08/14/2025
By Kwok Fan Chow

The Kennedy College of Science, Department of Chemistry, invites you to attend a Ph.D. Research Proposal defense by Mahsa Aghaei entitled “Investigating the Disulfide Bond Structures of Two Therapeutic Proteins.”

Degree: Doctoral
Location: Perry, Room 415
Date: Monday, Aug. 25, 2025
Time: 10:30 a.m.

Committee:

  • Chair: Jin Xu, Department of Chemistry, University of Massachusetts Lowell
  • Matthew Gage, Department of Chemistry, University of Massachusetts Lowell
  • Dongming Xie, Department of Chemical Engineering, University of Massachusetts Lowell
  • Carl Lawton, Department of Chemical Engineering, University of Massachusetts Lowel

Abstract:
This research proposal presents a comprehensive investigation into the structural and functional integrity of disulfide bonds within therapeutic proteins, emphasizing their critical role in maintaining protein stability, activity, and safety. Disulfide bonds are covalent linkages essential for proper folding and function, but their dynamic nature can lead to bond shuffling and mispairing under certain conditions, impacting the quality and efficacy of biologic drugs. Understanding these processes is crucial for enhancing the design, manufacturing, and storage of complex biotherapeutics, especially as they become increasingly sophisticated in structure.

The first aim of the study focuses on evaluating the disulfide bond dynamics in a fusion protein composed of a knob-into-hole (KiH) Fc and the IdeS protease, which has an intrinsic free cysteine residue in its active site. This aim will involve analyzing how three distinct configurations of this IdeS-Fc fusion protein affect disulfide bond architecture and cysteine status: (1) a monovalent KiH Fc-fusion protein with IdeS fused to the N-terminus of the knob chain, (2) a monovalent KiH Fc-fusion protein with IdeS fused to the C-terminus of the knob chain, and (3) a homodimeric divalent Fc-fusion protein with IdeS fused to the N-terminus of each chain. The constructs are stored for 28 days at 4 °C and 25 °C to enable a time-course study of potential disulfide bond shuffling, which may not be detectable without stressing. Using a validated LC-MS-based platform that combines reduced and non-reduced peptide mapping, the study aims to monitor disulfide bond rearrangements and free cysteine modifications with high sensitivity. Findings from this aim will provide insights into the thiol-related stability of a free cysteine-containing enzyme fused with a KiH Fc, helping to inform rational designs for improved stability and therapeutic performance.
The second aim extends this investigation to assess how manufacturing variables, such as different host cell systems and bioprocessing parameters, influence the disulfide bond configuration of a scFv-TCR fusion protein with an identical amino acid sequence. By systematically comparing proteins produced in various cell culture environments, this study will elucidate the impact of upstream process conditions on redox state, disulfide bond formation, and overall structural integrity. This knowledge is essential for optimizing production workflows to ensure consistent quality and functional stability of biotherapeutics, thereby reducing variability and potential immunogenicity.

Overall, this research aims to elucidate the mechanisms underlying disulfide bond stability and dynamics in complex therapeutic proteins. The findings will have broad implications for biopharmaceutical development by informing the design of more stable constructs, refining analytical techniques for quality control, and establishing bioprocessing standards that minimize undesired post-translational modifications that compromise drug efficacy and safety. Ultimately, the study will advance the safe and effective application of biologic therapeutics, fostering innovation in protein engineering, and supporting regulatory science for biomanufacturing.

All interested students and faculty members are invited to attend.