Comprehensive investigation of a structural variant in a bi‐specific, N‐and C‐terminal Fc‐fusion molecule, and its monitoring with LC‐MS based method

Abstract

There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical.Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N-and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N-and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.

Sodium chloride impacts glycosylation and N‐ and O‐glycan site occupancy of an Fc‐fusion protein

“…Several mitigation strategies have been developed including changes in bioprocess conditions (culture temperature and time, partial pressure of CO 2 [Chakrabarti et al, 2016; Ghorbani Aghdam et al, 2019; Menthe et al, 2022]) or change in cell culture media composition (addition of ferric citrate, or protease inhibitor cocktail [Chakrabarti et al, 2016; Hou et al, 2019]). Other recent studies on complex N‐ and C‐ terminal Fc‐fusion proteins (Datola et al, 2023) showed that LMW proteoforms may be caused by reshuffled disulfide bridges and can lead to protein misfolding responsible for aggregation and precipitation (Zhang et al, 2011), further affecting the biological activity and safety of the therapeutic molecule.…”