Accelerating Semaglutide Manufacture with Recombinant Enterokinase

In the early 2000s, GLP-1 analogs revolutionized diabetes treatment. Semaglutide, a leading GLP-1 analog, has demonstrated in clinical trials—when paired with oral hypoglycemics—to control blood sugar, support weight loss, lower systolic blood pressure, and enhance β-cell function. Its three products—Ozempic (injectable, diabetes), Rybelsus (oral, diabetes), and Wegovy (injectable, weight loss)—generated over $20 billion in 2023, and demand continues to grow.

Bioproduction of Semaglutide Intermediates

The semaglutide backbone is produced as either a 29-amino-acid intermediate peptide (Arg³⁴-GLP-1(9–37)) or a 27-amino-acid intermediate (Arg³⁴-GLP-1(11–37)). These intermediates can be made by chemical synthesis or bioproduction. Compared with chemical synthesis, bioproduction breaks through capacity limits, is better suited for large-scale continuous manufacturing, and further lowers production costs—leading to greater economic benefits

Table 1. Bioproduction Strategies

Step	Fusion-Protein Strategy	Tandem-Expression Strategy
Gene synthesis	Synthesize a fusion-protein gene that includes: • A fusion tag • An enterokinase cleavage site (DDDDK) • The target peptide sequence	Synthesize a tandem-repeat GLP-1(9-37) gene
Host strain construction	Transform into E. coli	Transform into E. coli
Fusion-protein expression	Optimize fusion tag for pI and hydrophilicity → high-density fermentation → inclusion bodies → refolding	High-density fermentation → inclusion bodies of tandem GLP-1(9-37) → refold to soluble tandem peptide
Intermediate peptide recovery	Enterokinase cleavage + purification → Arg³⁴-GLP-1(9–37)	Kex2 + Carboxypeptidase B cleavage + purification → GLP-1(9–37)

Enterokinase or Kex2 protease, used together with Carboxypeptidase B, serve as the key cleavage enzymes for semaglutide backbone bioproduction. Their efficiency and cost significantly impact overall manufacturing.Yeasen Biotech is committed to providing pharmaceutical companies with reliable enzyme reagents and comprehensive QC standards to help you increase capacity and batch consistency, lower costs, and accelerate market entry.Yeasen Biotech’s Integrated Semaglutide Solutions

Recombinant Enterokinase (rEK) for Precision Cleavage

Recombinant enterokinase (rEK) is a high-purity bovine enterokinase light-chain subunit that matches the specific cleavage activity of native enzyme at the Asp-Asp-Asp-Asp-Lys site. It efficiently removes N-terminal fusion tags with minimal off-target cuts, making it the protease of choice for N-terminal tag removal. Yeasen offers a range of rEK products—varying in expression system, quality grade, and His-tag presence—to fit different production needs.

Table 2. Yeasen Recombinant Enterokinase Product Selection Guide

Cat. No.	20395ES	20396ES	20401ES	20400ES
Product Name	Recombinant Enterokinase, expressed in Pichia pastoris (His-tag)	Recombinant Enterokinase, expressed in Pichia pastoris (His-tag)–GMP	Recombinant Enterokinase, expressed in E. coli (tag-free)	Recombinant Enterokinase, expressed in E. coli (tag-free)–GMP
Grade	R&D	GMP	R&D	GMP
Expression Host	Pichia pastoris	Pichia pastoris	Escherichia coli	Escherichia coli
Activity	5 U/µL	5 U/µL	≥ 5 U/µL	≥ 5 U/µL
Tag	His-tag	His-tag	None	None
Enzyme Removal	Easily removed using a Ni²⁺ affinity column.		Purified and removed using anion exchange resins (e.g., DEAE-FF).
Enzymatic Activity	One unit of activity is defined as the amount of enzyme needed to achieve 95% cleavage of 500 µg fusion substrate in 12–16 h at 25 °C (25 mM Tris-HCl, pH 8.0).

Product Features

• Animal-Free
  fully recombinant, no animal-derived contaminants.
• High Specificity
  Cleaves only at the tetrapeptide Asp–Asp–Asp–Asp–Lys (DDDDK) recognition site.
• High Purity
  Free of contaminating proteases; no non-specific cleavage.
• Stable Quality
  large-scale recombinant workflow ensures lot-to-lot uniformity.
• Ample Production Capacity
  A 500 L fermenter can yield ~50 MU of enterokinase; Yeasen operates fermenters from 5 L to 1,500 L to meet both R&D and commercial demands
• R&D & GMP grades (ISO 13485 compliant)

Demonstrated Performance

1. Enterokinase Activity Assay

This substrate is specifically designed for semi-quantitative or qualitative activity assays of recombinant or natural enterokinase.

2. Purity Assessment of Recombinant Enterokinase

 Figure 2. Yeasen Recombinant Enterokinase (20395ES) Exhibits Purity Greater Than 95%

Note: The theoretical molecular weight of recombinant enterokinase is 22.7 kDa. Due to glycosylation resulting from Pichia pastoris expression, SDS-PAGE analysis shows the apparent molecular weight to be approximately 40 kDa.
Glycosylated proteins typically display slightly smeared upward bands on SDS-PAGE (as seen in the right panel).
Yeasen also offers the deglycosylation enzyme Endo H (Cat#20414), allowing customers to perform deglycosylation prior to electrophoresis for more accurate purity assessment (as seen in the left panel).

3. Enterokinase Cleavage Specificity Test

 Figure 3. Yeasen Recombinant Enterokinase (20395ES) Shows Lower Non-Specific Cleavage Compared to Imported Competitor N

Note: Under identical conditions, recombinant enterokinase from Yeasen (20395ES) and an imported competitor N were used for enzymatic digestion tests.
The results indicate that, after purification, Yeasen’s enterokinase produces significantly fewer non-specific cleavage byproducts compared to the imported competitor.

 4. Freeze-Thaw and Accelerated Stability Testing of Recombinant Enterokinase

 Figure 4. Yeasen Recombinant Enterokinase (20395ES) Maintains Enzymatic Activity After Repeated Freeze-Thaw Cycles and Accelerated Stability Testing

Note: Two random batches of Yeasen recombinant enterokinase (20395ES) were subjected to 10 and 20 freeze-thaw cycles, with no significant change in enzymatic activity observed compared to the initial activity.
Additionally, the enzyme was incubated at 25°C for 7, 16, and 32 days, and at 37°C for 7 and 14 days, with no noticeable loss of enzymatic activity throughout the testing period.

 

Recombinant Kex2 Protease and Carboxypeptidase B

Table 4. Yeasen Recombinant Kex2 Protease & Carboxypeptidase B Product Selection Guide

Cat. No.	20418ES	20417ES
Product	Recombinant Kex2 Protease, expressed in yeast	Recombinant Carboxypeptidase B (CPB), expressed in E. coli
Grade	GMP	GMP
Expression Host	Pichia pastoris	Escherichia coli
Activity	≥ 10.0 units/mg protein	≥ 170 USP units/mg protein
Cleavage Specificity	Recognizes and cleaves C-terminal of paired basic residues (e.g. Arg–Arg, Lys–Arg)	Specifically hydrolyzes C-terminal basic amino acids (Lys, Arg, His)
Applications	1. Process-scale peptide drug de-tagging and maturation 2. Protein digestion for peptide mapping and sequencing	1. Production of recombinant insulin and analogs 2. C-terminal amino acid analysis of proteins 3. Removal of C-terminal His-tags 4. Processing of other recombinant peptide therapeutics 5. Enzymatic synthesis of specialty compounds

Using Kex2 protease in tandem with Carboxypeptidase B meets the demands of tandem-expression workflows, delivering high yields of Semaglutide intermediate peptides through efficient sequential cleavage.

 

Advanced GMP Enzyme Manufacturing Base

Yeasen’s ISO-certified Ultra-Clean Molecular Enzyme Production Facility (UCF.ME) is equipped with industrial-grade AKTA purification systems, analytical instrumentation, high-density fermentation units (100 L class), ISO Class 8 cleanrooms, and fully automated production lines. The facility supports scale-up fermentation and purification from 5 L to 1,500 L, enabling seamless transition from R&D to pilot-scale and large-scale commercial manufacturing. It is designed to meet diverse enzyme raw material needs across all stages of biopharmaceutical development.

  

Yeasen Biotech: Your Reliable Enzyme Partner for Semaglutide Production

With a strong R&D team and state-of-the-art enzyme manufacturing facilities, Yeasen continuously improves the quality and yield of key enzymes such as recombinant enterokinase through ongoing innovation and process optimization. We offer flexible, tiered pricing across multiple specifications to meet the diverse needs of customers at different stages of semaglutide production.

promotion product information

Product Name	Cat No.	Specification
Recombinant Enterokinase, His, Expressed in Yeast	20395ES60/76/90/92/94	100 U/500 U/ 5000 U/ 100 KU/ 1 MU(1000 KU)

Related Product information

Product Name	Cat No.	Specification
Enterokinase,Recombinant,Expressed in E.coli	20401ES60/76/90	100 U/500 U /5000 U
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	20414ES92/97	10000 U/ 50000 U