Peptide synthesis and production: From discovery to manufacturing
Peptide synthesis is becoming increasingy important in pharmaceutical research and development, due to the growing popularity of peptide therapeutics.
Peptides’ high specificity and potency make them extremely useful in targeted therapies and personalised medicine.
Today, there are more than 200 peptide therapeutics in clinical development , and the estimated value of the global peptide therapeutics market is $117.26 billion.
But what does it take to successfully research, synthesise, and manufacture these valuable therapeutics?
Keep reading to learn more about how biotechnology and pharmaceutical organisations can maximise success at every stage of peptide production, from peptide discovery and synthesis to purification and manufacturing.
Initiating peptide production: Identifying the best candidates
The journey of peptide production begins with peptide discovery.
Much like for other therapeutics, scientists begin by identifying receptors or pathways associated with a particular disease.
The discovery process involves high-throughput screening and computational modelling to identify and select the most promising candidates.
Many peptides are naturally occurring, for example, insulin is derived from the pancreas, and carfilzomib, a tetrapeptide epoxyketone used in treating multiple myeloma, is derived from a product isolated from Actinomycetes bacteria.
In addition to their natural occurrence, advancements in peptide discovery, including machine learning and high-throughput screening technologies, continue to aid scientists in accelerating the identification of novel peptide candidates.
Once a promising candidate is identified, the peptide undergoes optimisation to improve stability, binding affinity, and pharmacokinetic properties, overcoming limitations like weak membrane permeability and poor in vivo stability. Common optimisation techniques include the incorporation of non-natural amino acids and peptide cyclisation, both of which can improve stability and biological activity.
Methods for peptide synthesis
Two major techniques have emerged for peptide synthesis: solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS).
Over time, SPPS has become more common because it reduces the number of peptide synthesis steps for longer peptide chains.
In solid-phase peptide synthesis, amino acids are sequentially added to a chain on a solid resin support.
This method allows for high purity and yield for more complex peptides relative to LPPS, and automated and microwave-assisted SPPS has enabled even greater efficiency and control in the synthesis of longer peptide chains.
- Resin selection and attachment: The resin serves as the solid support on which the peptide chain is built.
- Deprotection: The protecting group is removed to expose the amine group.
- Activation and coupling: The next amino acid in the chain is activated and coupled to the free amine, and so on.
- Cleavage from solid support: The peptide is cleaved from the solid resin once the sequence is complete.
- Final purification: The peptide product is purified to remove unwanted byproducts or reagents.
Liquid-phase peptide synthesis, on the other hand, is useful for synthesising simple peptide sequences with high levels of precision.
However, it requires intermediates to be purified at each peptide synthesis step, making it more time-consuming and less feasible for longer peptide chains.
- Functional group protection: The N-terminal and side chains are first protected to avoid unwanted side reactions.
- Activation and coupling: The carboxyl group of one amino acid is activated, then coupled to the free amine of the other.
- Intermediate purification: The product is purified after each coupling step.
- Deprotection: The protecting group is removed before the next coupling step, then coupling and deprotection are repeated to form the sequence.
- Final purification: The peptide product is purified to remove unwanted byproducts or reagents.
Today, strategies that combine the strengths of SPPS and LPPS have also emerged.
Scientists can use SPPS in early steps where purity is most critical, then turn to LPPS as the chain length increases and SPPS becomes less efficient.
This hybrid approach can be useful in creating high-qualtiy, large peptide chains while containing costs.
Peptide purification: Supporting high purity and quality
Following synthesis, whether LPPS or SPPS is used, peptides must be purified to remove unwanted byproducts or unreacted reagents.
High-performance liquid chromatography (HPLC) is considered the gold standard in peptide purification due to its precision and effectiveness.
HPLC separates peptides based on hydrophobicity and polarity.
Reverse-phase chromatography involves hydrophobic interactions between the peptide and stationary phase, usually a silica-based grafted with C-4 or C-18 carbon chains.
After purification through chromatography, lyophilisation is utilised to remove excess water and stabilise the purified peptide for storage.
Though less common, crystallisation may also be used for peptides that are less complex and have been synthesised using LPPS.
Maximising success in peptide production
When transitioning peptide production from the lab scale to the commercial scale, there are several important considerations for scientists and engineers.
Early on, scientists must ensure that the process is optimised to maximise quality and yield both at the small scale and at the commercial scale, with the necessary equipment and controls in place.
The availability of large-scale synthesis, chromatography and lyophilisation equipment for peptide purification at scale is also essential.
Finally, ongoing monitoring involving tests during peptide production is imperative for quality control, so it is critical to ensure that robust analytical methods have been developed from the start.
Strong analytical development is critical in supporting chemistry development from the very start of the development program, more so than in small molecule development, due to the greater number of potential impurities that could form during a peptide synthesis and the greater challenge, both chemistry and engineering, of purification post synthesis.
Early identification of related impurities using advanced MS techniques is required to inform chemistry of degradation or synthetic pathways producing impurities that would carry through the process, incorrectly coupling, which if not adequately controlled, would reduce both yield and increase the demand on the purification steps in the process.
Alongside impurity identification, strong separation methods are required (HPLC/UPLC) to give not only analytical resolution for quantification of impurities, but also form the basis for the conditions of the large scale chromatographic purifications.
These separations become more challenging with increasing peptide chain length, as the relative structural differences between the target and related compounds decrease.
Release testing is more complex than for small molecule release, as techniques complementary to assay and purity by HPLC/UPLC are required to confirm product quality.
These can include confirmation of peptide sequence by LC-MS/MS, amino acid analysis by GC-MS / GC-FID, counter ion by IC, and other physical tests
After peptide manufacturing: Storage and transportation
Peptides come with stringent storage and handling requirements to maintain quality and prevent degradation.
Generally, peptides are stored at -20°C to -80°C, requiring cold chain logistics to remain within ideal temperature ranges during transportation.
Lyophilisation, or freeze drying, is necessary to stabilise peptides for long-term storage and transportation.
Lyophilised products tend to be more stable and require less stringent temperature controls than liquid formulations, supporting greater reliability throughout the supply chain.
Sterling: Supporting peptide production from discovery to manufacture
As peptide synthesis and manufacturing remains a key area of innovation in the biopharmaceutical industry, working with an experienced partner who can support every stage of peptide development is integral to capitalising on the immense opportunity these therapeutics represent.
At Sterling, we deliver comprehensive peptide services to supply all global markets.
Our early phase peptide discovery and development capabilities span robust analytical method development, product characterisation, stability studies, and chemical process development.
We also provide more than 25 years of expertise in small and large-scale peptide manufacturing and purification.
Our equipment includes one of the largest HPLC columns in the world at 1.6 metres in diameter, which is capable of purifying peptides at scale.
If you’re interested in learning more about how we can support your peptide synthesis and manufacturing programme, speak to an expert or check out our whitepaper, The power of peptide therapeutics.
You can also explore frequently asked questions on peptide production and synthesis here.
