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Optimising Process Development And Large Scale Peptide Manufacturing: Process Modelling In Peptide Synthesis
Ipsen Manufacturing Ireland Ltd.
Purification of Synthetic Peptides by Reversed Phase Chromatography
Three distinct approaches were systematically explored for the efficient total chemical synthesis of human insulin lispro (Humalog). Native chemical ligation of peptide-thioester segments generated by Fmoc chemistry SPPS turned out to be optimal. The 3D-structure and correct disulfide pairing of the insulin lispro protein molecule were confirmed by high-resolution X-ray crystallography, and the synthetic protein was fully active in an insulin receptor binding assay
The development of models to predict the optimum conditions for solid phase peptide synthesis will be presented and scale up principals and pitfalls will be discussed. The effective deployment of novel modelling tools will be demonstrated for both the process development and commercial manufacture of peptides and the use of these tools to control unwanted side reactions will be examined.
The formation of deletion sequences is one of the most serious problems encountered in solid phase peptide synthesis (SPPS). Deletion sequences only differ by the lack of one or a few amino acids from the desired target peptide and are typically challenging to separate in later purification steps. The longer the peptide sequence the more difficult the separation of these impurities usually becomes. This can be regarded as a major drawback of stepwise linear SPPS. In this presentation, causes for and experiments regarding the formation of deletion sequences will be discussed. Different strategies to prevent the formation of deletion sequences are applied and will be presented accordingly.
Method development for further advancing the efficiency of SPPS is of the utmost importance. Microwave irradiation provides simplified optimization, higher peptide purity, and an overall “greener” process. Compared to conventional heating methods, microwave irradiation provides rapid and direct energy exchange with the reagents.
Our previous research improved coupling efficiency and speed. The result, difficult and long sequences are effectively synthesized in a fraction of the time using much less solvent . Advances have been made which further reduce the cycle time to under 4 min, offer an overall solvent reduction greater than 90 % compared to other SPPS processes, and are readily scalable to generate up to 200 mg purified peptide. As an example, a 20mer at 0.3 mmol scale, can be synthesized in little as an hour. This unique chemistry, which is ideal and readily applicable for developing peptide vaccines for personalized medicine, will be discussed.
Rapid scale-up for clinical trials and peptide production has been accomplished using similar technology. Crude purity from R&D to production scale is preserved if not improved and unwanted side reactions such as epimerization and aspartimide formation are easily controlled. The result, easier purification and reduced labor cost. Cycles times at the production scale range from 10 – 60 min with the capability to produce up to 1 KG crude peptide in a single batch. Several examples, including process development, will be presented.
A divergent protein synthesis strategy was executed to effectively synthesize Bowman-Birk protease inhibitor analogues using native chemical ligation of peptide hydrazides. The crystal structure of a synthetic BBI analogue co-crystallized with a‑chymotrypsin confirmed the correct protein fold and showed a similar overall structure to unmodified BBI in complex with a‑chymotrypsin. Dynamic light scattering showed that C-terminal truncation of BBI led to increased self-association.
Recently, DMF, DCM and NMP, which are the most used solvents in Solid-Phase Peptide Synthesis have been classified as hazardous chemicals.
Herein, we will discuss our work focused to substitute DMF by green solvents. The use of N-formylmorpholine, 2-MeTHF, isosorbide dimethyl ether, γ-valerolactone and α,α,α-trifluorotoluene has been shown promising for SPPS.
Characterizing large peptide impurities with confidence is a challenge. Affordable High resolution mass spectrometry (HRMS) systems, coupled to Ultra High performance Chromatography open new opportunities. This presentation will show, through real case studies, how in-house HRMS technology allows fast identification and enables chemists to build up knowledge for better manufacturing processes.
HILIC is often overlooked as a method for peptide separations, due to concerns about peptide solubility and retention. We turned to HILIC for the purification of peptides consisting of only acidic and hydrophobic residues. For this class of peptides, yields in conventional RP-HPLC methodology are often very low and the HILIC method presented here has helped us overcome these problems.
The purification of synthetic peptides (25-55 amino acids) is still a challenge. The unwanted by-products of these peptides are often peptides with only one wrong amino acid in the sequence. Therefore, the peptide and the by-products elute at the same time during the chromatographic separation. The reversed phase chromatography is in many cases the method of choice. Sometimes orthogonal reversed phase methods with two chromatographic steps and two different column selectivities are needed to increase the purity to more than 95%. Chromatographic experience, a thorough method development and up scaling is needed for successful separations. Partial automation of the process leads to a remarkable throughput, which is particularly important in the field of research.
The presentation will describe the beneficial use of Doped Reversed Phase packings in the repulsive-attractive mode compared to non-doped RP packings on crude peptides. The novel orthogonal Doped Reversed Phase materials combine the dual action of strong IEX groups (acidic or basic) and Reversed Phase ligands like octyl chains on the packing surface.
It can be shown that in the majority of all cases tested so far, improved selectivities and increased resolution at decreased retention time and solvent consumption can be obtained.