Synthesized peptides are extensively used in applications such as enzyme design, drug testing, and antibody formation. Many scientists may prefer to outsource creating custom peptides to professional service providers rather than making them themselves. Plenty of factors contribute to the increased demand for large-scale peptide API manufacturing. The development, diversity, and evolution of peptide therapeutics that generate the need for large-scale peptide manufacturing also drive the complexity and heterogeneity of the synthetic approaches, scale-up strategies, and controls that need to be implemented.
Are you wondering how to determine the best peptide synthesis service? Keep reading to find the best tips that can help you select a suitable service.
What are the different approaches to peptide synthesis?
There are two approaches to peptide synthesis: solid-phase synthesis and liquid-phase synthesis. The C-terminus covers the more traditional solid-phase synthesis by attachment to a solid resin, simplifying separating the peptide from the reaction mixture.
Liquid-phase synthesis, also known as synthesis in solution, is more time-consuming and labor-intensive. Still, it benefits several rounds of purification and the possibility of convergent synthesis, in which synthesized peptides form larger ones by adding more. Most custom peptide synthesis services have the following options: addition of modifications (such as biotinylation, glycosylation, cyclization, phosphorylation, methylation, or attachment to carrier proteins or dye labels), quantity, purification, and additional testing.
How to choose the best peptide synthesis service?
Choosing a peptide synthesizer to fulfill your precise requirements can be difficult. Here are some main factors to consider to synthesize high-quality peptides in the quantity and scale needed for your applications:
- Is it better to be manual, semi-automated, or fully automated?
Many companies market peptide synthesizers without specifying the level of automation, but there can be a significant difference between peptide synthesizers. This difference is the most crucial consideration when selecting a peptide synthesizer.
A standard solid-phase peptide synthesis procedure for those who have never produced a peptide before would consist of deprotection cycles, multiple washes, and more multiple washes for each amino acid addition to their peptide. A total of ten washes and deprotection cycles are commonly used to ensure adequate synthesis, characterized by the high purity of the desired peptide being synthesized. Gene synthesis is also one of the popular services that some manufacturers give.
What does having a fully automated peptide synthesizer imply? Most peptide sequences are 20 to 30 amino acids long, and users would like to set up their peptide synthesizer, walk away, and return to a completed peptide. This process ensures that the synthesizer can completely automate the completion of a synthesis cycle for each amino acid and proceed to the following amino acid without any user interference, such as adding more solvents, reagents, or amino acids.
- The Size of the Reaction Vessel.
The reaction vessel size determines the whole synthesis scale that a peptide synthesizer can perform. The synthesis scale determines the amount of peptide material that is generated per synthesis batch. Most peptide synthesizers consider the solvent and amino acid consumption dependent on the reaction vessel size to ensure good unattended automation.
For example, users would not want a reaction vessel sized on a synthesizer that needs solvent addition every 30 minutes. The amounts that one consumer needs at various stages of the study determine the scope of the vessel. The amount of peptide generated in any given reaction vessel is determined by multiple factors such as resin substitution, chemistry, the number of amino acid additions, and expected growth.
- Chemistry Versatility
Most peptide users want to generate their target peptides. An easy-to-use peptide synthesizer is preferable to have many features, which can improve versatility and add complexity. For the advanced peptide chemist, it may be desirable to have a high degree of versatility in performing various types of chemistry to increase synthesis yield or to be able to evolve a method for scale-up. This versatility could include performing double couplings, cappings, Tboc synthesis, DIC coupling, Fmoc synthesis, or HBTU coupling within the same peptide.
There are several comprehensive factors to consider that will drive this versatility, most of which are related to the software interface and the performance of each system function; however, the most critical factor is whether the system has dedicated reagent bottles available. Conducting DIC coupling on one amino acid addition, followed by HBTU coupling on the following amino acid addition in the peptide series, the dedicated reagent bottles must be completely automated.
- Purification Process.
RP-HPLC is by far the most commonly used tool for peptide purification. To get rid of the impurities produced during synthesis, this process occurs. Using a gradient of increasing organic solvent vs. a buffer aqueous solution aids in their removal. Racemized organisms, deletions, insertions, and incompletely omitted to protect classes are examples of these. Many of these impurities will elute very close to or even coelute with the central product peak.
Finally, the purification process must be consistent and eliminate these process-related impurities to appropriate standards that meet “Agency” criteria and current trends to meet ICH 3b requirements. Contaminants from degradation (oxidation, pyroglutamate formation, aspartimide, aggregation species, etc.) must also be eliminated and regulated.
- Initiatives to Go Green.
Due to all of the repetitive washing steps during each period of chain elongation, peptide manufacturing, especially SPPS, generates massive amounts of chemical waste. Furthermore, all reactive side chains of the amino acid shielding groups obtain the desired peptide sequence (very low atom efficiency). The use of RP-HPLC often produces a significant amount of aqueous waste containing MeCN or other organic solvents.
Finally, lyophilization consumes a large amount of energy, which is both financially and environmentally expensive. Solvent recycling in DMF and MeCN synthesis and purification is one step toward addressing the waste aspect.
Recently, the use of green solvents such as 2-MeTHF and cyclopentyl methyl ether for synthesis has been investigated. There have also been reports of RP-HPLC purification methods that use ethanol instead of MeCN.
Another advancement is the substitution of spray-drying or crystallization for lyophilization. The capital cost of implementing green policies affects production costs, but over time will help the industry wisely meet the needs to reduce energetically inefficient processes and accumulate toxic chemicals that future generations will have to contend with.
Over the last five decades, peptide processing services have matured immensely. Improvements in synthesis, purification, and isolation technology continue to drive the economic aspects of the industry’s continued growth. The inclusion of the given five considerations while choosing a peptide synthesis service will benefit the end-user.