Removal of Cysteinylation from an Unpaired Sulfhydryl in the Variable Region of a Recombinant Monoclonal IgG1 Antibody Improves Homogeneity, Stability, and Biological Activity

Banks, Douglas D.; Gadgil, Himanshu S.; Pipes, Gary D.; Bondarenko, Pavel V.; Hobbs, Vipa; Scavezze, Joanna L.; Kim, Jun; Jiang, Xu‐Rong; Mukku, Venkat R.; Dillon, Thomas M.

doi:10.1002/jps.21014

Cited by 76 publications

(55 citation statements)

References 24 publications

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“…A similar antibody cysteinylation event was previously described by Gadgil et al, 11 and the biophysical impact of the modification was described by Banks et al 12 Though the authors' antibody, MAB007, and our mAb 001 are both clearly negatively affected by cysteinylation, there are several key differences between the two events. MAB007 was found to be cysteinylated on an additional, unpaired cysteine residue in the heavy chain CDR 3, compared to the light chain CDR 3 of our mAb 001.…”

Section: Discussionsupporting

confidence: 81%

“…As shown previously, antibody cysteinylation can be partially reversed under mild reduction conditions, at least in some situations. 12 To test whether the cysteinylation of mAb 001 could be reversed, the highly-modified, low-activity mAb 001 antibody lot 159218 was exposed to mild reducing conditions (50 mM cysteine in PBS, pH 8.0 for 90 minutes at ambient temperature). SEC and Biacore testing indicated that mock treatment of the mAb 001 lot (no cysteine addition) did not affect its aggregation levels or activity (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…However, when antibody cysteinylation has been observed, the effect on antibody function was significant. 11,12 To examine the effect of this oxidation event on the stability and activity of the antibody in question, HIC chromatography, MS and plasmon resonance-based potency testing were used to show the impact of this modification on the antigen binding capabilities of the antibody, independent of antibody aggregation or degradation. Molecular modeling was then applied in order to better understand and support the analytical data, leading to an improved understanding of this post-translational modification and its effects.…”

Section: Introductionmentioning

confidence: 99%

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Cysteinylation of a monoclonal antibody leads to its inactivation

McSherry

Ozaeta

et al. 2016

mAbs

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Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism.

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Section: Discussionsupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cysteinylation of a monoclonal antibody leads to its inactivation

McSherry

Ozaeta

et al. 2016

mAbs

View full text Add to dashboard Cite

show abstract

“…3A). Banks et al 21 also observed a similar CEX-HPLC chromatogram shift from multiple peaks to a more basic homogenous peak after cysteine treatment of a mAb that contained unpaired cysteine residues (MAB007). To identify the peaks in the CEX-HPLC profile of Cys-treated THIOMAB, fractions were collected, concentrated, deglycosylated and analyzed using LC/MS.…”

Section: Introductionmentioning

confidence: 70%

Charge-based analysis of antibodies with engineered cysteines

Chen¹,

Nguyen²,

Jacobson³

et al. 2009

mAbs

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“…Each subclass of IgG molecules has welldefined homogenous disulfide bond structures; however, free cysteine residues, scrambled disulfide bonds and the presence of trisulfide bonds and thioether bonds have also been widely reported. [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.…”

Section: Introductionmentioning

confidence: 99%

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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Removal of Cysteinylation from an Unpaired Sulfhydryl in the Variable Region of a Recombinant Monoclonal IgG1 Antibody Improves Homogeneity, Stability, and Biological Activity

Cited by 76 publications

References 24 publications

Cysteinylation of a monoclonal antibody leads to its inactivation

Cysteinylation of a monoclonal antibody leads to its inactivation

Charge-based analysis of antibodies with engineered cysteines

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

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