Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody

Liu, Hongcheng; Gaza-Bulseco, Georgeen; Tang, Xiang; Chumsae, Chris

doi:10.1016/j.molimm.2007.07.012

Cited by 77 publications

(61 citation statements)

References 35 publications

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“…The peptides for which a change in exchange profile was identified are located close to the two methionine residues in the Fc region and had residues in the interface between the C H 2 and C H 3 domains in the crystal structure. Comparison of the exchange profiles of the native and oxidised IgG1 samples enabled us to demonstrate that methionine oxidation resulted in structural changes to the IgG1s when the glycan is present, which is in contrast to the results reported by H. Liu et al, 18 we however, used a more direct measure of protein structure.…”

Section: Resultscontrasting

confidence: 68%

“…The relative levels of oxidation of these two residues were in agreement with previous reports. 18,21 The masses of the intact mAb were also measured for the oxidised and non-oxidised samples, shown in supporting information. By comparison of the weighted average of the two spectra it was calculated that an average of 3.0 methionine residues had been oxidised in each mAb during the accelerated oxidation.…”

Section: Resultsmentioning

confidence: 99%

“…[17][18][19] Liu et al analysed oxidised recombinant human IgG1 Fc that had been expressed in E. coli using a variety of biophysical and biochemical techniques. 17 Analysis by CD and NMR of an isotopically labelled Fc fragment showed that methionine oxidation resulted in detectable secondary and tertiary structural changes in the interface between the C H 2 and C H 3 domains.…”

Section: Introductionmentioning

confidence: 99%

“…H. Liu et al studied the effects of deglycosylation and methionine oxidation on a recombinant monoclonal antibody by limited tryptic and chymotrypic proteolysis. 18 The structural changes that occurred as a result of deglycosylation or methionine oxidation were investigated using the indirect probe of susceptibility of the molecules to trypsin and chymotrypsin proteolysis. It was reported that oxidation of the methionines in the Fc region did not result in significant structural changes, while deglycosylation resulted in an increase in susceptibility of the molecules to proteolysis, which was interpreted as evidence for conformational changes.…”

Section: Introductionmentioning

confidence: 99%

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Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Burkitt¹,

Domann²,

O’Connor³

2010

Protein Science

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Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247-253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C H 2 and C H 3 domains.

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Section: Resultscontrasting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Burkitt¹,

Domann²,

O’Connor³

2010

Protein Science

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“…Studies on mAb during storage revealed clear sites of oxidation [170][171][172]. Recent findings suggest the existence of "redox-activated" autoantibodies that are not detectable by conventional immunoassays [173,174].…”

Section: Igsmentioning

confidence: 99%

Oxidation of proteins: Basic principles and perspectives for blood proteomics

Barelli

Canellini

Thadikkaran

et al. 2008

Proteomics Clinical Apps

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Protein oxidation mechanisms result in a wide array of modifications, from backbone cleavage or protein crosslinking to more subtle modifications such as side chain oxidations. Protein oxidation occurs as part of normal regulatory processes, as a defence mechanism against oxidative stress, or as a deleterious processes when antioxidant defences are overcome. Because blood is continually exposed to reactive oxygen and nitrogen species, blood proteomics should inherently adopt redox proteomic strategies. In this review, we recall the biochemical basis of protein oxidation, review the proteomic methodologies applied to analyse redox modifications, and highlight some physiological and in vitro responses to oxidative stress of various blood components.

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Animal Cell Culture Media

Vijayasankaran¹,

Li²,

Shawley³

et al. 2010

Encyclopedia of Industrial Biotechnology

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Fed‐batch cell culture processes are the standard platform technology for the synthesis of high value biopharmaceutical proteins. Recent advances in cell line development and in the optimization of culture medium have enabled the operation of fed‐batch processes at significantly higher volumetric productivities than previously realized. While a robust basal medium is essential to ensure high productivity, a well‐developed feed medium coupled with an appropriate feeding strategy are essential for sustaining it over the course of the culture duration. Furthermore, optimization of cell culture medium needs to be cognizant of specific goals for its use. Different optimization strategies are adapted for developing platform media applicable for multiple cell lines and customized media developed for specific cell lines. While volumetric productivity continues to be an important parameter for cell culture medium optimization, the developed medium should also be robust during implementation in biopharmaceutical manufacturing facilities. The developed medium should be easy to prepare and have a long shelf life to ensure ease of process implementation in multiple commercial and clinical‐scale manufacturing sites. Due care should be taken to ensure that optimization of media does not lead to unacceptable alterations in the quality of the expressed protein. This article will address all these aspects that ensure the efficient optimization and implementation of animal cell culture media.

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Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody

Cited by 77 publications

References 35 publications

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Oxidation of proteins: Basic principles and perspectives for blood proteomics

Animal Cell Culture Media

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