Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry

Yan, Boxu; Valliere-Douglass, John; Brady, Lowell J.; Steen, Sean; Han, Mei; Pace, Danielle; Elliott, Susan; Yates, Zac; Han, Yi-Hong; Balland, Alain; Wang, Wei Chun; Pettit, Dean K.

doi:10.1016/j.chroma.2007.06.063

Cited by 96 publications

(71 citation statements)

References 33 publications

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“…The initial site of radical attack was at the interchain disulfide bond connecting the two heavy chains at Cys 231 and yielded a thiyl radical at one of the Cys 231 side chains, which initiated an electron/radical transfer to the hinge residues and yielded protein backbone fragmentation. This mechanism is consistent with a number of studies that have shown that preferential cleavage can be induced selectively by radicals at specific sites within a protein (32,54,55).…”

Section: Discussionsupporting

confidence: 91%

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Yan

Yates

Balland

et al. 2009

Journal of Biological Chemistry

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Recombinant human monoclonal antibodies (mAbs)2 have been the main products for the biotechnology industry for more than a decade (1-3). A key strength of antibodies as therapeutics is that their clinical potential can readily be increased by improving their existing properties through a range of antibody engineering technologies (4, 5). As therapeutic agents, mAbs are produced in large scale cell culture, purified, and stored under various conditions and administered to patients (6, 7). Exposure to these production/storage conditions may reduce the stability and efficacy of the mAb by increasing the chance for introducing undesirable modifications such as oxidation, proteolytic cleavage, deamidation, and isomerization. A better understanding of the whole spectrum of possible degradation pathways, particularly new pathways, could facilitate the engineering of mAbs with improvement in the production of stable, efficacious, and safe biotherapeutics.A recent study indicated that antibodies have the intrinsic capacity to convert molecular oxygen into hydrogen peroxide (H 2 O 2 ) (8) and in this process to produce some short lived hydroxyl radical species (HO ⅐ ) at the interface of the light and heavy chains (9 -12). These observations were further supported by a more recent observation that the light chains (three and three types) from the urine of six patients who had multiple myeloma and light chain proteinuria were found capable of generating H 2 O 2 (13). Substantial evidence suggests that the production of H 2 O 2 is an important signaling event triggered by the activation of various cell surface receptors, such as antibody-receptor interaction (14 -19). It has been demonstrated that H 2 O 2 -mediated redox chemistry can regulate the biological function of proteins through interactions with specific residues such as cysteine (Cys) (20 -23); thus, H 2 O 2 may represent a key signaling molecule in mammalian systems. Stamler and Hausladen (20) have proposed a continuum of H 2 O 2 -mediated Cys-SH modifications that constitute important biological signaling events on the one hand and irreversible hallmarks of oxidative stress on the other. Quite commonly, Cys-SH reacts with H 2 O 2 and yields oxidized forms of reversible or irreversible modified residues; reversible modified groups can be stabilized within the protein environment and recycled (21). Irreversible oxidation can lead to the degradation of proteins via a hydroxyl radical mediated mechanism to cleave a peptide bond at the ␣-carbon position through either the diamide or ␣-amidation pathways (24 -27). Although extensive studies have been conducted, because of the transient nature of a radical reaction and unstable intermediate products, the mechanisms underlying the formation of some specific reaction products is still not fully understood. It is well known that Cys residues of an IgG molecule form the intrachain or interchain disulfide bonds (1, 5); thus, the effect of H 2 O 2 -mediated Cys redox chemistry on the structure and stability of a human antibo...

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Section: Discussionsupporting

confidence: 91%

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Yan

Yates

Balland

et al. 2009

Journal of Biological Chemistry

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“…Domain-specific characterization can then be achieved by subsequent analyses. This approach has been demonstrated using proteolytic enzymes such as papain and Lys-C. 12,13 Pepsin is another enzyme that can generate F(ab') 2 fragment; however, Fc is degraded into smaller peptides upon pepsin digestion. When using papain and Lys-C to generate antibody fragments, the digestion condition needs to be carefully controlled to achieve high yields of the desired cleavage products and avoid over-digestion.…”

Section: Introductionmentioning

confidence: 99%

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

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“…As part of protein drug development, intact mass analysis supports the characterization package for regulatory filings and may be used to evaluate lot-to-lot consistency on a whole molecule level. Mass analysis of intact or reduced antibodies has been used to evaluate the degree of processing of C-terminal lysine on the heavy chain subunit [1]; evaluate N-terminal heterogeneity such as pyroglutamic acid formation [2,3]; profile N-linked carbohydrate heterogeneity [3,4]; and to detect instabilities in the molecule such as oxidation [5], succinimide formation from aspartic acid [6], glycation [7], internal cleavage [8], and thioether formation [9]. Reversed-phase HPLC (rpHPLC) separation followed by electrospray ionization-mass spectrometry (ESI-MS) analysis is the typical method for analyzing antibodies under both non-reduced and reducing conditions.…”

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confidence: 99%

Molecular mass analysis of antibodies by on-line SEC-MS

Brady

Valliere-Douglass

Martinez

et al. 2008

J. Am. Soc. Mass Spectrom.

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Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS. This method allows extremely efficient desalting of proteins under acidic conditions that are optimal for subsequent mass analysis using standard ESI conditions. Furthermore, this technique is significantly faster and more sensitive than rpHPLC methods, typically considered the standard chromatography approach for mass analysis of proteins. This method is flexible and robust, and should prove useful for applications where a combination of speed and sensitivity are required. M ass analysis of recombinant proteins is a key characterization assay used to evaluate the entire amino acid sequence of the molecule and the presence of post-translational modifications. As part of protein drug development, intact mass analysis supports the characterization package for regulatory filings and may be used to evaluate lot-to-lot consistency on a whole molecule level. Mass analysis of intact or reduced antibodies has been used to evaluate the degree of processing of C-terminal lysine on the heavy chain subunit [1]; evaluate N-terminal heterogeneity such as pyroglutamic acid formation [2,3]; profile N-linked carbohydrate heterogeneity [3,4]; and to detect instabilities in the molecule such as oxidation [5], succinimide formation from aspartic acid [6], glycation [7], internal cleavage [8], and thioether formation [9]. Reversed-phase HPLC (rpHPLC) separation followed by electrospray ionization-mass spectrometry (ESI-MS) analysis is the typical method for analyzing antibodies under both non-reduced and reducing conditions. This method is highly resolving and advantageous for the detection of minor product impurities and the resolution of different amino acid sequences or heterogeneous post-translational modifications. However, rpHPLC of antibodies has several distinct disadvantages. Due to the large size and relatively hydrophobic nature of antibodies, high temperatures are typically employed to improve elution and peak shape profiles [1,4]. However, high temperatures may lead to artifactual degradation of the sample during analysis. Inclusion of TFA as a mobile phase additive in rpHPLC is often necessary to obtain good chromatography [1], but may result in decreased ionization [10,11]. Additionally, reduction of antibodies and analysis of the constituent light and heavy chains often results in tailing of the heavy-chain component and the potential for carry-over problems. These issues can lead to lengthened run times or the inclusion of blank runs between samples to increase confidence in the results.We optimized the use of a commercially available polyhydroxyet...

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Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry

Cited by 96 publications

References 33 publications

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

A new tool for monoclonal antibody analysis

Molecular mass analysis of antibodies by on-line SEC-MS

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