Structural Identification of a Non-Glycosylated Variant at Ser126 for O-Glycosylation Site from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis

Byeon, Jaehee; Lim, Yu-Ri; Kim, Hyong-Ha; Suh, Jung Keun

doi:10.14348/molcells.2015.2256

Cited by 13 publications

(9 citation statements)

References 28 publications

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“…Relative amounts for unglycosylated sites Asn24 and Ser126 were as high as 14.7 and 19.8%, respectively, for sample EPO RP–. This is in line with results from a previous study, which described detection of nonglycosylated Ser126 sites in fractions eluting late from RP columns (similar to EPO RP−). However, expected ionization deficiencies for glycosylated peptides cast doubt on the absolute values.…”

Section: Results and Discussionsupporting

confidence: 91%

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography–Mass Spectrometry Peptide Mapping

et al. 2020

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Recombinant human erythropoetin (EPO) is an important biopharmaceutical mainly used for the treatment of anemia. It is highly heterogeneous because of common amino acid chemical degradations known to occur in protein therapeutics (e.g., oxidation and deamidation) and its complex glycosylation profile. Recently, multi-attribute monitoring (MAM), i.e., the quantification of multiple post-translational and chemical modifications in a single peptide mapping liquid chromatography–mass spectrometry (LC-MS)-based method, has received increased attention for the analysis of antibody-like biotherapeutic proteins. In this study, an MAM method for examination of residue-specific glycan profiles of EPO was established. The MAM method, by virtue of the increased sensitivity and selectivity provided with LC-MS, yielded additional site-specific information not afforded by the conventional quality control (QC) methods. Low abundant glycans as well as additional post-translational and chemical modifications could also be simultaneously detected by the MAM method. Our results demonstrate that desialylated N-oligosaccharides (DeNO) and N-acetylneuraminic acids (Neu5Ac) could be monitored by the developed MAM approach with data readout highly comparable to QC methods, while differences were observed for charge isoform distribution. In summary, the comparative data obtained demonstrate that MAM by LC-MS peptide mapping can, in principle, adequately replace selected QC methods and would add value to the in-process control and release testing strategy of EPO.

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Section: Results and Discussionsupporting

confidence: 91%

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography–Mass Spectrometry Peptide Mapping

et al. 2020

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“…43 Its fourth glycosylation site is located at Ser126, where O-linked mucin-type core 1 glycans are present. 44 The kinds of N-and O-linked glycans identified on EPO are thought to be the most common forms of these glycans on human glycoproteins. 45,46 Human EPO is 166 amino acids in length and is thus an average-sized soluble human protein.…”

Section: ■ Chemical Synthesis Of Glycoproteinsmentioning

confidence: 99%

“…The protein carries three N-glycans at Asn24, -38, and -83, which are mainly core-fucosylated and sialic acid-terminated complex-type N-glycans with di-, tri-, and tetra-antennary structures (Figure ). Its fourth glycosylation site is located at Ser126, where O-linked mucin-type core 1 glycans are present . The kinds of N- and O-linked glycans identified on EPO are thought to be the most common forms of these glycans on human glycoproteins. , Human EPO is 166 amino acids in length and is thus an average-sized soluble human protein.…”

Section: Chemical Synthesis Of Glycoproteinsmentioning

confidence: 99%

Using Chemical Synthesis To Study and Apply Protein Glycosylation

Chaffey

Guan

2018

Biochemistry

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Protein glycosylation is one of the most common post-translational modifications and can influence many properties of proteins. Abnormal protein glycosylation can lead to protein malfunction and serious disease. While appreciation of glycosylation's importance is growing in the scientific community, especially in recent years, a lack of homogeneous glycoproteins with well-defined glycan structures has made it difficult to understand the correlation between the structure of glycoproteins and their properties at a quantitative level. This has been a significant limitation on rational applications of glycosylation and on optimizing glycoprotein properties. Through the extraordinary efforts of chemists, it is now feasible to use chemical synthesis to produce collections of homogeneous glycoforms with systematic variations in amino acid sequence, glycosidic linkage, anomeric configuration, and glycan structure. Such a technical advance has greatly facilitated the study and application of protein glycosylation. This Perspective highlights some representative work in this research area, with the goal of inspiring and encouraging more scientists to pursue the glycosciences.

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“…This analytical method provides detailed information of primary structure for a given protein and enables the control of the protein sequence down to the level of single amino acids by coupling with mass spectrometry [44][45][46]. Based on the analysis of peptide mapping, it is possible to confirm genetic stability (correct translation), identify post-translation modification, and demonstrate the integrity of disulfide bonds [47][48][49][50].…”

Section: Peptide Mapping Of Mabmentioning

confidence: 99%

Characterization of Biopharmaceuticals Focusing on Antibody Therapeutics

Kim¹,

Kang²,

Suh³

2018

Biopharmaceuticals

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Biopharmaceuticals are highly complex molecules and also require high quality for safety and efficacy in human uses. For well-characterized products, the desired level of quality should be monitored and controlled during the manufacturing processes. A series of workflow for analytical characterization should be applied for product quality throughout those processes. In this chapter, several analytical techniques are introduced for assessing characteristics of biopharmaceuticals focusing on monoclonal antibodies (mAbs). Analytical characterization for primary structure was performed by mass spectrometry (MS), and assessment of post-translational modifications (PTMs) was done by conventional approaches. The analytical assessments were also done by multi-attribute method (MAM) approach using mass spectrometer (MS), and the performance of MAM was compared to conventional approaches. on the market results in gaining interest for drug development industry, and biopharmaceuticals are considered as fast growing and promising area for drug development [3][4][5][6].The approval of mAb-related products is dramatically increased in the recent years [6,7]. Over 90 mAb-related products are approved by European Medicines Agency (EMA) and US Food and Drug Administration (FDA). Those can be classified into mAb, Fc-fusion, Fab, antibody-drug conjugate (ADC), bispecific mAb (bsAb), and bispecific T cell engager (BiTE). Among them, mAbs are the major product, consisting of 77% of total. Others represent rest 23% of total, Fc-fusion (12%), ADC (5%), Fab (3%), bsAb (2%), and BiTE (1%), respectively. After the first approval of full-length mAb in 1998, mAbs are major product in the biopharmaceutical industry. This increasing number gives high revenue for pharmaceutical companies, and seven mAb-related products are positioned in top 10 drugs in the world, 2017, including Humira, Enbrel, Rituxan, Remicade, AVASTIN, Herceptin, and Lantus [8].Mylotarg is the first approved ADC in 2000, which combined a mAb targeting leukemic blast cells with a bacterial toxin (calicheamicin) [7,9]. ADC is a complex generated between a mAb and small molecule or a peptide, and mAb gives the selective delivery for targeting of cytotoxic drugs [1,[9][10][11]. Since the first approval, four additional ADC products are approved in Europe and USA. bsAb has two different antigen binding sites recognizing two different epitopes in a single mAb, and this dual specificity gives more specific targeting and higher efficacy [12][13][14]. Currently, three bsAbs are approved by EMA or US FDA. The first bsAb, Removab, was approved in 2009 but voluntarily withdrawn in 2013. Fc-fusion proteins are fusions of the IgG Fc domain with a desired linked protein, enhancing pharmacokinetic properties (serum half-life) and pharmacodynamics properties (ADCC and CDC) [6,15]. Following the first approval of Fc-fusion protein, Enbrel in 1998, eight Fc-fusion proteins are authorized for the marketing in the region of Europe and USA.Biosimilars, known as follow-on biologics, which follow t...

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Structural Identification of a Non-Glycosylated Variant at Ser126 for O-Glycosylation Site from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis

Cited by 13 publications

References 28 publications

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography–Mass Spectrometry Peptide Mapping

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography–Mass Spectrometry Peptide Mapping

Using Chemical Synthesis To Study and Apply Protein Glycosylation

Characterization of Biopharmaceuticals Focusing on Antibody Therapeutics

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