IgG1 Thioether Bond Formation in Vivo

Abstract

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.

Cysteine Racemization on IgG Heavy and Light Chains

“…The thioether bond formed at this position can be detected as a non-reducible species possessing the combined mass of the LC and HC by reducing capillary electrophoresis or reducing RP-HPLC/MS, or can be detected by peptide mapping (5). Peptide mapping under non-reducing conditions has been used to characterize this species as well as quantify the relative levels (10). Digestions of a previously stressed IgG1 antibody containing a light chain (IgG1) with the protease Lys-C generates a thioether-linked peptide of the sequence (H)SC*DK/(L)SFNRGEC*, where the asterisks represent positions of the modified cystine linkage, also called a lanthionine, and the letters in parentheses represent the antibody polypeptide chains (H for heavy chain, L for light chain).…”

“…Recently, we explored the base-catalyzed thioether bond formation in human IgG1s (10). Formation rates were faster in antibodies containing light chains over those with the more common light chains.…”

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

“…Bottom-up LC-MS, at reducing and non-reducing conditions [86][87][88] Aggregates and fragments SEC with light scattering detection, nanoparticle tracking analysis, light obscuration, micro-flow imaging Large size range needs to be covered. Multiple methods are required [102] [47] This direct connection between the amount of sialylated glycoforms present for a given antibody sample and the anti-inflammatory effect of IgG is also supported by the fact that steady-state serum IgG shows sialylation to a higher extent than serum IgG produced in response to a challenge with a specific antigen.…”

“…The state of the art methods for analyzing these variants are RP-LC and MS-based peptide map both under non-reducing conditions. [86] Using a mass tag or a fluorescent dye for labeling before the tryptic digest of the protein also allows the detection of free SH-groups in non-reduced peptide map. [87][88][89] …”

“…[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. Other Variants…”