Basics of Peptide Synthesis & Purity
How peptides are synthesized and purified, and what tests can be used to analyze and characterize them
Peptide Synthesis
Peptide synthesis is the process of creating a desired sequence of amino acids. There are many reasons to synthesize peptides, whether you are researching signaling pathways, receptors, enzymes, diagnostics, biomaterials, or a potential drug candidate. You will want to make sure you have not only the sequence you desire, but also data supporting the identity and purity of the peptide and its desired form for use. (Amblard et al., 2006; Sharma et al., 2022)
Solid-Phase Peptide Synthesis
One technique used to synthesize peptides is solid-phase peptide synthesis (SPPS). During SPPS, you attach the growing peptide chain to a solid, insoluble material, typically a resin. Protected amino acids are then coupled onto the growing chain one at a time. (Amblard et al., 2006; Behrendt et al., 2016)
Each peptide synthesis cycle consists of a few general steps: deprotect, couple, wash, and repeat until you have the full sequence. You can then cleave the peptide from the solid support and remove any protecting groups that remain as appropriate. (Amblard et al., 2006; Behrendt et al., 2016)
Protecting Groups
Amino acids can have multiple reactive sites that you may want to protect while synthesizing a peptide. Protecting groups can temporarily shield selected areas of an amino acid so unwanted reactions are reduced. Fmoc is one protecting-group strategy commonly used in modern solid-phase peptide synthesis. (Amblard et al., 2006; Behrendt et al., 2016)
Purifying Your Peptide
Once your peptide is synthesized, you will most likely have a crude mixture containing your desired peptide along with other impurities. Impurities can include deletion sequences from incomplete coupling, missing amino acids, unwanted modifications, oxidation products, by-products, and other unwanted materials from the synthesis process. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Purification allows you to isolate your desired peptide from other molecules that may be present in the crude mixture. Reversed-phase high-performance liquid chromatography (RP-HPLC) is commonly used to purify peptides. Because molecules interact differently with the column and mobile phase, the compound of interest can be separated based on its properties, including hydrophobicity. (Mant et al., 2007; Sharma et al., 2022)
What Types of Analyses Can You Run to Characterize Your Peptide?
Type of Analysis / Question You May Ask / What It Can Tell You
Analytical HPLC/UHPLC – How many peaks can I see? – This can give you an idea of what your chromatographic purity may be. It can also allow you to see peaks that elute separately from your compound of interest. (Sharma et al., 2022)
Mass spectrometry – Does my molecule have a mass that is consistent with my sequence? – This can help support identity. Mass differences may indicate modifications or impurities. (John et al., 2004; Sharma et al., 2022)
LC-MS – What peaks am I seeing, and what masses do they have? – Liquid chromatography-mass spectrometry can provide more information about sample identity and purity. (John et al., 2004; Sharma et al., 2022)
UV detection – What UV-absorbing material is coming off my column? – Can be used with HPLC to detect UV-absorbing compounds as they elute from the column and to support peak-area measurements. (Sharma et al., 2022)
Amino-acid analysis – What amino acids are in my sample? – Can provide additional information about the sample when appropriate. (Sharma et al., 2022)
Purity of Your Peptide on HPLC
If you run your sample on an analytical HPLC, you will get a chromatogram. You can compare the peak area of interest with the total integrated peak area to get an estimate of chromatographic purity under the method used. (Sharma et al., 2022)
Again, this is not everything you will want to know about your sample. Other molecules can sometimes travel with, or co-elute with, your compound of interest. You may also have compounds that are not detected well by the detection method being used. Therefore, an HPLC purity result only tells you what that particular method can detect under its analytical conditions. (Sharma et al., 2022)
Using Mass Spectrometry to Help Identify Your Peptide
Mass spectrometry can be used to help determine whether your molecule has the mass you expect. By comparing the observed mass information from your sample with the theoretical mass expected from the intended sequence, you can gather evidence about whether your compound of interest is present. (John et al., 2004; Sharma et al., 2022)
Using HPLC and mass spectrometry together can provide more information about your sample. HPLC can show that you have a major peak eluting from the column, while mass spectrometry can help determine whether that component has the mass you expect. (John et al., 2004; Sharma et al., 2022)
Identity vs. Purity vs. Content vs. Potency vs. Sterility
Identity – Evidence that your molecule is the compound you think it is.
Purity – How pure your molecule is based on the analytical method being used.
Content/Assay – The amount of your specified compound that is present in a sample.
Potency – The biological or pharmacological activity of your compound, when applicable.
Sterility – Whether the sample is free from viable microorganisms under an appropriate test.
Endotoxins – A separate test for bacterial endotoxins that may be present in the sample; endotoxin testing is distinct from chemical purity and sterility.
(Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Impurities You May See When Working With Peptides
Deletion sequences from incomplete coupling. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Incomplete coupling or chain assembly. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Modifications from reactive side chains. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Oxidation. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Other degradation products. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Unwanted protecting groups that were not removed. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Leftover solvents, reagents, counterions, or other processing-related materials. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Degradation associated with storage or handling. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Degradation associated with inappropriate temperature exposure. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Quality Control
Whether you are buying a peptide or making one yourself, you want to know what data are associated with the peptide. You should know what sequence you are trying to obtain and what analytical data are available for the sample. This could include the sequence, mass data such as theoretical and observed mass, lot or batch number, HPLC data, the method used to determine purity, and mass spectrometry data. These records should be traceable back to the sample being evaluated. (U.S. Food and Drug Administration, 2021)
If you are purchasing a peptide from a vendor, you may receive a Certificate of Analysis (CoA). You want to know what data are included on that CoA and what tests were performed on the sample.
Things to Look for on a CoA
Does the molecule name and sequence match what you are looking for?
What is the expected mass of the peptide, and is there analytical data supporting the observed mass?
Look at the HPLC trace if it is provided.
Know what method is being used to determine purity.
Know what lot or batch number applies to your sample.
Do not assume a peptide is sterile or suitable for use in humans just because it has been tested for chemical purity.
Always remember that a statement such as “99% pure” does not mean that the sample contains no endotoxins, is sterile, or is suitable for human injection. Chemical purity, identity, content, sterility, and endotoxin status are separate quality attributes. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
Take-Home Messages
There are many things to consider when synthesizing a peptide, from the initial synthesis through final purification and characterization. Knowing what tests have been run on a sample and what those tests can tell you is important. Once you understand the differences among identity, purity, content, potency, and sterility, it becomes easier to understand peptide research and analytical information. (Sharma et al., 2022; U.S. Food and Drug Administration, 2021)
More Information
There are many resources that can help you learn more about peptide synthesis and purification. Textbooks, peer-reviewed articles, and other reputable scientific resources can provide additional information.
These are general concepts related to peptide synthesis and purity. Always follow your laboratory's approved procedures and safety requirements when working with these types of samples.
References
Amblard, M., Fehrentz, J.-A., Martinez, J., & Subra, G. (2006). Methods and protocols of modern solid phase peptide synthesis. Molecular Biotechnology, 33(3), 239–254. https://doi.org/10.1385/MB:33:3:239
Behrendt, R., White, P., & Offer, J. (2016). Advances in Fmoc solid-phase peptide synthesis. Journal of Peptide Science, 22(1), 4–27. https://doi.org/10.1002/psc.2836
John, H., Walden, M., Schäfer, S., Genz, S., & Forssmann, W.-G. (2004). Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography-mass spectrometry. Analytical and Bioanalytical Chemistry, 378, 883–897. https://doi.org/10.1007/s00216-003-2298-y
Mant, C. T., Chen, Y., Yan, Z, Popa, T. V., Kovacs, J. M., Mills, J. B., Tripet, B. P., & Hodges, R. S. (2007). HPLC analysis and purification of peptides. Methods in Molecular Biology, 386, 3–55. https://doi.org/10.1007/978-1-59745-430-8_1
Sharma, N., Kukreja, D., Giri, T., Kumar, S., & Shah, R. P. (2022). Synthetic pharmaceutical peptides characterization by chromatography principles and method development. Journal of Separation Science, 45(13), 2200–2216. https://doi.org/10.1002/jssc.202101034
U.S. Food and Drug Administration. (2021). ANDAs for certain highly purified synthetic peptide drug products that refer to listed drugs of rDNA origin: Guidance for industry. https://www.fda.gov/media/107622/download