The recombinant approach to produce Semaglutide api

The recombinant production of semaglutide is a "semi-recombinant" process that combines genetic engineering fermentation and chemical modification, and the core is to express intermediate peptides through microorganisms, and then enzymatic digestion, chemical modification and purification to obtain the final product. Here are the key process steps and technical points:

1. Construction and fermentation of genetically engineered bacteria

Main chain design
The backbone of semaglutide is the 29-amino acid intermediate polypeptide Arg34GLP-1 (9-37) (sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly). This peptide is expressed in E. coli or yeast by genetic recombination technology.

Fusion protein optimization
To increase expression, intermediate peptides are combined with fusion peptides (e.g., patentedly designed sequence MATKAVSVL... ) is linked by the enterokinase recognition site DDDDK to form a fusion protein (structure: fusion peptide-DDDDK-Arg34GLP-1 (9-37)). Optimizing the isoelectric point and hydrophilicity of the fused peptide can significantly increase the expression of inclusions (up to 20–30 g/L).

Fermentation process
Engineered bacteria (such as Escherichia coli BL21) were fermented at high density at 37°C, and IPTG (final concentration 0.1 mM) was added to induce expression when the OD value reached 150.
The fusion protein is expressed in the form of inclusions, which are collected by centrifugation, homogenized and crushed at high pressure, and then washed.

2. Enzymatic digestion process releases intermediates P29

Enzyme selection

Single enzyme digestion (recombinant enterokinase rEK):
The cutting site DDDDK↓ has strong specificity, and the non-specific cutting impurities are lower than those of imported competitors. Enzyme digestion conditions: 35°C, 24 hours, buffer system with pH 8.0 Tris solution.

Double enzyme digestion method (Kex2 enzyme + carboxypeptidase B):
It is suitable for tandem expression processes, but it is expensive (Kex2 enzyme is expensive) and requires the optimal pH of the dienzyme to be coordinated (Kex2: pH 9.0; carboxypeptidase B: pH 7.5–9.0).

Enzyme cleavage efficiency:
Recombinant enterokinase activity is defined as the amount of enzyme required to cleavage 95% of the fusion protein within 24 hours at 25°C (50–500 μg of substrate per unit of digestion). Suppliers such as Yisheng Biotech provide GMP-grade enzymes with ≥95% purity and no animal-derived risks.

3. Chemical modifications extend the HR

• Sidechain Modification:

The intermediate Arg34GLP-1 (9-37) needs to be linked to the lipophilic side chain (structure: 18-carbon fatty acid + polyethylene glycol ligation arm + glutamic acid) at the Lys26 site to enhance albumin binding capacity and prolong the half-life of the drug.

• One-pot synthesis innovation:

Traditional processes require separation of intermediates, which are cumbersome steps (e.g., isoelectric precipitation, centrifugation, and resolutization).

The new process uses a mixed solvent system (e.g. NMP/water = 2:1) and is completed directly and sequentially:

1. Side chain modification: Intermediate reacts with Ste-Glu(AEEA-AEEA-OSu)-OH acylation;

   2. Backbone modification: add double Fmoc protective dipeptide Fmoc-His(Fmoc)-Aib-OH (condensation after activation);
   3. Deprotection: The piperidine solution removes the Fmoc group to obtain semaglutide crude peptide.

Advantages: Feed volume is reduced to 1–3 equivalents (traditionally 8–10 equivalents are required), yields are increased, and intermediate separation is avoided.

4. Purification and quality control

• Chromatographic purification:

Crude peptides after digestion or chemical modification are purified by ion exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) with a mobile phase of acetonitrile/water (containing 0.1% trifluoroacetic acid) and gradient elution.

• Lyophilization and Quality Inspection:

The purified peptide solution was freeze-dried into powder, the purity of HPLC was ≥98%, and the molecular weight was verified by mass spectrometry (theoretical molecular weight of semaglutide ≈4113 Da).

5. Technical difficulties and innovation

• Enzyme Digestion Specificity:

Intermediates contain multiple lysine (Lys26) and arginine (Arg34/36) and should avoid non-specific cleavage. High-purity recombinant enterokinase reduces impurities.

• Scale Challenges:

◦ Fermentation: The 500L fermentation tank can produce about 500,000 units of enterokinase to meet the needs of industrialization.

◦ Chemical modification: The one-pot method simplifies the process, reduces the amount of organic solvents, and is more suitable for scale-up production.

summary

The recombinant method realizes the efficient production of semaglutide through four steps: genetic engineering expression of intermediate peptides→ enzymatic cleavage→ chemical modification→ and purification, with the following key advantages:

1. Biological method breaks through capacity bottlenecks: fermentation replaces solid-phase synthesis and reduces large-scale costs;
2. Innovation in digestion process: highly specific recombinant enterokinase to improve yield;
3. Simplified chemical modification: The one-pot method avoids the separation of intermediates and optimizes atomic economy.
   The process has propelled semaglutide to become a "blockbuster drug" with annual sales of more than $20 billion, and continues to reduce costs through technological innovation.

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