Improving Sustainability and Efficiency in Peptide Synthesis: Liquid Phase Approach with Proprietary Tagging Technology

The current pharmaceutical landscape is marked by rapidly growing demand for peptide-based therapeutics, which are commonly manufactured via solid-phase synthesis (SPPS). In this approach, an amino acid is bound to insoluble polymeric resin beads, and the peptide is assembled through iterative deprotections and couplings of amino acids. The assembled peptide is then cleaved from the resin and purified via chromatography. While SPPS is a well-established methodology, it has several undesirable features, including the use of large volumes of problematic solvents, which increase manufacturing costs and decrease sustainability. Recently, tag-assisted liquid-phase peptide synthesis (LPPS) techniques have gained popularity for synthesizing peptides of various lengths while addressing several concerns with SPPS.i,ii LPPS can generate complex peptides and reduce costs and environmental impact through solution-based reactions that do not require the use of solid supports. Compared to SPPS, LPPS offers potential for the following improvements:

  • Reduced material usage: lower reaction and rinse solvent volumes, fewer equivalents of coupling reagents and amino acids
  • Improved reaction rates and yields
  • Improved reaction monitoring and in-process controls
  • Higher crude purity
  • Simplified processes: continuous processing capability, normal agitation rates, shorter purification
  • Ease of scale-up, larger maximum batch sizes
  • Readily available equipment
  • In-house production of peptide support tags, tag recoverability
peptide molecule

At Veranova, we have extended our chemical expertise to LPPS with the development of proprietary tag compound technology. Our unique tag scaffolds are soluble in low-polarity solvents but insoluble in polar solvents. Amino acids are coupled to the tags, and peptides are assembled through iterative couplings. The engineered solubility of the tag enables us to perform coupling reactions in solution, then precipitate the intermediate tag-peptide adducts by the addition of polar anti-solvents. The desired intermediates are isolated as solids, while the byproducts and unreacted starting materials remain in solution and are removed by filtration. The targeted peptides can then be cleaved under simple conditions with high crude purities. Performing these couplings in solutions allows lower reaction wash volumes and reagent loadings than those required in traditional SPPS.

LPPS can produce peptides via linear synthesis and can be incorporated into convergent syntheses to produce complex peptides (Figure 1). Small fragments produced by SPPS, LPPS, or liquid-phase synthesis without tags (limited to ~8-mer) can be combined by fragment condensation to access high molecular weight targets.

Figure 1: Peptide Manufacturing Approaches

Case Study: Liquid Phase Peptide Synthesis of Octreotide using Proprietary Tagging Technology

Octreotide is a cyclic octapeptide that serves as a more potent growth hormone inhibitor than its natural counterpart, somatostatin. This synthetic hormone is longer acting and more selective than the naturally occurring hormone. Octreotide is used to treat acromegaly, a disorder marked by the production of excess growth hormone, and it was proven to be over 20 times more active than somatostatin in initial in vivo models.iii Employing the LPPS approach, we have demonstrated the utility of our proprietary tag via liquid-phase synthesis of the commercial API, octreotide.

The liquid-phase synthesis of octreotide was performed utilizing a 6 + 2 fragment condensation approach, with the 6-mer fragment synthesized on tag (Scheme 1) and the dipeptide synthesized via general solution-phase. LPPS couplings and deprotections were carried out in dichloromethane (DCM) to ensure solubility of the tagged intermediates, but other solvents with lower environmental impact compatible with our technology have been identified.

2-mer Trt-tBu-Protected Octreotide Portion Synthesis: Fmoc-Cys(Trt)-OH was coupled with t-Bu-protected threoninol to form the 2-mer (H-Cys(Trt)-L-3(O-tBu)threoninol). After Fmoc-deprotection, the isolated solid was ready for coupling with the 6-mer portion described below.

6-mer N-Boc-Protected Octreotide Portion Synthesis (Scheme 1): After attaching threonine to the tag, the subsequent coupling reactions were performed with N,N-Diisopropylcarbodiimide (DIC)/Oxyma in DCM before the solvent was reduced and the product was precipitated by the addition of acetone as the antisolvent. Byproducts and unreacted starting materials were acetone-soluble, allowing for the isolation of the product by simple filtration. Fmoc-deprotection reactions were carried out in a piperidine/DCM system. After deprotection, the solvent was reduced, and the product was precipitated using acetonitrile as the antisolvent.

Diagram to show synthesis of Octreotide

 

Scheme 1: Synthesis of Octreotide

Octreotide Fragment Condensation Synthesis (Scheme 1): Once the 6-mer was assembled on the tag, it was cleaved using basic conditions to preserve the acid-labile amino protecting groups. Cleavage from the tag furnished the N-Boc-protected 6-mer acid (Boc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-OH), which was subsequently coupled with the 2-mer (H-Cys(Trt)-L-3(O-tBu)threoninol), followed by a global deprotection to produce linear octreotide. Cyclization to the ultimate target octreotide was performed using aqueous conditions under an atmosphere of open air.

Purification: Final purification of crude octreotide was performed using medium-pressure flash chromatography to afford octreotide as the acetate salt with a final purity of 92%. The purity was determined by liquid chromatography (LC, relative peak area with UV detection), and octreotide identification was confirmed by mass spectrometry (MS) of the main peak (Figure 2).

Figure 2. Chromatography of Octreotide Post-Purification. (a) Chromatogram for LC with UV Detection (Full-Scale Shown with Zoomed-in Inset) and (b) Mass Spectrum for Main Peak by LC-MS

We have successfully designed and synthesized an effective tag anchor for liquid-phase synthesis and demonstrated its utility with the synthesis of a cyclic commercial octapeptide, octreotide. Unlike peptide synthesis on solid support, liquid-phase synthesis of peptides on tags has no upper scale limitation, and we endeavor to achieve additional, customizable process development goals, including solvent optimization. We are confident in the advantages of liquid-phase reactions, including the use of lower solvent volumes and less expensive, more sustainable methodology for generating peptide APIs. We look forward to expanding our commercial and custom peptide portfolio using our proprietary tag compounds to deliver products with a more sustainable, efficient, and cost-effective approach.

References

i. Sharma, A.; Kumar, A.; de la Torre, B. G.; Albericio, F. Chem. Rev. 2022, 122, 13516−13546.

ii. Frederick, M.; Boyse, R. et al. Kilogram-Scale GMP Manufacture of Tirzepatide Using a Hybrid SPPS/LPPS Approach with Continuous Manufacturing. Org. Process Res. Dev. 2021, 25, 1628-1636.

iii. Bauer,W.; Briner, U.; Doepfner, W.; Haller, R.; Huguenin, R.; Marbach, P.; Petcher, T.; Pless, J. SMS 201–995: A very potent and selective octapeptide analogue of somatostatin with prolonged action. Life Sciences 1982, 31 (11), 1133-1140.

About the author

Alexander KovalAlexander B. Koval, Ph.D., is a Senior Scientist in the Process Research & Development group at Veranova. He received his doctoral degree in chemistry from Temple University (Philadelphia, PA) in 2019, authoring papers on natural product analog synthesis, anti-cancer compound synthesis, and peptide synthesis. Alex started his industry career in 2019 and joined Veranova (formerly Johnson Matthey Health, West Deptford, NJ) in 2021. He is an inventor on two patents and has expertise in custom synthesis, process development, HPAPI synthesis, SPPS, and LPPS, among others.

Special thanks to Cale Weatherly, Associate Director of Process Research & Development, and Dan Coughlin, Technical Fellow, for their technical contributions to this project.

 

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