How Is Semaglutide Made?

Overview of Pharmaceutical Peptide Manufacturing

Semaglutide is manufactured using solid-phase peptide synthesis (SPPS), a sophisticated chemical process that builds the peptide chain one amino acid at a time. This technology, developed in the 1960s by Bruce Merrifield (who won the Nobel Prize in Chemistry for this work in 1984), revolutionized peptide production and made therapeutic peptides like semaglutide commercially viable. The manufacturing process is complex, highly regulated, and requires specialized facilities, equipment, and expertise to ensure consistent quality, purity, and potency.

The production of pharmaceutical-grade semaglutide involves multiple stages: synthesis of the peptide backbone, attachment of the fatty acid side chain, purification to remove impurities and byproducts, formulation into the final drug product, and rigorous quality control testing. Each stage must meet stringent specifications established by regulatory agencies like the FDA and EMA. The entire process, from raw materials to finished product, can take several months and involves hundreds of individual steps and quality checks.

Solid-Phase Peptide Synthesis: Building the Backbone

The synthesis of semaglutide begins with solid-phase peptide synthesis, a method where the peptide is assembled while attached to an insoluble polymer support (the "solid phase"). This approach offers several advantages over solution-phase synthesis: reactions can be driven to completion using excess reagents, intermediate products don't need to be isolated and purified, and the process can be automated.

The Resin and First Amino Acid

Synthesis begins with a solid support, typically a polystyrene resin with reactive groups on its surface. The first amino acid (the C-terminal amino acid of the final peptide) is covalently attached to this resin through its carboxyl group. For semaglutide, this would be arginine at position 37 (position 34 in the active peptide, counting from the N-terminus of GLP-1(7-37)). The amino acid is protected at its amino group to prevent unwanted reactions—typically with a Fmoc (9-fluorenylmethoxycarbonyl) protecting group in modern synthesis.

Iterative Coupling Cycles

The peptide is then built through repeated cycles of deprotection and coupling. In each cycle, the protecting group on the amino terminus of the growing peptide is removed (deprotection), exposing the free amino group. The next amino acid (protected at its amino group but activated at its carboxyl group) is then added and coupled to the free amino group, forming a peptide bond. This process is repeated 30 times to build the complete 31-amino acid semaglutide backbone.

Each coupling reaction must be highly efficient (>99.5% yield) because even small inefficiencies compound over 30 cycles. If each coupling were only 95% efficient, the final yield would be less than 20%. Modern coupling reagents and conditions achieve >99.9% efficiency, but even so, synthesis of long peptides like semaglutide is challenging. The coupling reagents (such as HBTU, HATU, or DIC/HOBt) activate the carboxyl group of the incoming amino acid, making it reactive toward the amino group of the growing peptide.

Special Considerations for Semaglutide

Semaglutide synthesis has several special considerations. First, the aminoisobutyric acid (AIB) at position 8 is a non-proteinogenic amino acid not found in natural proteins, requiring specialized synthesis or procurement. Second, the lysine at position 26 (where the fatty acid will be attached) must be protected with a different protecting group than other lysines in the sequence, allowing selective deprotection and fatty acid attachment later. Third, the peptide contains multiple potentially reactive side chains (lysines, glutamic acids, aspartic acids, serines, threonines) that must be protected during synthesis to prevent unwanted reactions.

Monitoring Synthesis Progress

Throughout synthesis, the process is monitored using various analytical techniques. UV spectroscopy can detect the Fmoc group released during each deprotection step, confirming that deprotection was successful. Mass spectrometry can analyze small samples cleaved from the resin to verify that the correct amino acids have been incorporated. If problems are detected, the synthesis can be repeated from the problematic step or abandoned and restarted.

Fatty Acid Attachment: Creating the Albumin-Binding Moiety

After the peptide backbone is complete, the fatty acid side chain must be attached to create the albumin-binding moiety that gives semaglutide its extended half-life. This is a critical step that distinguishes semaglutide from native GLP-1.

Selective Deprotection

The lysine at position 26 was protected during synthesis with a protecting group that can be selectively removed without affecting other protecting groups or cleaving the peptide from the resin. This selective deprotection exposes the epsilon-amino group of this specific lysine while leaving all other reactive groups protected. This selectivity is crucial—if the fatty acid attached to the wrong position, the resulting molecule would have different properties and would not be semaglutide.

Spacer and Fatty Acid Coupling

The fatty acid (stearic acid, an 18-carbon saturated fatty acid) is not attached directly to the lysine but through a spacer molecule—specifically, two gamma-glutamic acid residues. This spacer is important for optimal albumin binding and receptor activation. The spacer is built onto the lysine using the same coupling chemistry as peptide bond formation, then the fatty acid is coupled to the end of the spacer.

The fatty acid coupling is more challenging than amino acid coupling because fatty acids are hydrophobic and less reactive. Special coupling conditions and reagents are required to achieve efficient coupling. The reaction must be carefully controlled to ensure complete coupling without causing side reactions or peptide degradation.

Verification

After fatty acid attachment, analytical techniques verify that the modification was successful. Mass spectrometry confirms the correct molecular weight (the fatty acid and spacer add approximately 600 Da to the peptide mass). HPLC (high-performance liquid chromatography) confirms that the product has the expected retention time, which changes significantly with fatty acid attachment due to increased hydrophobicity.

Cleavage and Deprotection

Once the complete semaglutide molecule (peptide backbone plus fatty acid modification) is assembled on the resin, it must be cleaved from the solid support and all protecting groups must be removed to yield the final peptide.

Cleavage Cocktail

Cleavage is typically performed using a strong acid cocktail, commonly trifluoroacetic acid (TFA) with scavengers like water, triisopropylsilane, and ethanedithiol. This cocktail simultaneously cleaves the peptide from the resin and removes most protecting groups from amino acid side chains. The reaction is performed for several hours at room temperature, then the peptide is precipitated by adding cold ether, which causes the peptide to come out of solution while leaving most impurities dissolved.

Crude Peptide

The product at this stage is "crude peptide"—it contains the desired semaglutide but also numerous impurities including deletion sequences (peptides missing one or more amino acids due to incomplete coupling), truncation sequences (peptides where synthesis was terminated early), and various side products from incomplete deprotection or side reactions. The crude peptide typically has purity of 50-70%, meaning that only about half to two-thirds of the material is the correct product. Extensive purification is required to achieve pharmaceutical-grade purity.

Purification: Achieving Pharmaceutical-Grade Purity

Purification is one of the most critical and challenging steps in semaglutide manufacturing. Pharmaceutical-grade peptides must be >95% pure, with individual impurities below specified limits. Achieving this from crude peptide requires sophisticated chromatographic techniques.

Reversed-Phase HPLC

The primary purification method is preparative reversed-phase high-performance liquid chromatography (RP-HPLC). This technique separates molecules based on their hydrophobicity. The crude peptide is dissolved and injected onto a column packed with hydrophobic stationary phase (typically C18-modified silica). A gradient of increasing organic solvent (usually acetonitrile) is applied, causing peptides to elute from the column at different times based on their hydrophobicity.

Semaglutide, with its fatty acid modification, is quite hydrophobic and elutes relatively late in the gradient. Impurities (deletion sequences, truncation sequences, peptides with incorrect modifications) have slightly different hydrophobicity and elute at different times. By carefully collecting only the fractions containing pure semaglutide, high purity can be achieved. However, because many impurities have very similar hydrophobicity to semaglutide, the separation is challenging and may require multiple purification passes.

Ion Exchange Chromatography

Some manufacturers use ion exchange chromatography as a complementary purification technique. This method separates molecules based on charge rather than hydrophobicity, providing orthogonal selectivity that can remove impurities not separated by RP-HPLC. Semaglutide has multiple charged amino acids (glutamic acids, aspartic acids, lysines, arginines) that interact with charged stationary phases, allowing separation from impurities with different charge distributions.

Lyophilization

After purification, the semaglutide is typically in aqueous solution with organic solvents and buffers. To create a stable solid product, the solution is lyophilized (freeze-dried). The solution is frozen, then placed under vacuum, causing the ice to sublimate directly from solid to vapor, leaving behind dry peptide powder. Lyophilization must be carefully controlled to prevent peptide degradation or aggregation. Excipients (additional ingredients like sugars or amino acids) may be added before lyophilization to protect the peptide during drying and storage.

Quality Control and Analytical Testing

Pharmaceutical-grade semaglutide must undergo extensive quality control testing to ensure it meets all specifications for identity, purity, potency, and safety. These tests are performed on both the active pharmaceutical ingredient (API) and the final drug product.

Identity Testing

Multiple techniques confirm that the product is indeed semaglutide with the correct structure. Mass spectrometry determines the exact molecular weight, which must match the theoretical weight of semaglutide. Amino acid analysis determines the amino acid composition, confirming the correct amino acids are present in the correct ratios. Peptide mapping (enzymatic digestion followed by HPLC-MS analysis of fragments) confirms the correct sequence and modifications.

Purity Testing

HPLC with UV detection quantifies the purity, typically requiring >95% purity with individual impurities below 1%. The identity of major impurities must be characterized—are they deletion sequences, oxidation products, aggregates? Mass spectrometry helps identify impurities. Some impurities may have biological activity (if they can still bind GLP-1 receptors), while others are inactive. Regulatory agencies require detailed characterization of impurities above certain thresholds.

Potency Testing

Biological potency assays confirm that semaglutide can activate GLP-1 receptors. Cell-based assays measure cAMP production in cells expressing GLP-1 receptors after semaglutide treatment. The potency must fall within specified ranges relative to a reference standard. This ensures that the semaglutide not only has the correct chemical structure but also the correct biological activity.

Safety Testing

Multiple safety tests are performed. Endotoxin testing (using the Limulus Amebocyte Lysate test) ensures bacterial endotoxins are below safe limits. Sterility testing confirms absence of viable microorganisms. Residual solvent testing quantifies any remaining organic solvents from synthesis and purification, which must be below ICH limits. Heavy metal testing ensures toxic metals are absent. These tests protect patients from contamination that could cause adverse effects.

Stability Testing

Stability studies determine how long semaglutide remains potent and pure under various storage conditions. Real-time stability studies store the product at recommended conditions (typically 2-8°C for semaglutide) and test it periodically over months to years. Accelerated stability studies use elevated temperatures to predict long-term stability more quickly. These studies establish the product's shelf life and storage requirements.

Formulation and Drug Product Manufacturing

The purified semaglutide API must be formulated into the final drug product—the pre-filled pens or vials that patients use. Formulation is crucial for stability, ease of use, and patient safety.

Formulation Components

Semaglutide injection contains several components beyond the active ingredient. The formulation includes buffers (to maintain pH), tonicity agents (to match the osmolality of body fluids), and preservatives (for multi-dose formulations). For Ozempic and Wegovy, the formulation includes disodium phosphate dihydrate, propylene glycol, phenol, and water for injection. Each component serves a specific purpose and must be pharmaceutical grade.

The pH is carefully controlled (around pH 7.4) to maximize stability and minimize injection site reactions. The formulation is isotonic (similar osmolality to blood) to prevent pain or tissue damage at the injection site. For multi-dose pens, preservatives prevent microbial growth after the pen is opened and used multiple times.

Filling and Packaging

The formulated solution is filled into pre-filled pens or vials under aseptic conditions to maintain sterility. Automated filling equipment precisely dispenses the correct volume into each container. The containers are sealed, labeled, and packaged. For pre-filled pens, the pen device (a sophisticated mechanical device that allows patients to select and inject precise doses) is assembled with the drug-filled cartridge.

Final Product Testing

The finished drug product undergoes additional testing. Dose accuracy is verified—does each pen deliver the labeled dose? Sterility is confirmed. Particulate matter is assessed (the solution should be clear with no visible particles). Functionality testing ensures the pen device works correctly. Only after passing all tests is the product released for distribution.

Manufacturing Scale and Capacity

Manufacturing semaglutide at commercial scale is a massive undertaking. Novo Nordisk has invested billions of dollars in manufacturing facilities to meet global demand. The company operates multiple manufacturing sites in Denmark, France, and the United States, with additional capacity under construction.

Production Volumes

The exact production volumes are proprietary, but given that Novo Nordisk supplies semaglutide to millions of patients globally, production must be measured in kilograms to tons of API annually. Each patient using Wegovy 2.4 mg weekly requires approximately 125 mg of semaglutide per year. With millions of patients, total demand is enormous. The supply shortages that have plagued semaglutide since 2021 reflect the challenge of scaling production to meet unprecedented demand.

Manufacturing Timeline

From starting synthesis to finished product ready for distribution takes several months. Synthesis and purification might take 4-8 weeks. Quality control testing takes several weeks. Formulation, filling, and final product testing add more time. Stability testing is ongoing. This long timeline means that manufacturers must forecast demand months in advance and maintain substantial inventory to ensure continuous supply.

Cost of Goods

While exact costs are proprietary, peptide synthesis is expensive. The raw materials (protected amino acids, coupling reagents, solvents) are costly. The specialized equipment and facilities require substantial capital investment. The highly trained personnel needed to operate the equipment and perform quality control are expensive. The extensive testing and quality control add significant cost. Estimates suggest the cost of goods for semaglutide might be $50-100 per patient per month, though this is speculative. The high retail price ($1,000+ per month) reflects not just manufacturing costs but also research and development costs, regulatory costs, and profit margins.

Regulatory Oversight

Semaglutide manufacturing is subject to extensive regulatory oversight to ensure product quality and patient safety.

Good Manufacturing Practices (GMP)

All manufacturing must comply with current Good Manufacturing Practices (cGMP) as defined by regulatory agencies like the FDA and EMA. GMP regulations cover every aspect of manufacturing: facility design and maintenance, equipment qualification and calibration, personnel training, raw material testing, manufacturing procedures, quality control testing, documentation, and more. Compliance with GMP is not optional—it's a legal requirement for pharmaceutical manufacturing.

Regulatory Inspections

Regulatory agencies conduct periodic inspections of manufacturing facilities to verify GMP compliance. Inspectors review documentation, observe manufacturing operations, interview personnel, and test samples. Any deficiencies must be corrected, and serious violations can result in warning letters, production shutdowns, or product recalls. Novo Nordisk's facilities are regularly inspected and must maintain compliance to continue manufacturing semaglutide.

Batch Release

Each batch of semaglutide must be reviewed and approved by qualified personnel before release for distribution. This review verifies that all manufacturing steps were performed correctly, all specifications were met, and all documentation is complete and accurate. Only after this review and approval can the batch be shipped to pharmacies and patients.

Compounded Semaglutide: A Different Manufacturing Paradigm

The supply shortages of brand-name semaglutide have led to widespread use of compounded versions produced by compounding pharmacies. These products are manufactured very differently from FDA-approved semaglutide and raise important quality concerns.

Compounding vs. Manufacturing

Compounding pharmacies are not required to follow the same GMP standards as pharmaceutical manufacturers. They operate under USP (United States Pharmacopeia) compounding standards, which are less stringent. Compounding pharmacies typically purchase semaglutide API from suppliers (often overseas) and formulate it into injectable solutions. They are not required to perform the extensive testing that pharmaceutical manufacturers must perform.

Quality Concerns

Compounded semaglutide may vary in purity, potency, and sterility. The API may contain impurities or degradation products. The formulation may not be optimized for stability. Sterility cannot be guaranteed without proper testing. Dose accuracy may vary. These quality issues have led to reports of adverse events from compounded semaglutide, including infections from contaminated products and unexpected side effects from impurities.

Regulatory Status

During FDA-declared drug shortages, compounding pharmacies are legally permitted to compound semaglutide. However, compounded products are not FDA-approved and do not have the same regulatory oversight as brand-name products. The FDA has issued warnings about compounded semaglutide, urging patients to use FDA-approved products when available and to ensure any compounded product comes from a reputable pharmacy that follows proper standards.

Patient Considerations

Patients considering compounded semaglutide should understand the risks. While compounded products may be less expensive and more accessible during shortages, they lack the quality assurance of FDA-approved products. If using compounded semaglutide, patients should ensure the pharmacy is licensed, follows USP standards, provides certificates of analysis from independent laboratories, and uses pharmaceutical-grade ingredients. Even with these precautions, compounded products carry inherent risks not present with FDA-approved formulations.

Future Manufacturing Developments

As demand for semaglutide and related peptides continues to grow, manufacturing technology is evolving to improve efficiency, reduce costs, and increase capacity.

Continuous Manufacturing

Traditional peptide synthesis is a batch process—each batch is synthesized, purified, and tested separately. Continuous manufacturing, where synthesis and purification occur in a continuous flow rather than discrete batches, could improve efficiency and reduce costs. Several companies are developing continuous peptide synthesis technologies, though these are not yet widely adopted for commercial production.

Improved Synthesis Methods

New coupling reagents, protecting groups, and synthesis strategies continue to be developed, potentially improving yields, reducing synthesis time, and minimizing impurities. Microwave-assisted synthesis can accelerate coupling reactions. Flow chemistry can improve reaction control and scalability. These advances may make peptide synthesis more efficient and economical.

Alternative Production Methods

While semaglutide is currently produced by chemical synthesis, recombinant DNA technology (expressing the peptide in bacteria or yeast) is used for some therapeutic peptides. However, semaglutide's non-natural amino acid (AIB) and fatty acid modification make recombinant production challenging. Hybrid approaches combining recombinant production of the peptide backbone with chemical attachment of modifications are being explored.

Biosimilars

As semaglutide patents expire (expected in the 2030s), biosimilar versions will likely be developed. Biosimilars are highly similar versions of biologic drugs that must demonstrate comparable quality, safety, and efficacy to the reference product. Biosimilar semaglutide could increase competition and reduce costs, improving access for patients. However, developing biosimilars for complex peptides like semaglutide is challenging and requires substantial investment in manufacturing and clinical testing.