To provide more treatment options for diabetic patients, some new diabetes drugs emerged in the early 21st century, such as glucagon-like peptide-1 (GLP-1) analogs. Semaglutide, as one of the representative drugs of GLP-1 analogs, has been proven in multiple clinical trial studies to effectively control blood sugar when combined with different oral hypoglycemic drugs, and can help patients lose weight, reduce systolic pressure, and improve pancreatic β-cell function. Its three formulations (hypoglycemic injection Ozempic, oral hypoglycemic drug Rybelsus, and weight loss injection Wegovy) exceeded $20 billion in sales in 2023. Due to its outstanding performance in blood sugar reduction and weight loss, the industry expects Semaglutide to maintain strong sales momentum for some time, attracting many companies to compete for layout!

The main chain structure of Semaglutide is a 29-amino acid intermediate polypeptide Arg34GLP-1(9-37) or a 27-amino acid intermediate polypeptide Arg34GLP-1(11-37). The production and manufacturing of this intermediate polypeptide mainly include chemical synthesis and biopreparation methods. Compared to chemical synthesis, biopreparation has broken through the capacity bottleneck and is more suitable for large-scale continuous production, which can further reduce production costs and bring more significant economic benefits.

Table 1. Examples of biopreparation methods for semaglutide intermediate polypeptides

	Biopreparation method（Example 1）	Biopreparation method（Example 2）
Gene Synthesis	Synthesize a fusion protein sequence, including a fusion peptide, enterokinase cleavage site, and the target main molecule sequence	Synthesize a tandem expression sequence with the GLP-1(9-37) gene sequence
Engineering Bacteria Construction	Introduce into E. coli to construct recombinant engineered bacteria	Introduce into E. coli to construct recombinant engineered bacteria
Fusion Protein Expression	By optimizing the fusion peptide sequence, changing the isoelectric point and hydrophilicity of the protein, etc., the expression of the fusion protein is effectively increased	After high-density fermentation induction, a tandem expression inclusion body protein expressing GLP-1(9-37) is obtained, and the refolding yields a soluble protein
Intermediate Peptide Acquisition	After the fusion protein undergoes enterokinase cleavage, purification, and other steps, the semaglutide intermediate polypeptide Arg34GLP-1(9-37) can be obtained	The soluble protein can be obtained after undergoing two-step enzyme cleavage with Kex2 enzyme and carboxypeptidase B, and purification steps, to obtain the semaglutide intermediate polypeptide GLP-1(9-37)

 

Enterokinase or Kex2 enzyme combined with carboxypeptidase B is a key enzyme for the main peptide chain of Semaglutide (biopreparation method), and the enzyme cutting effect and cost are crucial for production. Yisheng Biology is committed to providing stable enzyme raw materials for pharmaceutical companies, offering high-quality enzymes and complete quality control standards to help customers improve production capacity and batch stability, reduce costs, and quickly enter the market.

Yeasen overall solution for Semaglutide

1.Recombinant Enterokinase

Recombinant enterokinase (rEK) is a high-purity bovine intestinal enterokinase light chain subunit, which has the same specific cutting enzyme activity as naturally extracted enterokinase, with a cutting site of Asp-Asp-Asp-Asp-Lys, capable of removing the fusion protein located at the N-terminus of the protein to remove unnecessary fusion tags, and is a commonly chosen protease for N-terminal fusion. It has a low level of irregular cutting on other residues.

Yisheng currently offers recombinant enterokinases from different sources, different grades, and whether or not with His tags for customers to choose from.

Yeasen Recombinant Bovine Enterokinase, His, Expressed in Yeast is a high-purity, high-activity, and highly specific bovine enterokinase expressed by Pichia pastoris secretion. It can effectively cleave fusion proteins within a wide pH range (4.5-9.5) and a wide temperature range, and still has partial activity under various detergents and denaturants. This product has a His tag, which can be easily removed through Ni²⁺ affinity column after the cutting reaction, greatly simplifying the subsequent purification process.

Features 

High specificity: A specific protease that cuts at the carboxyl end of lysine containing four aspartic acids: Asp-Asp-Asp-Asp-Lys;

High purity: No other proteases, no non-specific cutting;

No animal origin: Recombinant production, no exogenous viral contamination, no use of any animal-derived raw materials in the production process;

Stable quality: Batch production can ensure stable and continuous batch production; no batch-to-batch differences, stable quality;

Sufficient capacity: 500L fermenter can obtain ~50 MU enterokinase, Yeasen has 5L-1500L different system fermenters to meet different customer requirements at different stages for product batch requirements;

Different grade product supply: R&D grade (20395ES), GMP grade (20396ES), GMP grade products comply with ISO 13485 quality management system.

Test Data

Enterokinase activity test:

Figure 1. The enzyme cutting effect of Yeasen enterokinase (20395ES) is consistent with Competitor N

Note: The positive substrate (20391ES) is a fusion protein composed of two specific protein sequences connected by DDDDK (enterokinase recognition and cutting site), with an overall molecular weight of about 64.6 kDa, which can be specifically cut into two independent protein fragments by enterokinase, with molecular weights of about 27.9 kDa and 36.6 kDa, respectively. This product, as an enterokinase substrate, can be used for semi-quantitative or qualitative enzyme activity detection of recombinant or natural enterokinase.

Enterokinase purity test:

Note: The theoretical value of enterokinase is 22.7 kDa. Due to the glycosylation effect after expression in Pichia pastoris, the SDS-PAGE shows that the molecular weight of the target protein is about 40 kDa. Generally, the glycosylated bands will have a slightly diffuse band running upwards (as shown in the right figure). Our company can also provide deglycosylation enzyme Endo H (Cat#20414) for customers to deglycosylate and run gel to measure purity (as shown in the left figure).

Enterokinase cutting specificity test:

Figure 3. Yeasen enterokinase (20395ES) has less non-specific cutting than import Competitor N

Note: Under the same conditions, Yisheng enterokinase (20395ES) and import competitor N's enterokinase were used for enzyme cutting tests. The results show that after purification, the non-specific cutting impurities produced by Yisheng enterokinase (20395ES) are significantly lower than those of import competitor N.

Enterokinase repeated freeze-thaw, accelerated stability test:

Figure 4. Yeasen enterokinase (20395ES) repeated freeze-thaw, accelerated stability test shows that enzyme activity did not change significantly.

Note: Yeasen enterokinase (20395ES) randomly selected 2 batches, repeated freeze-thaw 10 times, 20 times, enzyme activity did not change significantly from the initial enzyme activity.

Storage at 25°C for 7, 16, 32 days, 37°C for 7, 14 days, enzyme activity did not change significantly from the initial enzyme activity.

2.Recombinant Kex2 Enzyme, Carboxypeptidase B

Table 2.  Yeasen Recombinant Kex2 Enzyme, Carboxypeptidase B Product Selection Guide

Product Number	20418ES	20417ES
Product Name	Recombinant Kex2 Protease, Expressed in Yeast	Recombinant CPB, Expressed in E.coli
Product grade	GMP grade	GMP grade
Source	Recombinant Expression from Pichia pastoris	Recombinant Expression from E. coli
Activity	≥10.0 units/mg pro	≥170 USP units/mg pro
Cleavage Site	Specifically recognizes and cleaves peptide bonds at the carboxylic acid terminal of dibasic amino acids such as Arg-Arg, Lys-Arg	Specifically hydrolyzes proteins at the amino-terminal side of basic amino acids (lysine, arginine, histidine) at the C-terminus
Product Application	1. Process enzyme cleavage in peptide drug production. 2. Enzymatic digestion of proteins and peptide mapping, sequencing, etc.	1. Production of recombinant insulin and its analogs. 2. Determination of amino acids at the C-terminus of proteins. 3. Removal of histidine tags at the C-terminus of proteins.  4. Production of other recombinant peptide substances.  5. Enzymatic synthesis of certain special compounds.

 

Kex2 enzyme combined with carboxypeptidase B: Tandem expression process requirements, high yield of semaglutide intermediate after enzyme cutting

Yeasen GMP Grade Enzyme Production Base

ISO Certified Ultra-Clean Molecular Enzyme Production Base - UCF.ME, equipped with industrial-grade AKTA purification and physicochemical analysis equipment, hundred-liter high-density fermentation units, ten-thousand-grade standard clean workshops, and automated production lines. It possesses fermentation and purification systems of various specifications, including 5L, 10L, 30L, 100L, 500L, and 1,500L, with the capability to scale up production from 5L to 1,500L in the field of molecular enzyme industrialization, fully meeting the core enzyme raw material needs of downstream customers at different stages such as research and development, pilot scale, and large-scale production.

Yeasen has a strong R&D team and advanced enzyme production base, continuously improving the quality and capacity of key enzymes for semaglutide production (recombinant enterokinase) through continuous technological innovation and optimization, offering gradient pricing for different specifications to meet the various needs of customers at different production stages. 

Promotional product information

Product Name	Product Number	Specification	List Price($）	Promotional Price($）
Recombinant Bovine Enterokinase, His, Expressed in Yeast	20395ES60/76/90/ 92/94	100 U/500 U/ 5000 U/ 100 KU/ 1 MU(1000 KU)	45/115/ 825/ 12000/ 360000	45/115/ 585/ 5600/ 19600

 

Related product information

Product Name	Product Number	Specification
Cleavage Control Protein（Enterokinase）	20391ES03/11	1 mg/ 4 mg
Recombinant Kex2 Protease, Expressed in Yeast	20418ES60	100 μg
Recombinant CPB, Expressed in E.coli	20417ES03/10	1 mg/10 mg
Endo H	20414ES92/97	10000 U/50000 U