Mainstream Synthesis Processes and Optimization Research of Polypeptide Active Pharmaceutical Ingredients (APIs)

The synthesis processes of polypeptide active pharmaceutical ingredients (APIs) are mainly divided into three categories: Solid-Phase Peptide Synthesis (SPPS), Liquid-Phase Peptide Synthesis (LPPS), and Fragment Condensation. Among them, SPPS has become the mainstream choice in the current industry due to its simple operation, high automation, and suitability for large-scale production, accounting for more than 85% of the global polypeptide API production. LPPS is mainly used for the synthesis of short peptides (≤10 amino acids) and special-structured peptides. Fragment Condensation is suitable for the synthesis of long-chain peptides (≥30 amino acids) and complex cyclic peptides, which can effectively reduce cumulative errors and impurity generation in long-chain synthesis.

1. Solid-Phase Peptide Synthesis (SPPS) and Its Optimization

SPPS uses solid-phase resin as a carrier to gradually construct peptide chains through a cyclic reaction of "deprotection-activation-coupling". Its core advantages lie in the homogeneous reaction system, easy product separation, and the ability to realize automated continuous production, which meets the requirements of GMP large-scale production. The core links of its process include resin selection, protected amino acid selection, condensation system optimization, cleavage and purification process optimization, and each link directly affects the product yield and purity.

1.1 Optimization of Core Process Links

Resin selection should be combined with the peptide chain length and amino acid composition. Common resins include Wang resin (suitable for C-terminal carboxyl peptides), Rink amide resin (suitable for C-terminal amide peptides), and 2-CTC resin (suitable for fragment condensation). It is necessary to focus on the molar substitution coefficient, swelling coefficient and stability of the resin to avoid peptide chain breakage caused by resin degradation. As starting materials, the quality of protected amino acids directly affects the quality of the final polypeptide product. It is necessary to strictly control impurities such as chiral isomers, incompletely protected amino acids, and dipeptide derivatives. High-purity (≥99%) Fmoc/Boc protected amino acids are preferred. Among them, racemization-prone amino acids such as His and Cys need to be paired with sterically hindered side chain protecting groups (such as Pmbom and StBu) to inhibit the generation of racemization impurities.

The condensation system is the core of SPPS process optimization, and it is necessary to select efficient and low-racemization condensation reagents and additives. Currently, the mainstream in the industry adopts the second-generation urea salt condensation reagents (HATU, HBTU) combined with HOAt/OxymaPure additives, which can control the racemization rate below 0.1%. The application of the third-generation ynamide-type condensation reagent (Zhao's reagent) has achieved racemization-free synthesis (racemization rate <0.01%), and can simplify the process steps and improve atom economy. DIEA (weak base) is preferred as the base reagent, and strong bases such as NMM/TEA should be avoided to prevent racemization and peptide chain degradation.

For the cleavage process, appropriate cleavage reagents should be selected according to the type of protecting group. For the Fmoc protection system, TFA cleavage solution (containing scavengers such as triisopropylsilane and ethanedithiol) is commonly used, which can effectively remove side chain protecting groups and reduce peptide chain oxidation. For the Boc protection system, TFA/trifluoromethanesulfonic acid cleavage is adopted, and the cleavage temperature and time must be strictly controlled to avoid oxidation of amino acids such as Trp and Met. The purification process is mainly based on preparative HPLC, and it is necessary to optimize the mobile phase composition (such as acetonitrile-water-TFA system), elution gradient and sample loading to ensure that the purity of the target peptide is ≥98% and reduce the generation of polymeric impurities during the purification process.

1.2 Frontiers of Process Optimization

In recent years, the SPPS process has been upgraded towards "greenization, automation and continuity". The application of mechanochemical (ball milling) solvent-free synthesis technology can reduce the usage of organic solvents by more than 40%, and at the same time inhibit racemization, which is suitable for large-scale production of blockbuster products such as semaglutide and liraglutide. The upgrade of automated peptide synthesizers has realized "one-click" deprotection, coupling and monitoring. Combined with online UV-AU monitoring technology, it can real-time monitor the endpoints of coupling and deprotection, reducing human errors. The application of continuous flow microreaction technology can precisely control the reaction temperature, time and reagent concentration, increase the single-step coupling efficiency to more than 99%, and significantly reduce impurity accumulation.

2. Liquid-Phase Peptide Synthesis (LPPS) and Its Optimization

LPPS adopts solution-phase reaction to construct peptide chains through step-by-step coupling. It is suitable for the synthesis of short peptides (such as pentapeptides and hexapeptides) and hydrophobic, aggregation-prone peptides. Its core advantages lie in high reaction selectivity, easy product purity control, and no need for resin separation steps. The focus of its process optimization is on solvent selection, reaction condition control and product separation and purification.

The solvent should be a system with moderate polarity and good solubility for amino acids (such as DMF, DCM, NMP), and strong polar solvents should be avoided to prevent peptide chain aggregation. The reaction temperature is controlled at 0-5℃ (for racemization-prone amino acids) or 25℃ (for conventional amino acids) to reduce racemization and peptide chain degradation. For the coupling reaction, a "pre-activation" strategy should be adopted, in which the protected amino acid is pre-activated with the condensation reagent before being added to the reaction system to improve the coupling efficiency. Separation and purification adopt methods such as extraction, recrystallization and column chromatography, which are suitable for small-scale R&D and customized polypeptide API production. In large-scale production, it needs to be combined with the SPPS process to achieve complementary advantages.

3. Fragment Condensation Process and Its Optimization

For long-chain peptides (≥30 amino acids) and complex cyclic peptides, the single SPPS process is prone to problems such as peptide chain aggregation, decreased coupling efficiency and impurity accumulation. The Fragment Condensation process has become the optimal choice. Its core idea is to split the long peptide chain into 2-4 short peptide fragments, which are respectively synthesized and purified by SPPS/LPPS, and then connected into a complete peptide chain through condensation reaction, which can effectively reduce the pressure of single-step reaction and improve the purity of the final product.

The focus of process optimization is on the selection of fragment cleavage sites and the control of condensation conditions: fragment cleavage should avoid racemization-prone amino acids (His, Cys) and regions with dense hydrophobic amino acids, and amino acids with high stability such as Gly and Ala should be preferred as cleavage sites; the condensation reaction should use efficient condensation reagents (such as PyBOP, COMU), control the reaction concentration and pH to avoid self-polymerization of fragments; the purity of fragments should be controlled above 99% to reduce the introduction of fragment impurities into the final product. In recent years, the application of orthogonal protection strategies (such as DNPBS/Fmoc dual protection system) has realized the precise deprotection and coupling of fragments, further improving the efficiency and purity of fragment condensation.

4. Process Validation and Compliance Requirements

The process validation of polypeptide APIs must comply with GMP requirements, covering Process Performance Qualification (PPQ), cleaning validation, material validation and other links. It is necessary to clarify key process parameters (such as coupling time, temperature, reagent dosage, purification gradient), and verify through 3 or more consecutive batches of production to ensure process stability; cleaning validation needs to formulate reasonable residue limits for residual peptides, reagents and solvents, and use methods such as HPLC-MS for detection to ensure no cross-contamination; material validation should cover all starting materials such as protected amino acids, resins and solvents, and establish a complete material quality standard in line with ICH Q11 requirements. Among them, resins are not recommended as starting materials, and detailed source and quality inspection reports should be provided.