the fact that many viral entry genes are not expressed in CHO cells (Xu et al., 2011).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Xu et al., 2011

In the paper

“This trend is mostly driven by the increased attention directed to posttranslational modifications of these biologics, in particular towards their glycosylation state.”

In the paper

“Proteins requiring proteolytic processing for maturation may not always be fully cleaved and active when expressed in CHO.”

In the paper

“Among the mammalian-based expression systems, CHO cells is by far, the most commonly used cell line.”

What the sources say

• ?Not checked — no full text of the sourcecites Durocher and Butler, 2009 source
• ?Not checked — no full text of the sourcecites Kim et al., 2012 source
• ?Not checked — no full text of the sourcecites Butler and Spearman, 2014 source

In the paper

“Similarly, co-expression of proprotein convertases allowed for the efficient maturation of human bone morphogenetic protein-7 (Sathyamurthy et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Sathyamurthy et al., 2015 source

In the paper

“Although CHO cells possess many advantages for glycoprotein productions, they are unable to produce some types of human glycosylation, such as α-2,6-sialylation and α-1,3/4-fucosylation (Patnaik and Stanley, 2006).”

What the sources say

• ?Not checked — no full text of the sourcecites Patnaik and Stanley, 2006 source

In the paper

“The most commonly used human cell lines to manufacture glycoprotein therapeutics are the HEK293 cells and the HT-1080, respectively from human embryo kidney and fibrosarcoma origin (Rasheed et al., 1974;Graham et al., 1977).”

What the sources say

• ?Not checked — no full text of the sourcecites Rasheed et al., 1974 source
• ?Not checked — no full text of the sourcecites Graham et al., 1977 source

In the paper

“The ZFN knockout approach was particularly successful for specific deletion of the GS and the DHFR genes in CHO cells, thus improving the selection stringency of the generated cell lines (Liu et al., 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Liu et al., 2010 source

In the paper

“Many reports have shown that including these MAR elements in expression vectors for recombinant proteins could significantly improve the expression levels (for an extensive review see (Harraghy et al., 2015)).”

What the sources say

• ?Not checked — no full text of the sourcecites Harraghy et al., 2015 source

In the paper

“This approach has recently shown improved yields for stable production of antibodies in CHO cell lines (Rajendra et al., 2016).”

What the sources say

• ?Not checked — no full text of the sourcecites Rajendra et al., 2016 source

In the paper

“When the need for a quick and economical approach prevails, transient expression remains the best choice for protein production (Rosser et al., 2005;Pham et al., 2006;Geisse, 2009;Chahal et al., 2011;Baldi et al., 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Rosser et al., 2005 source
• ?Not checked — no full text of the sourcecites Pham et al., 2006 source
• ?Not checked — no full text of the sourcecites Geisse, 2009 source

In the paper

“Fed-batch performance and stability of ACE vs random integration systems have been compared for IgG1 expression in CHO cells and have shown similar performance (Combs et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Combs et al., 2011 source

In the paper

“Despite the success of the selection systems described above, a main problem persists with these methods, being the fact that they are based on random plasmid integration and its expression cassette in the host genome.”

In the paper

“This implies screening a very large number of clones (generally multiple hundreds to a few thousands, depending on the cloning method used) in order to find those rare ones with stably integrated plasmid in highly transcriptionally active chromatin regions (”

In the paper

“Copy number of integrated plasmid as well as the integration site(s) within the host genome are some of the key factors when it comes to stable gene expression.”

In the paper

“These cells are able to produce high titers of IgG without requiring amplification of the incorporated gene (Jones et al., 2003).”

What the sources say

• ?Not checked — no full text of the sourcecites Jones et al., 2003 source

In the paper

“By skipping the lengthy selection process for the cells that have integrated the plasmid within their genome, transient transfection is much faster (Rita Costa et al., 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Rita Costa et al., 2010 source

In the paper

“Transgenes are likely to be inserted in heterochromatin regions, which results in very weak gene expression levels.”

In the paper

“This random integration creates very heterogeneous cell population that have variable expression levels amongst the different clones.”

In the paper

“Even if these tools could prove very useful for site specific integration, one of the key challenges is still to identify good hotspots in the host genome that will allow good expression levels and stability (Zhang et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Zhang et al., 2015 source

In the paper

“For stable expression, a gene marker is usually integrated in the expression plasmid along with the cDNA encoding the gene of interest, conferring a selective advantage to the cells that integrate the plasmid into their genome (Rita Costa et al., 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Rita Costa et al., 2010 source

In the paper

“The first one, originally developed by Celltech (now Lonza Biologics), uses complementation of a glutamine auxotrophy by a recombinant GS gene (Fan et al., 2013).”

What the sources say

• ?Not checked — no full text of the sourcecites Fan et al., 2013 source

In the paper

“However, the production rate relies on many factors including efficiency of the transfection phase, cytotoxicity of the transfection reagent and extensiveness of the feeding strategy applied to the culture post-transfection (Pham et al., 2006;Daramola et al., 2014).”

What the sources say

• ?Not checked — no full text of the sourcecites Pham et al., 2006 source
• ?Not checked — no full text of the sourcecites Daramola et al., 2014 source

In the paper

“However, these organisms do not have the ability to adequately glycosylate recombinant proteins, due to the absence of the required enzymatic machinery.”

In the paper

“As with other mammalian expression systems, transgenic animals often produce different glycosylation pattern compared to native human proteins (Durocher and Butler, 2009).”

What the sources say

• ?Not checked — no full text of the sourcecites Durocher and Butler, 2009 source

In the paper

“These systems are thus mainly limited to the expression of nonglycosylated proteins.”

In the paper

“Similarly, overexpression of ceramidetransfer protein (CERT) S132A mutant, which is responsible for the transfer of ceramide from the ER to the Golgi and for sphingomyelin synthesis, also improved production of t-PA, human serum albumin and monoclonal antibodies in industrial cell lines (Florin et al., 2009;Rahimpour et al., 2013).”

What the sources say

• ?Not checked — no full text of the sourcecites Florin et al., 2009 source
• ?Not checked — no full text of the sourcecites Rahimpour et al., 2013 source

In the paper

“Addition of these chemicals has resulted in increased productivity in numerous studies, especially in CHO cells (Durocher and Butler, 2009;Yang et al., 2014).”

What the sources say

• ?Not checked — no full text of the sourcecites Durocher and Butler, 2009 source
• ?Not checked — no full text of the sourcecites Yang et al., 2014 source

In the paper

“However, other studies observed either a negative or no effect on productivity (Mohan et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Mohan et al., 2008 source

In the paper

“Some reports have revealed positive effects in overexpressing XBP-1 (X-box binding protein 1), a transcription factor regulating the unfolded protein response (UPR) during ER stress (Iwakoshi et al., 2003;Tigges and Fussenegger, 2006;Becker et al., 2008;Ku et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Iwakoshi et al., 2003 source
• ?Not checked — no full text of the sourcecites Tigges and Fussenegger, 2006 source
• ?Not checked — no full text of the sourcecites Becker et al., 2008 source

In the paper

“Overexpression of the Hsp27 and Hsp70 chaperones in CHO cells also increased recombinant IFNγ production (Lee et al., 2009).”

What the sources say

• ?Not checked — no full text of the sourcecites Lee et al., 2009 source

In the paper

“Similarly, the heavy chain-binding protein (BiP) chaperone gave mixed results, with enhanced or decreased productivity (Mohan et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Mohan et al., 2008 source

In the paper

“However there were no obvious benefit observed in stable CHO-K1 cells expressing IFNy, EPO or a human mAb (Ku et al., 2008).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Ku et al., 2008

In the paper

“They have been employed by many groups as cell engineering tools but have given variable results (Mohan et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Mohan et al., 2008 source

In the paper

“Yet, most of these strategies still need to prove their effectiveness and robustness for increasing recombinant protein production in mammalian cells, especially at an industrial level.”

In the paper

“Thus, these various and sometimes contradictory results could be partly explained by the use of different CHO host strains.”

In the paper

“However, the use of these inhibitors may also have undesired secondary effects, such as blocking cell cycle or inducing apoptosis (Sung et al., 2004;Jiang and Sharfstein, 2008;Sunley and Butler, 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Sung et al., 2004 source
• ?Not checked — no full text of the sourcecites Jiang and Sharfstein, 2008 source
• ?Not checked — no full text of the sourcecites Sunley and Butler, 2010 source

In the paper

“Moreover, overexpression of the endoplasmic-reticulum ERp57 protein, an isoform of PDI, together with calnexin/calreticulin was able to increase thrombopoietin production in CHO cells (Mohan et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Mohan et al., 2008 source

In the paper

“For example, by controlling vesicle docking and fusion, expression of SNAP-23 and VAMP8 has been shown to improve production of SEAP and monoclonal antibodies in CHO-K1 cells (Peng et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Peng et al., 2011 source

In the paper

“Similarly, GADD34 can also be overexpressed in order to increase productivity of CHO cells (Omasa et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Omasa et al., 2008 source

In the paper

“In recent years, development of bioinformatic tools capable to predict the outcome of MAR sequences helped to improve MAR elements (Girod et al., 2007;Harraghy et al., 2012Harraghy et al., , 2015)).”

What the sources say

• ?Not checked — no full text of the sourcecites Girod et al., 2007 source
• ?Not checked — no full text of the sourcecites Harraghy et al., 2012 source
• ?Not checked — no full text of the sourcecites Harraghy et al., 2015 source

In the paper

“As for anti-apoptosis engineering, genes of the Bcl-2 family (Bcl-2, Bcl-xL and Mcl-1) were overexpressed in a variety of CHO cell lines (Kim and Lee, 2000;Tey et al., 2000;Meents et al., 2002;Chiang and Sisk, 2005;Kim et al., 2009b;Majors et al., 2009).”

What the sources say

• ?Not checked — no full text of the sourcecites Kim and Lee, 2000 source
• ?Not checked — no full text of the sourcecites Tey et al., 2000 source
• ?Not checked — no full text of the sourcecites Meents et al., 2002 source

In the paper

“It was reported that afucosylated Fc domain glycans of human IgG1 could enhance the binding to FcγRIIIa subtype receptors of natural killer cells (Shields et al., 2002;Shinkawa et al., 2003;Niwa et al., 2005;Iida et al., 2006;Subedi and Barb, 2016).”

What the sources say

• ?Not checked — no full text of the sourcecites Shields et al., 2002 source
• ?Not checked — no full text of the sourcecites Shinkawa et al., 2003 source
• ?Not checked — no full text of the sourcecites Niwa et al., 2005 source

In the paper

“Among these approaches, anti-autophagy engineering and metabolic engineering have been used in order to optimize recombinant protein production in mammalian cells (for a detailed review on the topic see (Kim et al., 2012;Kim et al., 2013)).”

What the sources say

• ?Not checked — no full text of the sourcecites Kim et al., 2012 source
• ?Not checked — no full text of the sourcecites Kim et al., 2013 source

In the paper

“Oppositely, down-regulation of pro-apoptotic genes like Bax, Bak, microRNA mir-mmu-miR-466h-5p and caspase-3,-7,-8 and -9 were also tested (Kim and Lee, 2002;Sung et al., 2007;Yun et al., 2007;Cost et al., 2010;Druz et al., 2013).”

What the sources say

• ?Not checked — no full text of the sourcecites Kim and Lee, 2002 source
• ?Not checked — no full text of the sourcecites Sung et al., 2007 source
• ?Not checked — no full text of the sourcecites Yun et al., 2007 source

In the paper

“Beside media and feeds, numerous small molecules have been identified from a chemical library that could enhance protein production (Allen et al., 2008).”

What the sources say

• ?Not checked — no full text of the sourcecites Allen et al., 2008 source

In the paper

“The introduction of the mTOR gene in CHO cells resulted in increased cell proliferation and productivity of therapeutic IgG, alkaline phosphatase and α-amylase (Dreesen and Fussenegger, 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Dreesen and Fussenegger, 2011 source

In the paper

“Moreover, chemical manipulation of this pathway can also enhance glycoprotein productions in these cells (Dadehbeigi and Dickson, 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Dadehbeigi and Dickson, 2015 source

In the paper

“Plants, yeasts and non-human cell lines can generate glycans that are often absent from endogenous human proteins (see section 2).”

In the paper

“To attenuate these effects, optimization of the addition timing and concentration has shown to be important (Rodriguez et al., 2005).”

What the sources say

• ?Not checked — no full text of the sourcecites Rodriguez et al., 2005 source

In the paper

“Some attempts of knocking down the different CHO sialidases by shRNA or siRNA also improved the sialylation level of recombinant hIFNγ, particularly for Neu2 and Neu3 sialidases (Ngantung et al., 2006;Zhang et al., 2010b).”

What the sources say

• ?Not checked — no full text of the sourcecites Ngantung et al., 2006 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Zhang et al., 2010b

In the paper

“Additionally, the same study presented a human ST6Gal1 knock-in experiment in ST3Gal4/6 knockout CHO cells, which resulted in considerable increase of α2,6 sialylation.”

What the sources say

• ?Not checked — no full text of the sourcecites Yang et al., 2015 source

In the paper

“Indeed, comparison of proteomic profiles of fast vs slow growing CHO clones has already identified the valosin-containing protein (VCP) as a potential candidate for enhancement of cell growth by cell engineering (Doolan et al., 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Doolan et al., 2010 source

In the paper

“The C12orf35 gene comprised within this region seems to be responsible for the phenotype (Ritter et al., 2016a).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Ritter et al., 2016a

In the paper

“Heterologous expression of the bacterial enzyme GDP-4-dehydro-6deoxy-D-mannose reductase (RMD) in CHO cells strongly reduced their fucosylation capacity (von Horsten et al., 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites von Horsten et al., 2010 source

In the paper

“Sequencing of the CHO-K1 genome allowed the identification and positioning of their genes and of most of human glycosylation-associated transcripts (Xu et al., 2011).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Xu et al., 2011

In the paper

“Higher recombinant protein production was obtained when overexpressing the miR-7 gene in CHO cells (Barron et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Barron et al., 2011 source

In the paper

“A CHO cell line stably expressing both 30Kc19 and human EPO showed that it could also significantly increase the EPO sialylation (Wang et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Wang et al., 2011 source

In the paper

“A functional analysis of 31 glycosyltransferases was even conduced for human EPO glycosylation optimization in six different mammalian cell lines, where enhanced expression of ST3GalII, ST3GalIV and ST6Gal1 could enhance sialylation in HEK293, Cos-7, 3T3 and NSO cells (Zhang et al., 2010c).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Zhang et al., 2010c

In the paper

“As mentioned earlier, the silkworm hemolymph antiapoptotic protein 30Kc19 was shown to increase protein production (Wang et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Wang et al., 2011 source

In the paper

“Finally, a multi-omics study combining transcriptomics and metabolo-mics data, identified variations in gene expression and in enzymatic reactions during the transition from a parental HEK293 cell line to a producer cell line (Dietmair et al., 2012b).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Dietmair et al., 2012b

In the paper

“As an example, CHO cells, where simultaneous disruption of FUT8, BAX and BAK gene by CRISP/Cas9 was achieved and confirmed by deep sequencing, showed increased resistance to apoptosis (Grav et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Grav et al., 2015 source

In the paper

“CHO cell lines producing recombinant IgG were also found to have decreased expression of miR-221 and miR-222 compared to their parental cell line DG44 (Lin et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Lin et al., 2011 source

In the paper

“An engineered CHO cell line stably expressing the human α2,3-sialyltransferase ST3Gal3, the rat mutated GNE/MNK-R263L-R266Q glycosylation enzymes and the Chinese hamster CMP-sialic acid transporter was successful in increasing tetra-sialylation of recombinant human EPO (Son et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Son et al., 2011 source

In the paper

“Thus, increasing the sialylation of proteins may decrease the frequency of injection or the amount of therapeutic protein used in a single dose.”

In the paper

“Finally, transient transfection of the Chinese hamster ST6Gal1 gene in CHO cells expressing a bispecific antibody, also considerably increased the level of α2,6 sialylation (Onitsuka et al., 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Onitsuka et al., 2012 source

In the paper

“Noteworthy, knockout of both ST3gal4 and ST3gal6 genes affected sialylation.”

What the sources say

• ?Not checked — no full text of the sourcecites Yang et al., 2015 source

In the paper

“Comparison of gene expression profiles between high and low producing CHO clones allowed the identification of a deletion in chromosome 8 telomeric region of the cells correlating with higher productivity (Ritter et al., 2016b).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Ritter et al., 2016b

In the paper

“In a next generation sequencing (NGS) study regrouping diverse cDNA libraries from CHO cell lines, 29 000 transcripts were assembled, which identified 13 187 mRNA transcripts (Becker et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Becker et al., 2011 source

In the paper

“Many chemical inhibitors have been synthesized to target these enzymes but their use in large-scale bioprocess productions is still very limited due to their elevated fabrication cost (Ferrari et al., 1998).”

What the sources say

• ?Not checked — no full text of the sourcecites Ferrari et al., 1998 source

In the paper

“This study revealed predominant roles for some enzymes into CHO cells glycosylation pathway.”

What the sources say

• ?Not checked — no full text of the sourcecites Yang et al., 2015 source

In the paper

“Some of these approaches are based on the supplementation of cell culture media with different precursors of the sialic acid pathway, such as CMP-sialic acid, ManNAc and NeuNAc, but their effects are limited and still debated (Gu and Wang, 1998;Baker et al., 2001;Hills et al., 2001;Bork et al., 2009;Hossler et al., 2009;Wong et al., 2010;Kildegaard et al., 2016).”

What the sources say

• ?Not checked — no full text of the sourcecites Gu and Wang, 1998 source
• ?Not checked — no full text of the sourcecites Baker et al., 2001 source
• ?Not checked — no full text of the sourcecites Hills et al., 2001 source