Characterization of trisulfide modification in antibodies

Gu, Sheng; Wen, Dingyi; Weinreb, Paul H.; Sun, Yaping; Zhang, Lihe; Foley, Susan F.; Kshirsagar, Rashmi; Evans, David R.; Mi, Sha; Meier, Werner; Pepinsky, R. Blake

doi:10.1016/j.ab.2010.01.019

Cited by 94 publications

(151 citation statements)

References 28 publications

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“…46 Trisulfide bonds were later detected in all subclasses of recombinant IgG antibodies, as well as in human IgG from patients with myeloma. 47 In all cases, higher levels of trisulfide bonds were observed between the cysteine residues that normally form the inter light chain and heavy chain disulfide bonds. 47 Trisulfide bonds in recombinant mAbs are believed to be formed during fermentation as a result of the reaction of an intact disulfide bond with dissolved hydrogen sulfide (H 2 S).…”

Section: Trisulfide Bond Formationmentioning

confidence: 99%

Disulfide bond structures of IgG molecules

Liu

May

2012

mAbs

360

204

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Section: Trisulfide Bond Formationmentioning

confidence: 99%

Disulfide bond structures of IgG molecules

Liu

May

2012

mAbs

360

204

View full text Add to dashboard Cite

“…[98] Trisulfide bond formation on the other hand, has been proposed to occur during fermentation, resulting from the interaction of an already formed disulfide bond with hydrogen sulfide dissolved in the surrounding media. [99,100] While it has clearly been reported for trisulfide bonds that no associated change in antigen binding and biological activity of antibodies could be observed, [100] for thioether linkage it has been proposed that the change in bond length might in certain cases lead to a change of the orientation of the Fab fragment, which could potentially impact antigen binding properties. [86] 3.5.…”

Section: Beta Elimination and Trisulfide Bond Formationmentioning

confidence: 99%

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so‐called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half‐life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core‐fucosylation of the N‐glycan and aggregation the effects are clear and should be monitored, but with others such as C‐terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

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“…Trisulfide bonds so far have mainly been reported at the inter-chain locations between the heavy and light chains (i.e., between the 2 Cys in the lightchain peptide SFNRGEC and heavy-chain peptide SCDK), which is consistent with what was observed in our study. 28 Overall, the full MS and MS/MS spectra confirmed that the new peak was due to a trisulfide bond in which a sulfur atom was inserted into the inter-chain disulfide linkage in the mAb. The level of trisulfide bonds in 3 antibody lots were further determined to be 1.6%, 18.2%, and 31.7%, respectively, by calculating the ratio of the extracted ion chromatogram (XIC) peak intensity of trisulfide-linked peptide versus the total intensities of both disulfide and trisulfide linked peptides ( Table 1).…”

Section: Confirmation Of Trisulfide Bonds By Peptide Mapping (Lc/ms/mmentioning

confidence: 55%

“…Trisulfide bonds are formed in Chinese hamster ovary cell-expressed mAbs likely due to the presence of hydrogen sulfide in the cell culture. 28 Cell culture process is very critical during the process development of biotherapeutics, and needs to be optimized to develop robust growth conditions that yield desired and consistent product quality. MAbs usually have very low levels of trisulfide bonds, but occasionally can contain high levels of trisulfide bonds as a function of the manufacturing conditions.…”

Section: Introductionmentioning

confidence: 99%

“…[24][25][26] More recently, trisulfide bonds have been reported in all 4 subclasses of human IgG molecules. [27][28][29][30][31] In a recent study of ADC development using inter-chain cysteinedirected linker chemistry as described above, trisulfide bonds have been reported to affect the reduction step by TCEP during the antibody-drug conjugation process and affect average DAR value. 30,32 This occurred because a fraction of TCEP reacted with trisulfide bonds to form a thiophosphine and a disulfide, therefore creating less free thiols available in the antibody for conjugation.…”

Section: Introductionmentioning

confidence: 99%

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The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

Liu

Chen

Dushime

et al. 2017

mAbs

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Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.

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Characterization of trisulfide modification in antibodies

Cited by 94 publications

References 28 publications

Disulfide bond structures of IgG molecules

Disulfide bond structures of IgG molecules

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

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