Downstream Processing/ Purification/ Isolation of Peptides

Many peptides are traditionally purified and isolated by chromatography and freeze drying/lyophilization. Depending on purity required, multiple chromatography operations may be needed. Large-scale chromatography remains a significant cost center, and buffers and solvents used for eluents can give a large PMI for the product. Lyophilization is energy intensive on a large scale.

Depending on purity requirements and stability characteristics, more mass efficient and less energy intensive technologies like multi-column chromatography and spray drying might be considered.

Jadhav, S.; Seufert, W.; Lechner, C.; Schönleber, R. Bachem – Insights into Innovative and Sustainable Peptide Chemistry and Technology by the Leading Independent Manufacturer of TIDES. Chimia 2021, 75, 476–479.

Affinity tags/peptide ‘easy clean’ technology

Traceless affinity tagging is another emerging technique to provide alternative, more selective, and sustainable isolation of peptides. There are several strategies that can be employed. Typically, a multi-functional tag is attached to the peptide. Also present are other groups that can be manipulated with orthogonal deprotection. In the case of the example below, acid cleavage exposes a hydroxylamine that can be used as a binding site for an affinity chromatography resin. Once purified, the affinity group and resin attachment group are removed by hydrogenation.

Reimann, O.; Seitz, O.; Sarma, D.; Zitterbart, R. A traceless catch‐and‐release method for rapid peptide purification. J. Pep Sci. 2019, 25, e3136.

Zitterbart, R.; Berger, N.; Reimann, O.; Noble, G. T.; Lüdtke, S.; Sarma, D.; Seitz, O. Traceless parallel peptide purification by a first-in class reductively cleavable linker system featuring a safety-release. Chem. Sci. 2021, 12, 2389–2396.

Kheirabadi, M.; Creech, G. S.; Qiao, J. X.; Nirschl, D. S.; Leahy, D. K.; Boy, K. M.; Carter, P. H.; Eastgate, M. D. Leveraging a “Catch–Release” Logic Gate Process for the Synthesis and Nonchromatographic Purification of Thioether- or Amine-Bridged Macrocyclic Peptides. J. Org. Chem. 2018, 83, 4323−4335.

Relevant Scale Up Examples

Kilogram-Scale GMP Manufacture of Tirzepatide Using a Hybrid SPPS/LPPS Approach with Continuous Manufacturing

A hybrid solid-phase peptide synthesis/liquid-phase peptide synthesis (SPPS/LPPS) approach for the synthesis of tirzepatide. Continuous manufacturing and real-time analytical monitoring give high-quality material. The use of nanofiltration avoided difficult precipitations during intermediate purification.

Org. Process Res. Dev. 2021, 25, 1628–1636.
9 kg scale

Copper(II) Lysinate and Pseudoproline Assistance in the Convergent Synthesis of the GLP‑1 Receptor Agonists Liraglutide and Semaglutide.

The synthesis of lipopeptides liraglutide and semaglutide using a stepwise condensation of three fragments in SPPS. The use of pseudoproline and copper(II) lysinate intermediates was also described.

Org. Process Res. Dev. 2021, 25, 1598–1611.
1g scale

Sustainable, cost-efficient manufacturing of therapeutic peptides using chemo-enzymatic peptide synthesis (CEPS).

H-22-39-NH2 and H-1-21-O-Cam-L-NH2 exenatide fragments for the enzymatic ligations were synthesized by SPPS methods and ligated by omniligase-1 enzyme.

Green Chem. 2019, 21, 6451-6467.
25 g scale

Improved Tag-Assisted LPPS: An Application to the Synthesis of Bradykinin Receptor-Antagonist Icatibant Acetate.

Soluble tag-assisted liquid-phase peptide synthesis using a hydrophobic benzyl alcohol as a support is applicable to the 100 g scale.

Org. Process Res. Dev.. 2019, 23, 2576–2581.
100g scale

Org. Process Res. Dev.. 2012, 16, 1279−1282.
1.4 kg scale flow process