Peptide purification technologies are important because of the rising demand for synthetic peptides in research. This page explains key steps in peptide purification during synthesis, such as common methods, strategies, and removing contaminants.
Peptides are complex molecules, and traditional methods used for purifying organic compounds often fail to work. This makes it necessary to focus on improving efficiency and yield to produce the purest peptides at the lowest cost. While crystallization works well for many substances. The peptide purification usually depends on advanced techniques like high-pressure reversed-phase chromatography.
As stated earlier, the final synthesized peptide must be as pure as possible for research purposes. The minimum purity level depends on the research goal. For example, in vitro studies often require a high purity standard (above 95%), while an ELISA for measuring antibody titers may need a lower standard (above 70%). In all cases, the required purity level must be achieved. Understanding the types of impurities and their characteristics is crucial to meet these purity standards. Once the impurities are identified, the appropriate purification method can be applied.
Examples of common impurities in peptide synthesis include labile amide bond hydrolysis, deletion sequences (often produced during solid-phase peptide synthesis or SPPS), diastereomers, insertion peptides, and by-products formed during the removal of protecting groups. Some by-products arise during chemical synthesis. Additionally, polymeric versions of peptides may form as by-products in cyclic peptide production involving disulfide linkages. References discussing these impurities are available in [PMC free article].
The purification process must effectively isolate the desired peptide from a complex mixture of chemicals and contaminants.
Peptide purification can be challenging. Ideally, it would take just a few steps to get the desired purity. However, combining two or more purification methods often gives better results. This works well when the methods use different chromatography principles. For example, ion-exchange chromatography, which uses ion-exchange resins, and reversed-phase chromatography can work together to improve separation and produce highly pure peptides.
The first step in purifying crude peptides removes most contaminants. These contaminants are often uncharged, low molecular weight by-products from the final stages of peptide synthesis. While this step clears many impurities, it is not always enough. A second step, called “polishing,” is often needed to reach higher purity. This step works best when it uses a different chromatography method from the first step.
Peptide purification systems consist of several key subsystems and components. These include buffer preparation systems, solvent supply systems, fractionation systems, data gathering systems, reagents, columns, and detectors. The column is the core of the purification system, and its specific features—such as sample volume capacity—are critical for efficiency. Columns can be made of glass or steel and may operate in either static or dynamic compression modes. These features can significantly affect the final purification outcome.
In addition, all purification processes must follow current Good Manufacturing Practices (cGMP).
This method uses a specific ligand coupled to a chromatographic matrix, often in conjunction with high-performance liquid chromatography (HPLC of peptides), to isolate peptides with wide applicability. Unbound material is washed away as the target peptide binds reversibly to the ligand.
The binding conditions are then adjusted to favor desorption, which can be achieved selectively using a competitive ligand or nonspecifically by altering pH, polarity, or ionic strength; urea can also be used to improve binding conditions.
The secondary structure of the purified peptide is subsequently collected with a high yield. Affinity chromatography (AC) offers both high sample capacity and high resolution.
This purification method takes advantage of charge variations between peptides in a mixture. When chromatographic media with the opposite charge is used, peptides of one orientation are separated. Peptides are fed into a column and bind; after that, the conditions are altered such that the bound compounds are eluted in diverse ways.
The amount of salt in the solution or the pH level of the solution are the variables that are changed. Salt (NaCl) is commonly used to elute the mixture. During the binding procedure, the required peptide is concentrated and collected in purified form. IEX is a method with a high resolution and capacity.
The principle of hydrophobicity is used in this technique. The interaction of a peptide with the hydrophobic surface of a chromatic medium allows for the isolation of the desired peptides. Furthermore, this contact is reversible, allowing for the concentration and purification of the peptide. In addition, a buffer with a high ionic strength improves the process, making HIC a highly effective purification approach to use after a salt elution purification procedure (like the IEX technique).
Samples in a high ionic strength solution bind together and are put onto a column during HIC. Following that, elution with lower salt concentrations leads to differential elution of the bound compounds. The use of ammonium sulphate to dilute the sample over a decreasing gradient is a common way of application. After that, the desired peptide is extracted in a concentrated and purified state. As a result, HIC has a high level of resolution and sample capacity.
By exploiting the molecular dimensions difference between the peptides and the contaminants, the gel filtration isolates peptides. Consequently, GF is used only in modest volume samples. But, on the other hand, this method offers a good outcome.
This purification method uses high resolution to separate peptides from impurities. It relies on chromatographic separation, which works by reversible interactions between target molecules and the hydrophobic surface of a medium. The peptides are purified by changing the elution conditions. This helps release the bound compounds at specific stages.
Reversed-phase chromatography (RPC) is a type of high-performance liquid chromatography (HPLC) often used for this. It requires organic solvents, like acetonitrile, to break hydrophobic interactions and allow elution. These solvents help remove impurities and isolate the peptides.
RPC is common for polishing peptide and oligonucleotide samples. It is also useful for peptide mapping and other analytical separations. However, this method is not ideal for processes where activity recovery or proper tertiary structure is needed. The organic solvents used can denature some peptides.
Observance of GMP
GMP compliance is critical during peptide synthesis and purification. Following these standards ensures that the final peptide is pure and meets high-quality standards. All chemical and analytical methods must be carefully documented to meet GMP requirements. Before manufacturing begins, test procedures and requirements must be clearly defined to ensure the process is controlled and repeatable.
The purification stage of peptide synthesis has strict GMP requirements. This step occurs late in the synthesis process and has a major impact on the final peptide’s quality. To meet these standards, key actions and parameters must be clearly defined, with set boundaries to ensure the process can be repeated consistently.
• Column loading is a critical parameter in the peptide purification process, as it directly affects the flow rate and overall efficiency of the separation.
• The rate of flow.
• The performance of the columns.
• Cleaning methods for the columns.
• The elution buffer’s composition.
• The amount of time spent in the process of storing data.
• Fractions are pooled.
Scientific Research
[1] Insuasty Cepeda DS, Pineda Castañeda HM, Rodríguez Mayor AV, García Castañeda JE, Maldonado Villamil M, Fierro Medina R, Rivera Monroy ZJ. Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology. Molecules. 2019 Mar 28;24(7):1215.
[2] McCue JT. Theory and use of hydrophobic interaction chromatography in protein purification applications. Methods Enzymol. 2009;463:405-14.
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