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Optimized Production of Fc Fusion Proteins by Sortase Enzymatic Ligation
Kyle D Apley1, Amy D Laflin1, Stephanie N Johnson1
1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045, United States.
Industrial & Engineering Chemistry Research
|April 22, 2024
Summary
Sortase-mediated ligation (SML) offers an efficient method for producing Fc-fusion drugs. This study demonstrates SML
Area of Science:
- Biotechnology
- Drug Development
- Protein Engineering
Background:
- Fc fusions, linking antibody Fc domains to proteins/peptides, present manufacturing challenges.
- Fc-insulin serves as a model candidate for evaluating Fc fusion synthesis strategies.
Purpose of the Study:
- To evaluate three synthetic approaches for generating Fc fusions.
- To optimize Fc-insulin synthesis using sortase-mediated ligation (SML) and chemoenzymatic methods.
Main Methods:
- Engineered human IgG1 was digested to create an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH6).
- Fc-insulin2 was synthesized via direct SML with triglycine-insulin and two chemoenzymatic strategies (using triglycine-azide or triglycine-DBCO).
- Reaction conditions were optimized for reagent concentration, equivalents, temperature, and time.
Main Results:
- Direct SML achieved 60-70% conversion of Fc-LPETGGH6 to Fc-insulin2.
- Optimized chemoenzymatic synthesis yielded 30-40% conversion of Fc-LPETGGH6 to Fc-insulin2.
- Direct SML demonstrated superior efficiency compared to chemoenzymatic approaches.
Conclusions:
- Sortase-mediated ligation (SML) is a practical and efficient method for Fc fusion synthesis.
- An optimized pathway for Fc fusion drug synthesis was established.
- This research provides valuable insights into manufacturing Fc-fusion therapeutics.
