Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Chumsae, Chris; Gaza-Bulseco, Georgeen; Sun, Joanne; Liu, Hongcheng

doi:10.1016/j.jchromb.2006.11.050

Cited by 174 publications

(175 citation statements)

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“…Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.…”

mentioning

confidence: 99%

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Liu

Gaza-Bulseco

Chumsae

2009

J. Am. Soc. Mass Spectrom.

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Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly. (J Am Soc Mass Spectrom 2009, 20, 2258 -2264) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry M ass spectrometry is one of the most indispensable techniques for the characterization of recombinant monoclonal antibodies because most of the modifications that result in heterogeneity and degradation are involved in molecular weight differences [1,2]. Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.Mass spectrometry is commonly coupled with reversedphase high-performance liquid chromatography (RP-HPLC), which desalts the samples and separates different components based on hydrophobicity. The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on...

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confidence: 99%

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Liu

Gaza-Bulseco

Chumsae

2009

J. Am. Soc. Mass Spectrom.

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“…50 Methionine residues can be readily oxidized to sulfoxide derivatives, but under extreme conditions the sulfone can also be generated. 51 Oxidation at methionine residues has been extensively studied and was found to result in detectable changes in the secondary and tertiary structure of the mAb, which correlated with decreased thermodynamic stability and increased aggregation rates. 52 Of special interest are the methionine residues in the Fc region that are Figure 6.…”

Section: Discussionmentioning

confidence: 99%

“…In the 3-dimensional structure of a recombinant mAb, 2 methionine residues close to the C H 2À ÀC H 3 interface were found to be easily oxidized. 51,53 Changes in the oxidation status of a mAb can affect its biological function. 32 Previous studies have shown that peroxide-induced oxidation at methionine residues can significantly decrease the binding affinity of the human IgG 1 to Fc receptors.…”

Section: Discussionmentioning

confidence: 99%

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

Morar-Mitrica

Puri

Sassi

et al. 2015

mAbs

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The physical and chemical integrity of a biopharmaceutical must be maintained not only during long-term storage but also during administration. Specifically for the intravenous (i.v.) delivery of a protein drug, loss of stability can occur when the protein formulation is compounded with i.v. bag diluents, thus modifying the original composition of the drug product. Here we present the challenges associated with the delivery of a low-dose, highly potent monoclonal antibody (mAb) via the i.v. route. Through parallel in-use stability studies and conventional formulation development, a drug product was developed in which adsorptive losses and critical oxidative degradation pathways were effectively controlled. This development approach enabled the i.v. administration of clinical doses in the range of 0.1 to 0.5 mg total protein, while ensuring liquid drug product storage stability under refrigerated conditions.

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“…According to the suggestion of ref. [18] these peaks can be assigned to the components formed by the oxidation of methionine.…”

Section: Study Of Solution and Thermal Stability Of Rituximabmentioning

confidence: 99%

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

G¹

2014

J Chromatogr Sep Tech

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This paper focuses on the applicability of capillary zone electrophoresis (CZE) using uncoated fused silica capillaries for the determination of heterogeneity of rituximab (MabThera, Roche) and for the study of its solution and thermal stability. The best resolution for the charge variants of the main component was obtained with a buffer electrolyte containing 800 mM 6-amino caproic acid, 2 mM triethylene tetramine and 0.05% hydroxypropyl methylcellulose at pH = 5.2. It was found that the pH and the components of the buffer used for the electrophoretic separation and for the dilution of the sample prior to the analysis are important to the stability of the rituximab. We demonstrated the rituximab is stable in the pharmaceutical product MabThera due to the stabilizing additives, but the dilution of the MabThera caused a slow formation of acidic variants, while the amount of the basic variants did not change. After incubation of the diluted rituximab at higher temperature several charge variants could be determined by CZE.

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Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Cited by 174 publications

References 38 publications

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

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