Recombinant vs synthetic peptide synthesis: reaching efficiency through hybridization

The demand for the manufacturing of polypeptides has radically increased since the recent introduction of new diabetes, obesity and cardiovascular disease peptide drugs. Consequently, peptide API manufacturing has seen a radical industrial intensification in line with increased commercialization to drive efficiency, improve quality and lower cost.

To achieve this at a commercial scale, peptide APIs can be produced through two principle processes: synthetic or recombinant.

Both methodologies offer unique advantages, with their own respective drawbacks. Thus, it’s vital to ensure that stakeholders are fully aware of the limits and strengths of each approach, in order to make a balanced and informed decision when selecting the most appropriate production method.

What is recombinant peptide synthesis?

As a biotechnological process, recombinant peptide synthesis uses genetically engineered organisms such as yeast, bacteria or mammalian cells, to produce peptides.

In recombinant peptide synthesis, the DNA sequence encoding the desired peptide is inserted into the genetic material of a host organism. Typically incorporated into a plasmid or other vector, this modified DNA instructs the host organism’s cellular machinery to produce the peptide as it grows and divides.

During this method of synthesis,  the host organism translates the recombinant DNA into the peptide chain, often with the capability to perform post-translational modifications. These are chemical changes made to the peptide after it has been synthesised, including phosphorylation, glycosylation, and the formation of disulfide bridges. These modifications are intrinsic to the functionality of many biologically active peptides. The peptides produced are subsequently harvested from the culture medium or cell mass and purified to remove any biological contaminants and unwanted byproducts. 

Advantages

1. Less costly for large-scale production

Recombinant synthesis is more cost-effective at scale than synthetic, due to the microbial or cell culture media required being cheaper in comparison to synthetic amino acids. This makes it appropriate for the production of large volumes of peptides in industrial processes.

1. Facilitates 3D protein folding and post-translational modifications

Recombinant technology has specific benefits in its ability to produce peptides with complex tertiary structures and specific post-translational modifications, which can be necessary for biological activity and drug efficacy.

1. Reduced waste footprint and greater sustainability

Utilising biological systems can mitigate chemical waste and energy consumption typical in synthetic processes, thus promoting green manufacturing principles and reducing environmental impact. However, an extensive purification process might reduce overall benefits.

1. Long-chain peptide production capabilities

In comparison to synthetic methods, such as solid-phase peptide synthesis (SPPS), the use of biological organisms and their ribosomal machinery makes recombinant polypeptide synthesis extremely efficient, even at chain lengths of hundreds of amino acids.

Challenges

1. Complex purification requirements and impurity profiles

The presence of host cell proteins, nucleic acids, and other impurities requires robust downstream processing, which is both costly and technically challenging, resulting in reduced overall process efficiency.

1. Risk of immunogenicity and biological contamination

The introduction of novel peptide sequences via recombinant synthesis may result in increased risk of eliciting an immune response in patients, which could inhibit the clinical utility of the produced peptides.

1. Lengthy development cycles and up-front costs

The creation and optimization of genetic constructs, along with the scale-up of fermentation processes, require prolonged time input, which can result in the initial production phase being delayed.

1. Limited ability to accommodate modifications

Chemical modifications including backbone stabilizations, artificial amino acids or unusual side chains are rarely able to be accommodated, due to their complexity.

What is synthetic peptide synthesis?

The construction of peptides without the use of biological organisms is referred to as synthetic peptide synthesis. This involves sequentially linking amino acids, forming a peptide chain through a series of chemical reactions, including techniques such as solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS).

In SPPS, the peptide is assembled on a solid support – usually a resin – which leverages the automation and purification of the synthesis process. 

Each amino acid is added sequentially to the growing peptide chain, with cycles of coupling (adding the amino acid) and deprotection (removing protective groups such as Fmoc or Boc) taking place until the desired sequence is complete. This method includes the incorporation of non-natural amino acids and various modifications, allowing for precise control over the peptide’s sequence and composition.

Advantages

1. Precision, customization, and chemical modifications

Synthetic peptide production facilitates the incorporation of various synthetic elements, such as non-proteinogenic amino acids and a range of biochemical or biophysical probes. This method is particularly suitable for incorporating post-translational modifications that increase peptide functionality, stability, or bioavailability.

1. Automation, scalability, and speed

One of the benefits of Automated Solid-Phase Peptide Synthesis (SPPS) is the quick turnaround in peptide production. The scalability of this particular technique is beneficial for keeping up with the rapid pace of demand in pharmaceutical applications. 

1. Elimination of risk of biological contaminants and immunogenic triggers

In comparison to recombinant methods, synthetic synthesis does not require the involvement of host organisms, thus bypassing the risk of contamination with harmful biological agents, including viruses or prions. This streamlines the regulatory compliance process, particularly concerning BSE and TSE safety. 

1. High-quality peptides with known impurities

Synthetic synthesis promotes a high degree of control over the peptide’s composition and purity, reducing variability between batches.

Challenges

1. Complex starting materials and expensive material costs

The starting materials for synthetic synthesis are costly. Building blocks such as synthetic amino acids with protective groups are, on top of specialised resins, reagents, and instruments.

1. Scale-up and process optimization complexities

Increasing scale can result in variations in mixing efficiency, temperature control, and the timing of reactions, which may affect the yield and purity of the final product. Such scale-up challenges require significant process development and optimization, which can increase time to market and cost, and often require iterative testing and modifications for the process to become commercially viable. 

1. High process mass intensity and organic solvent waste

Deprotection, repetitive coupling, and washing steps use up resources and create a significant amount of solvent waste. Synthesis and purification have the most notable impact on process-mass-intensity – which acts as a  metric for sustainability. Attempts to mitigate these impacts need advanced waste management and solvent recovery systems, which can increase operational complexity and cost even further.

1. Error accumulation is prohibitive for longer peptides

The process of synthesising peptides longer than 50 amino acids presents numerous barriers, including lower reaction yields and a higher chance of sequence errors or misfolding. These issues arise from the gradual accumulation of minor errors across each synthesis step, which are exacerbated as the peptide lengthens.

Chemical and hybrid synthesis working in tandem: Next generation peptide API manufacturing solutions

The ongoing success of new peptide drugs for various indications is mostly due to solving pharmacokinetic challenges such as peptide stability, bioavailability, half-life, efficacy, renal clearance, and low immunogenicity by chemical modifications of backbone, non-proteinogenic amino acids, or side chains.

Growing molecule complexity, steadily increasing demand for speed and technological innovations give chemical synthesis an advantage, making it the preferred choice for manufacturing next-generation drugs. 

Chemical synthesis is particularly effective at producing stable molecules, which aligns with the trend towards oral administration as well. It also results in a faster production time, shortening the time to clinic and market and aligning with the industry’s need for accelerated drug development to meet demand. Technological innovations such as soluble-anchor based Molecular Hiving for synthesis and MCSGP for continuous purification promote the scalability of commercial production, positioning chemical synthesis as an increasingly attractive option for pharmaceutical companies looking to lead peptide innovation.

However, choosing either synthetic or recombinant peptide synthesis techniques requires a balanced consideration of several factors, such as the specific peptide’s complexity, production volume, cost, and timeline for development. Peptide drug development in the future will likely see innovations that address the current drawbacks of both recombinant and synthetic methods, making sure that the pharmaceutical industry can satisfy the growing demand for peptide-based therapeutics.

New hybrid or semi-synthetic approaches offer promising steps forward in this regard, which utilise partially recombinant, partially synthetically produced (or modified) peptide fragments which get ligated or conjugated into the full-length complex API. These methods offer a welcome addition to the growing toolkits of drug manufacturers, and offer greater flexibility and efficiency.

The hybrid model of chemical and biological recombination synthetic methods allows for the efficient and reliable production of synthetic peptides on an industrial scale. These peptides can be further modified in a site-specific manner through chemical synthesis or genetic code expansion to enhance their stability and physiological activity.

Thus, while chemical and synthetic methods will provide temporary solutions for the near future, these developments will forge further innovation, driving competition and ultimately benefiting patients.