β-Elimination and Peptide Bond Hydrolysis:  Two Distinct Mechanisms of Human IgG1 Hinge Fragmentation upon Storage

Cohen, Steven L.; Price, Colleen; Vlasak, Josef

doi:10.1021/ja0705994

Cited by 141 publications

(156 citation statements)

References 16 publications

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“…8), which may correspond to a cleavage at the N-terminus of Cys49. These results suggested that heat treatment of VHHs causes fragmentation of polypeptide around the position of the disulfide bond as previously reported (30,31).…”

Section: A Series Of Vhhs With Various Numbers Of Disulfide Bondssupporting

confidence: 87%

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

2015

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Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heatinduced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.Keywords: antibody engineering/disulfide bonds/protein chemical modification/protein engineering/protein stability.Abbreviations: bLA, Bacillus cereus b lactamase; CD, circular dichroism; hCG, human chorionic gonadotropin; SPR, surface plasmon resonance; t 1/2 90 C , halflife of VHH at 90 C; T m , mid-point temperature of thermal unfolding; VHH, variable domain of camelid heavy chain antibody.The variable domain of the camelid heavy chain antibody (VHH) is known to be highly heat resistant.VHHs remain active even after incubation at high temperatures (>80 C) because of their ability to refold to their native structure from a heat-induced unfolded state (1 6). However, even for VHHs, a long incubation finally leads to denaturation: e.g. the half-life of VHH at 90 C (t 1/2 90 C ) was 3 h at neutral pH (7). Residual activity of VHH depends only on the incubation time at 90 C, and not on the number of heating (90 C)-cooling (20 C) cycles, indicating that folding and unfolding intermediates are irrelevant to the loss of activity. Heat denaturation of VHHs is independent of protein concentration; therefore, VHHs lose their activity in a unimolecular manner, not by aggregation. This strongly suggests that chemical modifications play an important role during VHH denaturation. Indeed, replacing the Asn residues, which are prone to chemical modification, increase the heat resistance of VHHs (7).VHHs typically have only one intramolecular disulfide bond. The introduction of a second non-canonical artificial disulfide bond connecting the amino acids at positions 49 and 69, with numbering according to Kabat et al. (8), results in a significantly higher midpoint temperature of equilibrium thermal unfolding (T m ) in comparison to wild-type protein (9 13). In spite of its superior equilibrium thermodynamic stability, the mutant with a second disulfide bond exhibited lower residual activity ...

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Section: A Series Of Vhhs With Various Numbers Of Disulfide Bondssupporting

confidence: 87%

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

2015

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“…The mass spectra of peaks a to c are shown in Figure 4b. The theoretical molecular weights of antibody-A heavy chain without C-terminal Lys and with a core-fucosylated biantennary complex oligosaccharide structure without terminal galactose ( linkage has been well documented in the literature [31][32][33] and reported for monoclonal antibodies [17,28]. No interpretable mass spectrum was obtained for the shoulder that eluted in front of peak a.…”

Section: On-line Sec-msmentioning

confidence: 99%

“…␤-Elimination is a common degradation pathway of recombinant monoclonal antibodies [17,28], which results in a nonreducible thioether linkage of the light chain and heavy chain. The linkage site was previously determined using material collected from SEC with 6 M guanidine hydrochloride in the mobile phase [28].…”

Section: Comparison Of Sec and Rp Chromatographymentioning

confidence: 99%

“…No interpretable mass spectrum was obtained for the shoulder that eluted in front of peak a. The small peak that eluted between peaks b and c had an observed molecular weight of ϳ26,932 Da, which corresponded to antibody-A heavy chain amino acids 225-450 with an addition of a pyruval group, which resulted from peptide bond cleavage after ␤-elimination of the inter heavy chain and light chain disulfide bond [17]. …”

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confidence: 98%

“…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.…”

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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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Molecular and Functional Characterization of Monoclonal Antibodies

Zhao

Stadheim²,

Chen

et al. 2009

Therapeutic Monoclonal Antibodies

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β-Elimination and Peptide Bond Hydrolysis: Two Distinct Mechanisms of Human IgG1 Hinge Fragmentation upon Storage

Cited by 141 publications

References 16 publications

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

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

Molecular and Functional Characterization of Monoclonal Antibodies

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