Improving crude product quality for complex synthetic peptides
Peptide manufacturing has matured over the past two decades. With reasonably priced Fmoc-based starting materials, taking on longer and more challenging peptides is where the industry is headed. As such, improvements in quality control techniques such as UPLC, SEC and even NMR has also contributed to raising the proverbial bar for the final API. Reaching high purity ranges of >98+% with no single impurity >0.1% becomes challenging when peptides reach lengths of >35 residues. Thus, we must look for improvements in the chemical manufacturing steps as critical to helping to achieve this result. This short review describes several of the key methods to improving the crude quality utilized in our industry which are employed at AmbioPharm Inc.
The resins used for solid-phase peptide chemistry are still a black box. Uniformity of the resins commonly used in most industrial applications has improved over the past two decades. The polystyrene-based resins have been cross-linked with 1% bis-divinyl benzene. This imparts a reasonable stability to mechanical shear forces as well as providing good swelling properties in the common solvents used in peptide chemistry. The resin beads are typically 75-150 µm (100-200 mesh) and 37-75 µm (200-400 mesh). The narrow size distribution of the resin either 100-200 mesh or 200-400 mesh is critical for providing uniform reaction conditions through out the insoluble polymeric support.
Derivatization of this polymeric support results in the manner which the C-terminal amino acid is joined to the resin. As the vast majority of solid-phase peptide synthesis (SPPS) is now utilizing an Fmoc-tBu strategy (1), the most common resins are either 2-Chlorotrityl chloride, Wang or HMPA, HMPB for acids and either Sieber, Rink-mBHA or Ramage tricyclic linker (Figure 1) (2). Following loading of the first amino acid to these r ...