1. Home
  2. Science & Research
  3. Angiotensin converting enzyme 2 fusion protein stability and spike protein variant binding
  1. Section N/A

Angiotensin converting enzyme 2 fusion protein stability and spike protein variant binding

Authors:
Poster Author(s)
Vincent Falkowski, FDA/CDER (Student); Alicia Matthews FDA/CDER (contractor); Xin Bush FDA/CDER (Student); Cyrus Agarabi FDA/CDER (Federal Employee); Talia Faison FDA/CDER (Federal Emplyee), Thomas Biel FDA/CDER (Mentor); Tongzhong Ju FDA/CDER (PI)
Center:
Contributing Office
Center for Drug Evaluation and Research

Abstract

Poster Abstract

COVID-19 is caused by the infection of the novel SARS-CoV-2 virus. Currently, there are no FDA approved vaccines or therapeutics that prevent or suppress SARS-CoV-2 infections, although three vaccines and many therapeutics have received emergency use authorization (EUA). Viral infectivity and entry into host cells is mediated by the binding of viral Spike protein to host cell Angiotensin Converting Enzyme 2 (ACE2). A novel strategy to suppress SARS-CoV-2 infections is to treat patients with an ACE2-Fc fusion protein to sequester the virus from binding and infecting host cells even if the SARS-CoV-2 has mutated overtime and the consequent mutants or variants often are of higher infectivity. Here we generated two ACE2-Fc expressing cell lines that secrete catalytically active and inactive ACE2-Fc fusion proteins and performed different chromatography methods to purify the fusion proteins. Using biolayer interferometry, the binding characteristics of ACE2-Fc and four spike protein variants was determined and compared. The Lambda variant of the spike protein was identified to have the highest binding affinity to the ACE2-Fc fusion proteins, and the lowest rate of dissociation. To investigate the protein stability, the purified ACE2-Fc fusion proteins in a preliminary formulation buffer were subjected to different storage conditions (buffer, time and temperature) to qualitatively and quantitively determine conditions that lead to protein instability using SDS-PAGE, immunoblotting, and capillary SDS. To identify excipients that suppress temperature induced protein instability, the ACE2-Fc fusion proteins were subject to temperature ramping studies in the presence and absence of different excipients to characterize the onset of protein denaturation and aggregation using dynamic light scattering (DLS), static light scattering (SLS) and full spectrum differential scanning fluorimetry (DSF). These findings will be used to develop a storage buffer that can support the structural integrity of the ACE2-Fc fusion protein to facilitate additional characterization studies and production of experimental COVID-19 therapeutics with high quality.

Back to Top