In this section, we will discuss the four main peptide synthesis methods. These include:

• • Solid-phase synthesis (SPPS)
  • Liquid-phase peptide synthesis (LPPS)
  • Chemo-enzymatic peptide synthesis (CEPS)
  • Molecular Hiving™

What are the main peptide synthesis methods?

Table 11 highlights the primary peptide synthesis technologies employed by Bachem. Alongside the widely used methods of solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS), also known as solution-phase or classical peptide synthesis, Bachem is leveraging innovative synthesis techniques developed in collaboration with industry partners.

Solid-phase peptide synthesis (SPPS)

How does it work?

In solid-phase peptide synthesis (SPPS), the growing peptide chain is anchored at its C-terminus to an insoluble polymer. This allows the sequential addition of protected amino acids, building the primary structure of the peptide in a C-to-N directional assembly (see Figure 5).

Figure 5: General scheme of Fmoc-SPPS X = O, NH AA = Amino Acid PG = Protecting Group P = Polymer Support.

A typical synthetic cycle includes the following steps:

• Cleavage of the α-amino protecting group.
• Washing to remove residual cleavage reagents.
• Coupling of the next protected amino acid.
• Washing to eliminate excess reactants and byproducts.

Since the growing peptide chain is anchored to an insoluble support, excess reagents and by-products can be efficiently removed through repetitive washing with suitable solvents. For deprotection and coupling steps, only solvents capable of adequately swelling the peptide resin are used. Washing protocols may also include shrinking steps to optimize resin handling.

Once the synthesis is complete, the desired peptide is cleaved from the resin. This cleavage process typically involves the use of acids of varying strength, depending on the specific peptide and protecting groups involved.

The solid support

In SPPS, the solid support plays a crucial role in anchoring the growing peptide chain and enabling efficient reactions.

Polystyrene resin: the standard carrier

Polystyrene, cross-linked with 1% divinylbenzene, remains the most popular carrier resin in SPPS. It is:

• Chemically inert under SPPS conditions.
• Easily derivatised to introduce a variety of anchoring groups.
• Efficiently swelled in solvents suitable for SPPS.

The anchoring group used depends on the synthetic strategy and the desired peptide’s C-terminus. Cross-linked polystyrene beads are typically employed as the resin material. At Bachem, we exclusively use polystyrene cross-linked with 1% divinylbenzene and offer two particle sizes:

• 200–400 mesh = 38–75 Mikrometer
• 100–200 mesh = 75–150 Mikrometer

(Note: The mesh number corresponds to the sieve openings used to separate beads; a higher mesh number indicates smaller particles.)

Swelling in Solvents

While the resin beads are insoluble in most solvents, they swell in specific ones, forming a gel (see Figure 6). Good swelling is essential in dimethylformamide (DMF) and/or N-methylpyrrolidone (NMP), the standard solvents in SPPS. The better the swelling, the more gel-like the beads become, facilitating faster reactions within the resin matrix.

Fmoc vs Boc SPPS

SPPS methods differ based on the temporary Nα protecting group used, leading to two main strategies:

• Fmoc-SPPS: Uses fluorenylmethyloxycarbonyl (Fmoc) protection.
• Boc-SPPS: Uses t-butyloxycarbonyl (Boc) protection.

Switching between these strategies during synthesis is not feasible, as they require different side-chain protecting groups. Due to its milder reaction conditions and superior raw peptide quality, Fmoc-SPPS is the preferred method and will be the focus here.

Common Resins in Fmoc-SPPS

For peptides with a free C-terminus, two resins are commonly used:

1. Wang resin (4-alkoxybenzyl alcohol resin)
2. 2-Chlorotrityl resin (2-chlorotrityl chloride resin)

Loading the C-terminal Fmoc-protected amino acid onto the resin can be challenging. To simplify this step, Bachem offers a wide range of pre-loaded resins, ensuring consistent quality and efficiency for your synthesis needs.

Figure 6: Swollen resin beads under a microscope. The color in the right image indicates unconverted amino groups and thus incomplete coupling.

With these resins, the Fmoc group is removed before coupling the next Fmoc-protected amino acid. The completion of the coupling process is verified using a colour test:

• Positive result: Free amino groups are dyed, indicating incomplete coupling.
• Negative result: No colour change, confirming no free amino groups remain.

The right image in Figure 6 illustrates how incomplete coupling appears under a microscope.

At the end of the synthesis, the peptide is cleaved from the resin using trifluoroacetic acid. Because of this, the side-chain protecting groups must also be compatible with this acid for “global deprotection.” The specific protecting groups used in Fmoc-SPPS are detailed in Table 12.

Table 12: Protecting groups for Fmoc-SPPS

Advantages of SPPS

SPPS stands out for its efficiency and suitability for automation. Key benefits include:

• Rapidity: The stepwise process enables faster synthesis compared to solution-phase methods.
• Automation:
  • Fully automated synthesizers can efficiently manufacture small to medium quantities of peptides (see Figure 7).
  • Semi-automated systems or manual methods are used for producing larger quantities, such as several kilograms of raw peptides.

The stepwise nature of SPPS, where the peptide chain is constructed from the C-terminus to the N-terminus by sequentially coupling amino acids, makes it ideal for automation.

Figure 7: A peptide “synthesizer”, a fully automated machine for SPPS. The left photograph shows reactors in which peptide synthesis is already proceeding.

Challenges with long peptides

As demand for peptides up to 80–100 amino acids increases, SPPS faces several Challenges:

• Impurity risk: With increasing peptide length, the stepwise process can result in impure crude products due to the accumulation of by-products, making purification difficult with standard chromatographic methods.
• Reagent excess: SPPS typically requires large excesses of reagents for each step, which leads to inefficiencies and increases solvent consumption. From a green chemistry perspective, these large volumes of solvents and reagents should ideally be minimized, as they contribute to environmental impact and increased costs.

Native chemical litigation (NCL)

One possible synthetic methodology is native chemical ligation (NCL), which was developed by Stephen Kent and co-workers as a viable alternative to stepwise SPPS for preparing very long peptides. The essential feature of NCL is the chemo-selective coupling of unprotected peptide fragments (e.g., obtained by SPPS), thus simplifying the subsequent purification to the removal of unreacted fragments.

With NCL, the chemical synthesis of even small proteins has become feasible, at least in research quantities (10–20 mg), through a combination of stepwise SPPS and chemical ligation. The synthesis of proteins by this convergent approach is a viable alternative to standard recombinant technologies using microorganisms, offering a plethora of additional options. Another option would be chemo enzymatic peptide synthesis (CEPS), which enables the regio- and stereoselective synthesis of peptides that cannot be efficiently manufactured by stepwise SPPS.

Liquid-Phase Peptide Synthesis (LPPS)

• Advantages:
  • LPPS allows for the intermediate purification of partial sequences, which are then assembled in the final steps.
  • This capability reduces impurity levels compared to SPPS, where purification occurs only at the end of synthesis.

Liquid-phase peptide synthesis (LPPS) 

Liquid-phase peptide synthesis (LPPS) requires more careful planning compared to solid-phase peptide synthesis (SPPS). While Fmoc-SPPS follows a standard synthetic protocol that works for most peptides (though with varying raw peptide quality), no such universal protocol exists for LPPS. This results in a greater diversity of strategies in solution synthesis, particularly in the selection of protecting groups, coupling reagents, solvents, and reaction conditions.

How does LPPS work?

Liquid Phase Peptide Synthesis (LPPS) builds peptides step by step in a liquid solution, but not every combination of protecting groups is compatible. For example, using an α-amino protecting group like Z, Boc, or Fmoc can limit side-chain protecting group options, especially if you aim to remove them all in a single deprotection step at the end.

In SPPS, the combination of Fmoc/tBu is particularly effective and commonly used because these groups can be cleaved selectively and in any sequence. In solution synthesis, a similar combination would be Boc/Bzl or Z/tBu, as detailed in Table 14.

An example of solution synthesis for a peptide modified at the C-terminus (a “peptide substrate”) is shown in Figure 7. This demonstrates the modular nature of LPPS. The same peptide fragment, Boc-Ala-Ala-Pro-OH, can be used to synthesize other peptide substrates, such as Boc-Ala-Ala-Pro-Ala-pNA, or inhibitors like MeO-Suc-Ala-Ala-Pro-Ala-chloromethyl ketone. The modular approach enables the reuse of frequently required fragments, which can be synthesized in bulk and stored for future use, thus saving time. Additionally, all intermediate products in this process can be isolated, characterized, and purified if needed, although this requires extra time.

In the example shown in Figure 8, Boc was used as the temporary Nα protecting group. Boc or Z groups are typically employed for temporary protection in solution synthesis.

Figure 8: Solution synthesis of Suc-Ala-Ala-Pro-Arg-pNA.

Reactive “active esters” of protected amino acids are also often used instead of a separate coupling reagent. Hydroxysuccinimide esters (OSu) are especially popular for solution synthesis (see Figure  9).

Figure 9: Solution synthesis using an active ester.

In solution synthesis, reactive “active esters” of protected amino acids are often used instead of traditional coupling reagents. Hydroxysuccinimide esters (OSu) are especially popular for this purpose, as shown in Figure 8. These active esters facilitate the efficient coupling of amino acids, streamlining the synthesis process.

Advantages of LPPS over SPPS

LPPS offers a key advantage over SPPS by allowing intermediate purification of partial peptide sequences. These fragments can be purified at each stage of synthesis, resulting in lower impurity levels. In contrast, SPPS only allows purification at the end of the process, leading to higher impurity accumulation.

LPPS operates in solution, using soluble supports that simplify workup and clean up after each step. This approach eliminates the need for large excesses of reagents and solvents, which helps reduce waste and minimizes the environmental impact associated with traditional peptide synthesis methods.

Green chemistry benefits

One of LPPS’s main strengths is its alignment with green chemistry principles. Unlike SPPS, which requires large volumes of toxic solvents and reagents, LPPS significantly reduces the use of these materials. It also minimizes the need for condensation agents and reduces isomerization and other byproducts. These improvements contribute to the production of high-purity peptides without relying on extensive chromatography.

Soluble tagging in LPPS

In LPPS, the growing peptide chain is supported on a soluble tag. This tag must have distinct properties from the reagents and byproducts generated during synthesis. These differences allow for easy separation of the tag and peptide from the reaction mixture through simple methods like precipitation, filtration, or extraction.

Cost efficiency and sustainability

Compared to SPPS, LPPS requires significantly less solvent, resulting in lower material and energy consumption. The process also has higher volumetric efficiency and can be automated, making it ideal for large-scale peptide production. By reducing the need for a large labor force and cutting material costs, LPPS offers a more sustainable and cost-effective approach to peptide synthesis.

*Convergent synthesis refers to the preparation of longer peptides by synthesizing and then combining several smaller fragments. This can be faster than a stepwise approach because several fragments can be constructed simultaneously. It is also possible to combine the SPPS of protected fragments with solution coupling (e.g., the synthesis of enfuvirtide).

Chemo-enzymatic peptide synthesis (CEPS)

As therapeutic peptides become increasingly longer and more complex, new methods are needed to produce them efficiently and sustainably. Chemo-enzymatic peptide synthesis (CEPS) is an advanced approach developed in partnership with EnzyTag, designed to synthesise cyclic and long peptides or small proteins that are challenging to produce using traditional recombinant methods. This cutting-edge technique is a greener, more sustainable solution for generating high-quality peptides, addressing limitations in current peptide manufacturing processes.

How does it differ from previous methods of peptide synthesis?

CEPS uses a specialised peptide ligating enzyme, known as a peptiligase, to join shorter peptide fragments into longer sequences. These fragments are produced using Fmoc-based solid-phase peptide synthesis (SPPS), a method capable of creating peptides up to 50–60 amino acids in length. While SPPS struggles with impurity issues for longer sequences, CEPS overcomes this limitation by assembling peptides exceeding 100 amino acids through enzymatic ligation.

The process operates under mild, aqueous conditions with the enzyme active in a near-neutral pH range of 7 to 8.5. The peptiligase enzyme itself is produced via an endotoxin-free biotechnological process using Bacillus subtilis, a microorganism that is generally regarded as safe (GRAS). This combination of enzymatic precision and environmentally conscious methods makes CEPS a state-of-the-art tool in peptide synthesis.

How it works

Peptiligase, a modified form of a serine protease, has been engineered to create peptide bonds instead of breaking them. This transformation was achieved by modifying the enzyme to enhance its ligation capabilities.Additionally, peptiligase has been tailored to recognise multiple amino acid sequences, eliminating the need for specific recognition motifs and making CEPS a traceless ligation method.

The enzyme performs efficiently even in challenging environments, such as in the presence of organic co-solvents or denaturing agents. This capability allows it to ligate hydrophobic or folded peptides effectively.

To synthesise a peptide using CEPS, at least two fragments are required. The first is a synthetic peptide with an oxo- or thioester group at its C-terminus. The process begins with the reaction between this ester and the active site cysteine’s thiol group (figure 10, step 1), which forms a covalent bond, releasing an organic alcohol (step 2). This creates a thioester linkage between the peptide and the enzyme. Next, an amine fragment (either synthetic or recombinant) replaces the thioester bond, forming the final peptide product (steps 3 and 4). The enzyme is then free to catalyse additional cycles.

If further extension is needed, the product of one cycle can serve as the amine fragment for subsequent reactions, provided the N-terminus is first deprotected. For cyclic peptides or cyclotides, the enzyme requires a minimum of 12 amino acids to complete the cyclisation, with six amino acids necessary for recognition within the enzyme’s active site and six for closing the loop.

Peptiligase accommodates a wide range of canonical amino acids in its active site, including hydrophobic ones, and can also incorporate non-canonical amino acids and non-peptide motifs outside of the active site. This flexibility enables the synthesis of diverse and complex peptide structures.

Figure 10: The CEPS process

The benefits of CEPS

• CEPS allows for regio- and stereoselective synthesis of peptides that cannot be efficiently produced through stepwise SPPS.
• By combining CEPS with SPPS, it is possible to create long peptides with more than 40 amino acids and cyclic peptides with over 12 amino acids, achieving high purity.
• Side-chain functionalities do not require protection, as the process eliminates side reactions and racemisation.
• CEPS is a more environmentally friendly approach, significantly reducing the use of organic solvents.
• This method is scalable and suitable for GMP manufacturing.
• It can synthesise a significant portion% of pharmaceutical peptides currently on the market.

Molecular Hiving

Molecular Hiving™ (MH) is an innovative technology pioneered by Professor Kazuhiro Chiba of the Tokyo University of Agriculture and Technology. It is exclusively implemented at scale by Bachem to help address these issues head-on. Combining the benefits of liquid-phase peptide synthesis (LPPS) with efficient, aqueous-based purification, MH presents a sustainable, streamlined alternative to SPPS. This new technology minimizes solvent usage and waste, and it aligns with Bachem’s long-standing commitment to green chemistry and sustainable manufacturing practices in the pharmaceutical industry.

How does molecular hiving work?

Molecular Hiving™ utilizes a unique liquid-phase synthesis method that allows continuous peptide assembly in solution instead of on a solid-phase resin. Tag-based approaches like MH streamline LPPS-based peptide manufacturing by eliminating isolation steps and removing excess reagents and building blocks through aqueous extraction. This process is only required after Fmoc cleavage, simplifying the workflow. The key stages of the MH process include:

• Tag attachment: A soluble, hydrophobic tag is attached to the first amino acid, forming the foundation for the peptide chain.
• Fmoc-based coupling: Fmoc-protected amino acids are sequentially coupled in a liquid-phase environment using standard Fmoc chemistry. This ensures compatibility with existing peptide synthesis workflows.
• Aqueous extraction: After each coupling, water is added to the reaction mixture. The excess reagents and impurities are transferred to the aqueous phase while the peptide-tag complex remains in the organic phase. This eliminates the need for multiple solid-phase filtrations, reducing solvent use and simplifying purification.
• Cleavage and final purification: Once the peptide is fully assembled, it is cleaved from the tag and any protecting groups. The peptide is then ready for final purification, streamlining the last steps of production.

This approach combines the benefits of LPPS with the platform technology of SPPS, offering a more efficient, streamlined peptide synthesis method.

Figure 11: Molecular Hiving™ schematic process

The Molecular Hiving manufacturing process

The Molecular Hiving™ process is designed to enhance peptide synthesis at a production scale by optimizing cycle time, yield, and product quality. The approach focuses on streamlining key steps to reduce solvent use, improve efficiency, and simplify purification. Here’s an overview of the manufacturing process:

• Reactions
  All reactions are performed in solution, including the loading of the tag, coupling of amino acids, and Fmoc cleavages. This eliminates the complexity of solid-phase resin and ensures more efficient peptide synthesis.
  • Unlike SPPS, where diffusion is a factor due to biphasic reactions, MH avoids this issue, leading to comparable reaction times at both lab and production scales. For example, the coupling of a 4-mer peptide achieves full conversion efficiently in both scales.
• Phase separations
  The removal of excess amino acid derivatives, reagents, and additives is critical for efficient peptide synthesis.
  • SPPS typically relies on multiple filtrations, requiring large volumes of organic solvents. Filtration can become a bottleneck at the production scale, requiring increased filter area and additional washing steps.
  • Molecular Hiving™ uses aqueous extractions instead of organic solvents, allowing the removal of undesired material more efficiently. Only 1-2 aqueous extractions are needed to achieve excellent separation. This method reduces solvent consumption and significantly enhances phase separation speed, making it scalable and efficient at production levels.
• Transfers
  Reducing the number of transfers between unit operations is key to minimizing the cycle time and improving efficiency at the production scale.
  • SPPS requires resins to remain in the reactor throughout the entire synthesis, with reactions, cleavages, and wash solvents filtered off after each step.
  • Molecular Hiving™ keeps the tag and tagged peptide solution in the same reactor for the entire synthesis. This eliminates the need for multiple transfers, simplifying the process. The peptide solutions are only filtered after the synthesis is complete.
• Isolation
  The isolation process for the full-length protected peptide is streamlined and highly efficient.
  • The peptide is directly precipitated from the organic phase after reducing the volume through evaporation, avoiding the need for solvent exchange.
  • The addition of a suitable anti-solvent facilitates precipitation, which is followed by fast filtration of the mother liquor and wash liquors.
  • Drying times are minimal, typically less than one day, and the remaining residual solvent does not impact subsequent steps, such as cleavage.
• Downstream Processing

Peptides synthesized via Molecular Hiving™ can undergo tailored downstream processes depending on specific requirements.

• • The peptide can be cleaved from the tag and side-chain protecting groups simultaneously using standard TFA cleavage cocktails, similar to SPPS.
  • Alternative cleavage conditions are also possible, including methods that avoid subsequent salt exchange steps.
  • The cleaved tag can be removed via precipitation and filtration or by extraction with suitable solvents. High-purity peptides can be obtained directly by precipitation or crystallization, eliminating the need for additional purification steps like preparative HPLC.

The advantages of Molecular Hiving (MH)

Scalability and efficiency

MH’s one-pot approach ensures seamless upscaling from small to large reactor sizes, with comparable reaction times observed across lab and production scales. This scalability, combined with faster reaction times and fewer reagents, optimises production cycles and lowers material costs. The process supports multi-kilogram peptide batches, with Bachem’s facilities demonstrating MH’s reliability for large-scale peptide synthesis.

Sustainability and environmental impact

MH significantly reduces solvent use and waste generation, achieving up to a 60% reduction in organic solvent consumption compared to traditional methods. This not only decreases the environmental footprint but also lowers waste disposal costs. By eliminating CMR solvents and generating mostly aqueous waste, MH aligns with strict safety standards and environmental regulations, making it suitable for applications requiring high environmental compliance.

Cost efficiency

The reduction in solvent and reagent consumption lowers material costs and waste, while faster reaction times boost throughput. Case studies show  that MH reduced the Peptide Manufacturing Index (PMI) from 293 to 88 for octreotide synthesis and from 1917 to 416 for liraglutide, highlighting the substantial efficiency gains.

Compatibility

MH integrates smoothly with standard Fmoc-based workflows, making it compatible with existing peptide synthesis systems. This flexibility facilitates broad application across various peptide manufacturing projects.

Quality control

MH’s scalability is complemented by real-time process monitoring, allowing for direct adjustments during production. This ensures high consistency and product quality across batches, making MH a reliable solution for large-scale peptide manufacturing.

Figure 12: Advantages of Molecular Hiving for next-generation large-scale synthesis.