Maximizing $$$ and Purity in Peptide Synthesis: The Hybrid Process

As the demand for longer, more complex synthetically produced peptides has increased, so too have the methods of producing these peptides adapted in order to achieve high results even at the commercial scale.   One such method that employs the Fmoc/t-butyl protecting scheme is the hybrid or fragment condensation method.   The hybrid process takes advantage of the relative ease of building peptide fragments on the solid phase, followed by coupling the fragments either both in solution, or one fragment on resin and one fragment in solution.


The hybrid process was described nearly 30 years ago for the synthesis of the 109 residue peptide, prothymosin4.  However,  it was not until more recently that the hybrid process was validated as a commercial success when it was used for the production of the anti HIV peptide FuzeonTM. This thirty six residue peptide was produced by a three fragment approach condensing the protected fragments in solution.  Fragments were built on 2-chlorotrityl chloride polystyrene support1 utilizing the Fmoc/t-butyl protecting scheme.  The process was run at the commercial scale, producing up to ton amounts2.


There are several advantages that are realised when using the hybrid process.  When employing the Fmoc/t-butyl protecting scheme and 2-chlorotrityl chloride resin for solid-phase synthesis of the fragments, analysis can be performed after each amino acid coupling.  Utilizing mild fluoro alcohols for cleavage3 allows one to cleave the peptide fragment from the resin without disturbing the protecting groups.  Analysis after each coupling allows for optimization and ultimately, the production of highly pure fragments to bring into the condensation step.


The economic advantages of the hybrid process over a stepwise approach can be found in several areas including: lower reagent stoichiometry, reduced solvent usage, fewer chromatography steps and improved overall yield due to optimized purity obtained from isolations.  Contrary to popular belief, the resin cost difference between the stepwise and fragment approach is insignificant.   The table below evaluates the differences between a stepwise (one “fragment”) and a fragment (e.g. three fragment) approach of a hypothetical 30 residue peptide.


Process Resin Loading Reagents & Solvents Isolations Chromatography

linear, 30 AA

Low (0.3mm/g) High excess 2 Extensive
Hybrid Process

3 fragments, 10 AA

High (1.0mm/g) Low excess