Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C

Abstract

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Streamlining the Characterization of Disulfide Bond Shuffling and Protein Degradation in IgG1 Biopharmaceuticals Under Native and Stressed Conditions

“…Likewise, disulfide bond shuffling is also frequently discussed with regards to proteins derived from E.coli cells because E.coli lack an endoplasmic reticulum. For proteins produced in mammalian cell lines, such as the CHO cell lines used to produce rituximab and bevacizumab, the endoplasmic reticulum acts a center for disulfide bond modulation, checking for the proper formation of bonds ( Zhang et al, 2011 ; Cai et al, 2021 ). Nevertheless, IgG1 therapeutics are not fully immune to disulfide bond shuffling.…”