Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins

Bosques, Carlos J.; Collins, Brian E.; Meador, James W.; Sarvaiya, Hetal; Murphy, Jennifer L.; DelloRusso, Guy; Bulik, Dorota A.; Hsu, I-Hsuan; Washburn, Nathaniel; Sipsey, Sandra F; Myette, James R.; Raman, Rahul; Shriver, Zachary; Sasisekharan, Ram; Venkataraman, Ganesh

doi:10.1038/nbt1110-1153

Cited by 140 publications

(100 citation statements)

References 10 publications

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“…In recent years, researchers have found that a CHO ortholog of N-acetyllactosaminide 3-a-galactosyltransferase-1 is active, and can affect glycosylated protein products produced in CHO. 39 They also observed a 1,3-Gal glycan form in a CTLA4-Ig product, which had an abundance of »0.2% of the mass of total N-linked glycans. 39 No data on this immunogenic glycan variant is provided in this study because we found that the N-linked glycans had an abundance below 0.1% (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…39 They also observed a 1,3-Gal glycan form in a CTLA4-Ig product, which had an abundance of »0.2% of the mass of total N-linked glycans. 39 No data on this immunogenic glycan variant is provided in this study because we found that the N-linked glycans had an abundance below 0.1% (data not shown). Further study of the Ig fusion proteins using additional analytical methods and preclinical and clinical trials of them need to be done in the future.…”

Section: Discussionmentioning

confidence: 99%

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Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu

Guo

Guo³

et al. 2014

mAbs

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These authors contributed equally to this work.Keywords: characterization, CTLA4-Ig fusion protein, glycan, glycosylation modification, intact protein, mass spectrometry, peptide mapping, similarity CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N-and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC-MS) is a versatile technique for characterizing N-and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N-and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu

Guo

Guo³

et al. 2014

mAbs

View full text Add to dashboard Cite

show abstract

“…Some reports have revealed that CHO cells do not produce α-1,3-galactosyltransferase [31] and have a pattern of glycosylation that differs from that of the Erbitux host cell line Sp2/0. However, data from other groups have identified the presence of the CHO ortholog of N-acetyllactosaminide 3-α-galactosy ltransferase-1 [32] , which is responsible for the synthesis of the α-Gal epitope. These groups also found that the amounts of terminal α-Gal in CHO clones ranged from 0 to 404 picomol per mg of protein; therefore, selection of a cell line without α-Gal expression is important for CHO-derived therapeutic antibody production.…”

Section: Wwwchinapharcom Yi Ch Et Almentioning

confidence: 99%

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

Ruan

Wang

et al. 2014

Acta Pharmacol Sin

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Aim: To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. Methods: The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. Results: The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. Conclusion: The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

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“…Glycosylation is one of very important post-translational modifications because it can have significant impact on antibody-dependent cell-mediated cytotoxicity (ADCC) activity [3,4], complement-dependent cytotoxicity (CDCC) activity [5], clearance [6] and immunogenicity [7,8]. Protein glycosylation can be significantly impacted by the host cell line, clone, media composition, feeding strategy and downstream processing conditions [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

Wang¹,

Peng²,

Sosic³

et al. 2017

J Anal Bioanal Tech

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Rapid and sensitive product quality analysis is important for real-time monitoring during biopharmaceutical development and manufacturing. However, low level of protein concentration and complex cell culture matrix pose challenges for product quality characterization at early stages of cell line selection and process development. Here, we describe a fast and simple microfluidic ZipChip CE-MS method to measure quality attributes of monoclonal antibody protein directly from cell culture supernatant. Cell culture supernatant samples were characterized with charge-based separation using microfluidic capillary electrophoresis coupled to a high-resolution mass spectrometer. Under sample reducing conditions, multiple protein glycosylation attributes were determined on the heavy chain, whereas titer information was obtained from comparison of light chain signal intensity following sample spiking-in with heavy labeled mAb. Therefore, the protein expression and product quality can be monitored using the same method with a single microfluidic device. A total volume of ten to fifty microliter of cell culture supernatant is needed, whereas analysis time is within three minutes per sample. In addition, comparison of new method with traditional RP-LC-MS method using a set of time-course bioreactor cell culture samples has been performed. A good correlation of the levels of N-glycosylation attributes between ZipChip CE-MS of crude samples and RPLC-MS analysis following Protein A (ProA) purification step has been demonstrated.

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Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins

Cited by 140 publications

References 10 publications

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

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