How to ensure optimal peptide purity & yield in SPPS

How to ensure optimal purity and yield in solid-phase peptide synthesis

Previous articles in this series have highlighted how peptide synthesis has enabled the development of a wide range of powerful vaccines and therapeutic agents. This has been made possible by the ability to synthesize peptides to high purity and in large scale. An almost limitless combination of sequences that often include modified amino acids, however, makes considerable demands on synthesis optimization.

Solid-phase peptide synthesis (SPPS) involves the successive addition of protected amino acid derivatives to a growing peptide chain, including deprotection and washing steps to remove unreacted groups and also side products. If every single step went to 100% completion, then only the full-length peptide would be produced. This is very rarely the case, however well a protocol is optimized, and the risk of side reactions and incomplete reactions means that peptide purity becomes a key issue in the context of the application.

Determining peptide purity

The purity of peptides is determined using analytical methods such as reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS). These methods provide a percentage of the sample which is detected using UV absorbance of certain chemical moieties (for HPLC) or charged species in the case of MS. This does not identify the contribution of water and salts in the sample, for example. Amino acid analysis can also be used to calculate yield or actual amount of peptide.

Incomplete deprotection and coupling even at a low level can soon result in a low yield of the final product. For example, the theoretical purity of a 70-mer peptide can be calculated as follows:

• 97% yield at every step (deprotection and coupling) gives 0.97¹⁴⁰ = 1.4% overall yield

Increasing the efficiency towards 100% has dramatic effects:

• 99% yield: 0.99¹⁴⁰ = 24% overall yield
• 99.5% yield: 0.995¹⁴⁰ = 50% overall yield

Purity to match your application

The first thing to do is to decide what level of purity you require, or rather, what impurities can be tolerated in your application. The most common impurities include:

• Deletion sequences
• Truncation sequences
• Incompletely deprotected sequences
• Sequences modified during cleavage (reattachment of protecting groups at other locations on the peptide)
• Other side-reaction products that are formed during synthesis (e.g. aspartimide formation, oxidation products)
• TFA (trifluoroacetic acid)
• Acetic acid

Depending on the level, one or more of these impurities can have a direct effect on your application. For example, specific truncation sequences can interfere with target-binding under investigation, or TFA can kill cells used in a bioassay. This means that the synthesis itself must be optimized to ensure the right molecular species are synthesized to sufficiently high levels and/or the levels of incorrect, problematic sequences are minimized. Alternatively, it might be necessary to look at downstream processing steps, such as including a salt exchange step after synthesis — for example acetate or citrate are commonly required for drug substance or assay testing. You may also need to examine the relative merits of freeze-dried vs. maintaining the peptide in solution, including stability testing.

With this in mind, there are general guidelines to the purity you can aim at for a particular application

1. Crude (>50% or >70%). This level is suitable for high-throughput screening of lead compounds. >70% may be sufficient for an antigen if you are studying immune response but specific contaminants may need to be removed.
2. Medium purity (>70% or >80%). This level is sufficient for the production, purification and testing of antibodies used in immunoassays <https://www.gyrosproteintechnologies.com/immunoassays-in-biopharmaceutical-development>, enzyme substrate studies, epitope mapping, affinity purification, bioassays or other immunological applications, and peptide screening. Achieving >85% purity may be necessary for biochemistry and semi-quantitative applications, such as enzymology, epitope mapping, or studying biological activity, enzyme-substrate interactions, or phosphorylation, and also peptide blocking in Western blotting and cell attachment.
3. High purity: >90% to >98% purity will be required for quantitative bioassays, quantitative in vitro receptor-ligand interaction studies, biological activity with ligand binding studies, quantitative blocking and competitive inhibition assays, quantitative phosphorylation and proteolysis studies, electrophoresis markers and chromatography standards.
4. Extremely high purity: >98% purity for in vivo studies, clinical trials, drug studies that use peptides as pharmaceuticals and structure-activity relationship studies. Examining protein structure by nuclear magnetic resonance studies or protein crystallography.

Optimize synthesis towards the required purity

Achieving the purity you need for a specific application means maximizing the efficiency of correct coupling and minimizing side reactions, deletions, racemization etc. The challenge can vary depending on the peptide sequence. Specific regions might cause aggregation during synthesis. Long peptides (>30 amino acids) can present challenges in themselves, as can complex peptides that include hydrophobic amino acids or modifications such as cyclic, or branched residues.

The optimization process can be broken down into key steps:

• Sequence analysis, for example using software, to determine if there are regions that might present particular challenges in synthesis. 
• An assessment of the risk for side reactions, such as racemization. There are a number of strategies that can reduce the risk of side reactions or help increase the efficiency of individual couplings, including pseudoprolines, dipeptide building blocks, additives, and alternative sidechain protection.
  
  
• Method screening and optimization that includes key aspects of SPPS:
  
  
  1. Resin selection – SPPS is normally conducted on polystyrene crosslinked with 1% divinylbenzene (DVB). Alternatives include polyethylene glycol (PEG) derivatives, and the choice of core can affect synthesis success and crude purity. Linkers attached to the bead provide a reversible linkage between the growing peptide and the solid phase support and determine the properties of the final product and the chemistry that can be used. The choice of linker determines the C-terminal functional group in the final product.
  2. Coupling chemistry – Choice is generally dependent on the synthesis speed, with faster synthesis requiring more highly reactive (= unstable) coupling reagents (HCTU, HATU and COMU) than slower synthesis (DIC, HBTU). Reaction temperature can affect the choice of coupling chemistry.
  3. Reaction temperature – Increasing the temperature may speed up synthesis and improve purity, but not always. Note that microwave-based heating is just heating. Microwaves in themselves do not have a special effect on synthesis.
  4. Monitoring deprotection – Synthesis setup is not always a case of ‘press the button and walk away’. Being able to monitor the efficiency of, for example, the deprotection step by real-time ultraviolet monitoring can be important in optimizing protocols.
  5. Double couplings – Difficult couplings identified by the prediction software can be handled by doubling coupling, extending the coupling time, or adding more equivalents.
  6. Steric hindrance – Coupling reagents must be sufficiently rapid so that sterically hindered amino acids can be incorporated.
  7. Capping – This is done to permanently block any unreacted amino groups following a coupling reaction, or to acetylate the N-terminus of a completed peptide. It is useful for minimizing deletion products during the synthesis of difficult or long peptides and assisting the purification process.
  8. Solvent use – Always use fresh solvents. Re-using solvents reduces purity.

Choosing a peptide synthesizer that will quickly deliver peptides with the right purity

A major factor that affects how you can optimize SPPS to achieve the peptide purity you need is the choice of synthesizer. Here are a few tips to help you in your choice:

• Parallel reaction vessels to screen methods and reagents — As we have seen, optimization can involve looking at a large number of variables. The ability to run different reagent combinations in parallel will greatly speed up the optimization process.
• Deprotection monitoring — Being able to monitor the efficiency of the deprotection step during the run avoids guesswork that can lead to incomplete deprotections, deletions, and side reactions.
• Temperature control — Increasing the temperature can improve purity for some sequences. Being able to test the effect of different temperatures in parallel can boost productivity.
• Minimal cross contamination — A peptide synthesizer that provides multi-channel synthesis with no reagent or resin cross-contamination will speed up optimization and minimize the risk of resynthesis.
  
• No-prime single shots for rare monomers — Many of the monomers and reagents used in special chemistries can be expensive or precious, so some synthesizers offer prime-free ‘Single-Shot’ additions from any amino acid/monomer bottle position to ensure that nothing is wasted.
• Software designed for 21 CFR Part 11 compliance if peptides are being synthesized in a GxP facility.

You can find out more about designing a peptide synthesis and minimizing side reactions by downloading our recent summary of SPPS Tips for Success webinars:

• Designing a Synthesis: How to maximize the likelihood of success when developing an automated synthesis protocol for a new peptide sequence.
• Side Reactions: How to implement strategies to reduce the risk of unwanted side reactions that inevitably occur during the chemical synthesis process.
• Peptide Manufacture: A real-world example of the synthesis of 24 SARS-CoV-2 peptides with therapeutic potential, manufactured with a focus on cGMP and regulatory compliance.