Exposure of a Monoclonal Antibody, IgG1, to UV-Light Leads to Protein Dithiohemiacetal and Thioether Cross-Links: A Role for Thiyl Radicals?

Mozziconacci, Olivier; Kerwin, Bruce A.; Schöneich, Christian

doi:10.1021/tx100193b

Cited by 37 publications

(66 citation statements)

References 18 publications

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“…Human IgG1 has 4 interchain disulfide bonds and 12 intrachain disulfide bonds. The only thioether linkage that was observed in this study, as well as in a previous report (16,29,30), was between the HC and LC, although thioether linkages have been reported at other sites of IgG1 exposed to UV light (24). In this study we report that thioether is a naturally occurring modification, with 11% of endogenous IgG1 and 5% of endogenous IgG1 containing thioethers at this one position in the molecule.…”

Section: Discussionsupporting

confidence: 68%

“…Mozziconacci et al (24) has demonstrated that UV light could generate a thioether cross-link between the LC cysteine 214 (Eu numbering) close to the C terminus and a HC cysteine 226 (Eu numbering) instead of the HC cysteine 220 (Eu numbering) where original HC-LC disulfide formed in IgG1. Moreover, thioether formation at this position was only observed under intense photoirradiation (24) and was not detected in the nonirradiated control.…”

Section: Characterization and Quantification Of Thioether In Igg1-mentioning

confidence: 82%

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IgG1 Thioether Bond Formation in Vivo

Zhang

Schenauer

McCarter

et al. 2013

Journal of Biological Chemistry

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Background: Thioethers have been observed in therapeutic antibodies, with increasing levels upon storage. Results: IgG1 thioether bond formation is naturally occurring, but the formation rate depends on light chain type. Conclusion: Slower thioether bond formation on IgG1 is caused by dehydrogenation impairment through its light chain. Significance: Safety concerns associated with thioether control on therapeutic antibodies are diminished by its natural production.

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Section: Discussionsupporting

confidence: 68%

Section: Characterization and Quantification Of Thioether In Igg1-mentioning

confidence: 82%

IgG1 Thioether Bond Formation in Vivo

Zhang

Schenauer

McCarter

et al. 2013

Journal of Biological Chemistry

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“…38 A similar loss of bioactivity with the loss of Trp in the CDR was also observed for an IgG1 mAb by Wei et al 43 Methionine oxidation to methionine sulfoxide in the Fc was also noted by Liu et al 42 and Lam et al 41 33 In those studies the mAb was photoirradiated with light at 254 nm rather than the broad spectrum light noted in the previous studies. They identified a series of dithiohemiacetal and thioether cross-links formed following photoirradiation and suggest the involvement of thiyl radicals in the processes.…”

Section: Prior Work On Protein Photodegradationsupporting

confidence: 64%

“…demonstrated that exposure to 254 nm light lead to the formation of dithiohemiacetal and thioether cross-links. 33 Such products are potential candidates also for our experimental conditions (302 nm light) but must await further characterization.…”

Section: Discussionmentioning

confidence: 99%

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Effect of pH and Light on Aggregation and Conformation of an IgG1 mAb

2012

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Abstract:During the purification process, monoclonal antibodies may be exposed to parts of UV-C (200 to 290 nm), UV-B (290 to 320 nm) and visible light (400 to 760 nm) under a variety of buffer and pH conditions. Together, these conditions can promote both chemical and physical degradation which may result in conformational changes. To examine this possibility, the impact of UV light on an IgG1 mAb at pH 3.5, 5 and 8 was studied at multiple protein concentrations. Exposure to 302 nm light resulted in a pH-dependent formation of high molecular weight species where the degree of oligomerization increased with increasing pH.Characterization by SDS-PAGE under reducing and non-reducing conditions, and SEC-MALS, revealed that the predominant species were non-reducible dimeric, trimeric and higher order oligomeric species which occurred through processes other than intermolecular disulfide bond showing the greatest degree of change in the β-sheet structure. Exposure to UV light resulted in aggregation with pH-dependent yields decreasing in the following order, 8.0>5.0>3.5, while the opposite trend was observed for conformational changes, with pH-dependent extents decreasing in the following order:3.5>5.0>8.0. These pH-dependent trends suggest that different strategies will be required to stabilize the protein against these modifications during processing.

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Effect of ambient light on monoclonal antibody product quality during small‐scale mammalian cell culture process in clear glass bioreactors

Mallaney¹,

Wang²,

Sreedhara³

2014

Biotechnology Progress

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During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

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Exposure of a Monoclonal Antibody, IgG1, to UV-Light Leads to Protein Dithiohemiacetal and Thioether Cross-Links: A Role for Thiyl Radicals?

Cited by 37 publications

References 18 publications

IgG1 Thioether Bond Formation in Vivo

IgG1 Thioether Bond Formation in Vivo

Effect of pH and Light on Aggregation and Conformation of an IgG1 mAb

Effect of ambient light on monoclonal antibody product quality during small‐scale mammalian cell culture process in clear glass bioreactors

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