Integrated Genome and Protein Editing Swaps <i>α</i>‐2,6 Sialylation for <i>α</i>‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Chung, Cheng Yu; Wang, Qiong; Yang, Shuang; Yin, Bojiao; Zhang, Hui; Betenbaugh, Michael J.

doi:10.1002/biot.201600502

Cited by 38 publications

(45 citation statements)

References 30 publications

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“…In our laboratory, GIG has been used for studies of N-glycan profiling in tissue or blood samples derived from patients with prostate cancer 44 , pancreatic cancer 25 , ovarian cancer 45 and cardiac hypertrophy 46 , as well as samples from glycoengineered Chinese hamster ovary (CHO) cells 47 , and for studies of glycoforms of HIV gp 120 (ref. 48), glycoengineered sialylation of CHO cells 49 and N-gly-cosylation in cockroach allergen regulation of human basophil function 50 . GIG has been used for the extraction of N-glycans for isobaric labeling using iARTs 51 or QUANTITY 37 .…”

Section: Introductionmentioning

confidence: 99%

Simultaneous quantification of N- and O-glycans using a solid-phase method

Yang

Sokoll

et al. 2017

Nat Protoc

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Glycosylation has a pivotal role in a diverse range of biological activities, modulating the structure and function of proteins. Glycogens coupled to the nitrogen atom (N-linked) of asparagine side chains or to the oxygen atom (O-linked) of serine and threonine side chains represent the two major protein glycosylation forms. N-glycans can be released by glycosidases, whereas O-glycans are often cleaved by chemical reaction. However, it is challenging to combine these enzymatic and chemical reactions in order to analyze both N- and O-glycans. We recently developed a glycoprotei n immobilization for glycan extraction (GIG) method that allows for the simultaneous analysis of N- and O-glycans on a solid support. GIG enables quantitative analysis of N-glycans and O-glycans from a single specimen and can be applied to a high-throughput automated platform. Here we provide a step-by-step GIG protocol that includes procedures for (i) protein immobilization on an aldehyde-active solid support by reductive amination; (ii) stabilization of fragile sialic acids by carbodiimide coupling; (iii) release of N-glycans by PNGase F digestion; (iv) release of O-glycans by β-elimination using ammonia in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP) to prevent alditol peeling from O-glycans; (v) mass spectrometry (MS) analysis; and (vi) data analysis for identification of glycans using in-house developed software (GIG Tool; free to download via http://www.biomarkercenter.org/gigtool). The GIG tool extracts precursor masses, oxonium ions and glycan fragments from tandem (liquid chromatography (LC)–MS/MS) mass spectra for glycan identification, and reporter ions from quaternary amine containing isobaric tag for glycan (QUANTITY) isobaric tags are used for quantification of the relative abundance of N-glycans. The GIG protocol takes ~3 d.

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Section: Introductionmentioning

confidence: 99%

Simultaneous quantification of N- and O-glycans using a solid-phase method

Yang

Sokoll

et al. 2017

Nat Protoc

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“…MALDI‐TOF glycoprofile analysis of gpIgG1 and single amino acid mutants expressed from CHOK1 and CHO23KO cells. N‐glycan analysis of gpIgG1 from CHOK1 cells from our previous study (Chung et al, ) (panel a), gpIgG1F241A from CHOK1 cells (panel b), and gpIgG1 F241A from CHO23KO cells (panel c)…”

Section: Resultsmentioning

confidence: 94%

“…Knockout of the α‐2,3 sialyltransferase genes eliminates the further modification of the glycan to include sialic acid residues not typically present on IgGs. Next an IgG variant with all four amino acids mutated was generated (gpIgG14mu with amino acid substitution of F241A, F243A, V262E, and V264E) (Chung et al, ). Interestingly the inclusion of all the mutations sites simultaneously exhibited a N‐glycan pattern with G2F levels similar to that of gpIgG1F241A from CHO23KO cells (Figure b, panel b3).…”

Section: Resultsmentioning

confidence: 99%

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells

Chung¹,

Wang²,

Yang³

et al. 2017

Biotech & Bioengineering

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One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications.

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“…In a similar study, Chung et al achieved successful α‐2,6‐sialylated IgG by amino acids substitution in the IgG structure to allow access by the sialyltransferase enzyme. When expressed in CHO cells containing st3gal gene knockout and knock‐in of st6gal‐1 gene, IgG with four amino acid substitutions contained nearly exclusively α‐2,6‐sialylation with a 14‐fold increase in sialic acid content compared to wild type IgG obtained from unmodified CHO cells . While combining gene editing techniques with protein engineering for optimal α‐2,6‐sialylation may be necessary, effects of mutations in a mAb should be carefully considered as amino acid substitutions can elicit an anti‐inflammatory response as well as decreasing the antibody affinity toward the Fcγ receptor, leading to a decrease in ADCC …”

Section: Manipulating Glycosylation and Glycosaminoglycan Productionmentioning

confidence: 99%

Glycoengineering in CHO Cells: Advances in Systems Biology

Tejwani

Andersen

Nam

et al. 2018

Biotechnology Journal

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For several decades, glycoprotein biologics have been successfully produced from Chinese hamster ovary (CHO) cells. The therapeutic efficacy and potency of glycoprotein biologics are often dictated by their post-translational modifications, particularly glycosylation, which unlike protein synthesis, is a non-templated process. Consequently, both native and recombinant glycoprotein production generate heterogeneous mixtures containing variable amounts of different glycoforms. Stability, potency, plasma half-life, and immunogenicity of the glycoprotein biologic are directly influenced by the glycoforms. Recently, CHO cells have also been explored for production of therapeutic glycosaminoglycans (e.g., heparin), which presents similar challenges as producing glycoproteins biologics. Approaches to controlling heterogeneity in CHO cells and directing the biosynthetic process toward desired glycoforms are not well understood. A systems biology approach combining different technologies is needed for complete understanding of the molecular processes accounting for this variability and to open up new venues in cell line development. In this review, we describe several advances in genetic manipulation, modeling, and glycan and glycoprotein analysis that together will provide new strategies for glycoengineering of CHO cells with desired or enhanced glycosylation capabilities.

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Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Cited by 38 publications

References 30 publications

Simultaneous quantification of N- and O-glycans using a solid-phase method

Simultaneous quantification of N- and O-glycans using a solid-phase method

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells

Glycoengineering in CHO Cells: Advances in Systems Biology

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