Strategic feeding of NS0 and CHO cell cultures to control glycan profiles and immunogenic epitopes of monoclonal antibodies

Villacrés, Carina; Tayi, Venkata S.; Butler, Michael

doi:10.1016/j.jbiotec.2021.04.005

Cited by 12 publications

(8 citation statements)

References 85 publications

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“…The major O -glycan structures identified on rhCD45 (NS0) were core 1 structures containing glycolyl neuraminic acid (Neu5Gc) (Table ). This is consistent with the prior observation that murine myeloma cell lines (NS0, Sp2/0) produce Neu5Gc at significantly higher levels compared to other nonhuman cell lines like CHO or BHK-21. − Other observed species were Tn-antigen and asialylated, mono-, and disialylated core 1 containing Neu5Ac. Structures that could be extended core 1 or core 2 [HexNAc(2)Hex(1)NeuAc(1), HexNAc(2)Hex(2)NeuAc(1), and HexNAc(2)Hex(2)NeuAc(2)] were also annotated by Byonic software on the rhCD45 (NS0) glycopeptides.…”

Section: Resultssupporting

confidence: 90%

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans

et al. 2021

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Characterization of mucin-type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constraints, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain types of glycoproteins, or protein sequence bias. These factors limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. Peptide sequence selectivity and its impact on IMPa activity were interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/ threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomic workflow for glycoprofiling of the purified glycoproteins granulocyte colony-stimulating factor and receptor-type tyrosine-protein phosphatase C without the need for glycopeptide enrichment. In these examples, IMPa enabled both the identification of O-glycosites and the range of complex O-glycan structures at each site.

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Section: Resultssupporting

confidence: 90%

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans

et al. 2021

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“…This is consistent with prior observations that murine myeloma cell lines (NS0, Sp2/0) produce Neu5Gc at significantly higher levels compared to other non-human cell lines like CHO or BHK-21. 29–31 Other observed species were Tn-antigen, asialylated and mono-, di-sialylated core 1 containing Neu5Ac. Structures that could be extended core 1 or core 2 (HexNAc(2)Hex(1)NeuAc(1), HexNAc(2)Hex(2)NeuAc(1), HexNAc(2)Hex(2)NeuAc(2)) were also annotated by Byonic software on the rhCD45 (NS0) glycopeptides.…”

Section: Resultsmentioning

confidence: 99%

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

Vainauskas

Guntz

McLeod

et al. 2021

Preprint

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Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

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“…In a culture of NSO cells, Villacres et al. (2021) observed a 12% decrease in glycan content containing Neu5Gc when 7 µM manganese chloride was added to the culture medium. In NS0 culture supplemented with 7 µM manganese chloride and 10 mM galactose, the combined action of manganese chloride and galactose reduced Neu5Gc content by 17% and increased Neu5Ac by 58%.…”

Section: Methods To Reduce the Risk Of Neu5gcmentioning

confidence: 99%

“…Furthermore, the comparison of sodium carbonate or sodium hydroxide as a basis for pH control in a bioreactor showed that Neu5Gc could be reduced by 33% reproducibly using sodium hydroxide (Borys et al, 2010). In a culture of NSO cells, Villacres et al (2021) observed a 12% decrease in glycan content containing Neu5Gc when 7 µM manganese chloride was added to the culture medium. In NS0 culture supplemented with 7 µM manganese chloride and 10 mM galactose, the combined action of manganese chloride and galactose reduced Neu5Gc content by 17% and increased Neu5Ac by 58%.…”

Section: Study On Neu5gc Reduction In Mammalian Cellmentioning

confidence: 99%

N‐glycolylneuraminic acid in red meat and processed meat is a health concern: A review on the formation, health risk, and reduction

Liang,

Wu,

et al. 2024

Comp Rev Food Sci Food Safe

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One of the most consistent epidemiological associations between diet and human disease risk is the impact of consuming red meat and processed meat products. In recent years, the health concerns surrounding red meat and processed meat have gained worldwide attention. The fact that humans have lost the ability to synthesize N‐glycolylneuraminic acid (Neu5Gc) makes red meat and processed meat products the most important source of exogenous Neu5Gc for humans. As our research of Neu5Gc has increased, it has been discovered that Neu5Gc in red meat and processed meat is a key factor in many major diseases. Given the objective evidence of the harmful risk caused by Neu5Gc in red meat and processed meat to human health, there is a need for heightened attention in the field of food. This updated review has several Neu5Gc aspects given including biosynthetic pathway of Neu5Gc and its accumulation in the human body, the distribution of Neu5Gc in food, the methods for detecting Neu5Gc, and most importantly, a systematic review of the existing methods for reducing the content of Neu5Gc in red meat and processed meat. It also provides some insights into the current status and future directions in this area.

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Strategic feeding of NS0 and CHO cell cultures to control glycan profiles and immunogenic epitopes of monoclonal antibodies

Cited by 12 publications

References 85 publications

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

N‐glycolylneuraminic acid in red meat and processed meat is a health concern: A review on the formation, health risk, and reduction

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