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Peptide Purification
In the modern era, huge leaps forward in the scientific field of peptide synthesis have enabled the production of custom peptides on an immense scale. With the increased production of synthetic peptides for research, the implementation of effective peptide purification methods has only become more critical.
Peptides are complex molecules, and this complexity can render other purification methods that are effective on other organic compounds inefficient. During synthesis, special attention must be paid to maximizing both efficiency and yield in order to provide customers with the purest possible peptide at the lowest possible price. While purification processes based on crystallization are often effective with other compounds, many peptide purification processes utilize the principles of chromatography, such as high-pressure reversed phase chromatography.
Removing Specific Impurities From Peptides
As mentioned before, it is vital that the final synthesized peptide is as pure as possible for research use. Minimum acceptable purity levels can vary among different research purposes; for example, in vitro studies generally require a much higher standard of purity (greater than 95%) than, say, performing an ELISA standard for measuring titers of antibodies (minimum acceptable purity greater than 70%). Nonetheless, the minimum purity level must be achieved. In order to ensure that purity standards are met, it is vital to recognize the types of impurities than can arise as well as their nature. Then the appropriate purification method (or methods) can be implemented
During peptide synthesis, specific impurities that can occur include hydrolysis products of labile amide bonds, deletion sequences generated mainly in solid-phase peptide synthesis (SPPS), diastereomers, and insertion peptides and by-products formed during removal of protection groups. This latter impurity can occur in the last step of peptide synthesis. Additionally, polymeric forms of the peptide intended to be synthesized can also occur, often arising as a byproduct resulting from the formation of cyclic peptides that have disulphide bonds.
Certainly, the purification process employed must be able to effectively isolate the targeted peptide in a multifaceted mixture of compounds and potential impurities.
Peptide Purification Strategy
Ideally, the purification method should be as simple as possible, achieving targeted purity in as few steps as possible. Often, two or more purification processes conducted sequentially can give excellent results, particularly when each process operates through differing principles of chromatography. For example, ion exchange chromatography utilized in conjunction with reversed phase chromatography can result in a very highly pure final product.
Generally, the first step in peptide purification is a capturing step that removes the majority of impurities from the synthetic peptide mixture. Many of the impurities removed in this phase are produced in the final deprotection step of peptide synthesis and are mostly uncharged and have a small molecular weight. While a significant amount of impurities can be removed during this initial step, a second purification step can be added if a higher purity level is required. This second step can be referred to as a polishing step and is highly effective, especially when operating on a complementary chromatographic principle as previously mentioned.
Peptide Purification Processes
Peptide purification systems are composed of several integral subsystems and units, which can include buffer preparation systems, solvent delivery systems, fractionation systems, and data collection systems, along with the crucial columns and detectors. Indeed, the column is the heart of the purification system and its selected features can be critical to the process’s efficaciousness. A column may have features constructed of glass or steel along with static or dynamic modes of compression, any of which can affect the final purification outcome.
Additionally, it is vital that all purification methods are carried out in accordance with current Good Manufacturing Practices (cGMP) and sanitization is given the highest priority.
Throughout the processes of peptide synthesis and purification, special attention must be given to following GMP. This is to ensure that the final peptide is pure and of high quality. GMP requires that chemical and analytical procedures performed are well documented. Test methods and specifications are required to be established beforehand, ensuring that the manufacturing process is under control and reproducible.
GMP requirements for the purification phase of peptide synthesis are particularly rigorous. This is because this process is a late step in the overall synthesis process and has a large impact on the quality of the final peptide. Critical steps and parameters must be identified, along with limits for those parameters, so that the process is reproducible within those predetermined limits. Vital parameters of the peptide purification process can include column loading, flow rate, column performance, column cleaning procedures, composition of elution buffer, storage time in process, and pooling of fractions.
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