Multistep Denaturation and Hierarchy of Disulfide Bond Cleavage of a Monoclonal Antibody

Brody, Tom

doi:10.1006/abio.1997.2062

Cited by 16 publications

(13 citation statements)

References 28 publications

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“…However, even when these transitions overlap considerably, available software allows rapid deconvolution of the transition envelope to obtain estimates of the individual domains' transitions (Buchner et al, 1991;Martsev et al, 1995;Vlasov et al, 1996). The existence of denaturation intermediates has also been reported by Brody (1997), who detected a ladder of denaturation intermediates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after exposure of the IgG to sodium dodecyl sulfate, urea, or heat, and by Hughes and coworkers (Hughes and Richberg, 1993;Alexander and Hughes, 1995), who described IgG ladder formation by capillary electrophoresis. The different intermediates were formed, as proposed by Brody, due to differences in sensitivity of the disulphide bonds in the different IgG domains.…”

Section: Introductionmentioning

confidence: 56%

The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein

Vermeer

Norde

2000

Biophysical Journal

601

428

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The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the F(ab) and F(c) fragments of the IgG molecule. The F(ab) fragment is most sensitive to heat treatment, whereas the F(c) fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate-determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.

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Section: Introductionmentioning

confidence: 56%

The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein

Vermeer

Norde

2000

Biophysical Journal

601

428

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“…In theory, when analyzing an antibody by SDS-PAGE under non-reducing condition, there should be only one band: namely that of the intact molecule. However, bands with both higher and lower molecular weights than intact IgG molecules are frequently reported (Angal et al 1993;Brody 1997;Forrer et al 2004;Hunt and Nashabeh 1999;King et al 1992;Schauenstein et al 1996;Schuurman et al 2001;Taylor et al 2006;Vasilyeva et al 2004;Zhang and Czupryn 2002).…”

Section: Introductionmentioning

confidence: 98%

Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE

et al. 2007

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SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and beta-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by beta-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.

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“…The hypothesis that proteins are made up of a number of independent, compact globular regions, called domains, has become generally accepted (Hardie and Coggins, 1986). An important class of proteins that conforms to such a common subunit structure is the class of immunoglobulins (Tischenko et al, 1982;Goto et al, 1988;Brandts et al, 1989;Miller, 1990;Martsev et al, 1994;Lilie and Buchner, 1995;Brody, 1997;Vermeer and Norde, 2000a). In general, immunoglobulins, otherwise called antibodies, consist of continuous stretches of the polypeptide chain comprising approximately 100 amino acids, that show a characteristic fold (Chothia et al, 1985;Edmundson and Ely, 1986;Padlan, 1997).…”

Section: Introductionmentioning

confidence: 99%

The Unfolding/Denaturation of Immunogammaglobulin of Isotype 2b and its Fab and Fc Fragments

Vermeer

Norde

Amerongen³

2000

Biophysical Journal

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The unfolding and further denaturation of IgG and its F(ab) and F(c) fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F(ab) and F(c) units was retained after fragmentation of the IgG. The F(ab) fragment denatured at approximately 61 degrees C and the F(c) fragment at 71 degrees C. The structural transitions observed in the whole IgG is the sum effect of those determined for the isolated F(ab) and F(c) fragments.

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Multistep Denaturation and Hierarchy of Disulfide Bond Cleavage of a Monoclonal Antibody

Cited by 16 publications

References 28 publications

The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein

The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein

Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE

The Unfolding/Denaturation of Immunogammaglobulin of Isotype 2b and its Fab and Fc Fragments

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