Purifying Peptides And What You Should Know

by | Jun 30, 2022 | Knowledge Center

Advancements in today’s world have driven the scientific field of peptide synthesis to what it is today. Peptide synthesis has allowed for an immense production of custom peptides. Due to having more peptides for research, it is imperative and critical to have and implement effective peptide purification procedures. Here you will find information on the different aspects of peptide purification that takes place during the synthesis, different strategies, and methods for it as well as possible mistakes such as impurities that can actually be removed just by purification during the synthesis.

They are unique and complex. Due to this complexity, they can affect purification methods that work for other organic compounds. To maximize efficiency and yield to supply the purest peptides possible for an affordable cost, special attention must be made when they are in synthesis. Chromatography, specifically a high reverse phase chromatography, is what many purification processes like to utilize. However, purification based on crystallization is also effective with other compounds.

The BluePrint and Process for Peptide Purification

The purification process should be simple and have as little steps as possible to achieve purity.
You can still get your targeted results even with two or more purification processes being done right after one another. One example is when an ion exchange chromatography is used in alliance with a reverse phase chromatography. This can result in a highly pure product when completed.

The first step to be taken is the capturing step. This step will remove more than half of the impurities found in a synthetic mixture. The impurities that are eliminated are typically produced in the final deprotection stage of peptide synthesis. Those impurities are typically uncharged and are considered small due to their molecular weight.

If a higher purity level is needed, a second purification can be done as well to achieve the target purity. This second step is referred to as the polishing step. It is effective when working with a complementary chromatographic principle.

There are multiple subsystems and units that make up the system for peptide purification. These include:
1. Buffer preparation Systems
2. Solvent Delivery Systems
3. Fraction Systems
4. Data Collection Systems.
*There are also columns and detectors that could be pivotal.

The heart of the purification system is the column. The column’s features are vital to the effectiveness of the process. Columns have features that are made from glass or steel with static or dynamic modes of compression. Any of those features can affect the outcome of the final purification. It is crucial that every purification practice is carried out following the Good Manufacturing Practices (GMP), and priority is given to sanitation.

Purification Process Types:

1. Ion Exchange Chromatography (IEX)
The Ion Exchange Chromatography excels with different charges among peptides in a mixture. When peptides with opposite charges meet in a chromatographic medium, one charge begins to isolate. Columns and binds are what peptides are loaded into. So that bound substances are eluted differently, conditions are continuously changed. What is being manipulated during this is levels of salt concentration (which is used to elute the mixture) or the pH level. The peptide, during the binding process, is concentrated and then, in its purified form, collected. This process has high resolution and high capacity as well.

2. Hydrophobic Interaction Chromatography (HIC)
Hydrophobicity is the principle; that this purification process follows; the selected peptides will then isolate due to the interaction between a peptide and a hydrophobic surface of a chromatic medium. Due to this also being an action that can be reversible, a peptide can be concentrated and purified. HIC could be an effective purification to use after a previous purification method was utilized that consisted of salt in elution (IEX). To do this, a high ionic strength buffer is needed to magnify the process. During this process, they bind together while in a high ionic strength solution. As they bind, they are loaded onto columns. Following this, the salt concentration is decreased, which causes elution. This results in bound substances eluting differentially. Using ammonium sulfate is one way to typically implement this process. It is used to dilute samples on a reduced gradient. In the end, the peptide is collected in the final concentrated and purified form. HIC provides good levels of resolution and sample capacity.

3. Affinity Chromatography (AC)
The first note to make about Affinity Chromatography is that it offers high resolution and sample capacity. It isolates them through capitalizing the interaction between peptides and a particular ligand that is attached to a chromatographic matrix. The peptide then bonds to a ligand, and the unbound material is cleaned off. One crucial information to know about this binding is that it is reversible. This means the condition is changed to be more complementary to desorption. Desorption could also be performed specifically or not specifically. Specific desorption uses a competitive ligand, while nonspecific uses an altered pH, polarity, or ionic strength. Once that is completed, the peptide of choice is collected in its purified form.

4. Reversed Phase Chromatography (RPC)
This method separates peptides from contaminate by using reversible interactions between the selected molecules and chromatographic mediums on a hydrophobic surface. This method also offers high resolution. Sample could then be loaded on columns or even bind together. After they have done one of these steps, the conditions are changed to the bound substances elute differently. The organic solvents and additional additives are needed for elution. Due to the reversed phase matricis, initial bonding is considered to be strong. Typically to achieve elution, organic solvents concentration needs to be increased, known as acetonitrile. Through the binding process, the concentrated molecules are then gathered while it’s in its pure form. Note this method is primarily used as a clean up step with peptides and oligonucleotide samples. RPC is successful when working with analytical separations such as peptide mapping. It still is not the most perfect method to use as organic solvents can denature peptides. Thus, it is not always the preferred method as requirements may call for recovery of activity and need to return the peptides to a correct tertiary structure.

5. Gel Filtration (GF)
By taking advantage of different molecular sizes between the chosen peptides and impurities, this purification method can isolate peptides. This method, however, is only used when dealing with small volume samples. It does still offer good resolution as well.

The Importance Of Following The Good Manufacturing Practices

To ensure the final peptides are pure and high quality, researchers must ensure to follow the guidelines given by Good Manufacturing Practices (GMP). It is a requirement that chemical and analytical procedures that have been executed are properly documented. All testing and research methods and specifications needed must be known and accepted before any work is begun. This will promise that the process is reproducible and regulated correctly.

The requirements for purification of peptide synthesis are careful and diligent. This is partly due to the step having a huge impact on the final results of the peptides quality. Steps and parameters that are crucial must be pinpointed, and there must also be limits set for those parameters. This is required so that the process can be duplicated, and these steps could be replicated by others and produce the same results. Examples of the important parameters of the purification process include:
1. Column Loading
2. Flow Rate
3. Column Performance
4. Column Cleaning Procedures
5. Composition of Elution Buffer
6. Storage Time in Process
7. Pooling of Fractions

How To Rid Peptides Of Specific Impurities

The goal for synthetization is to have the purest peptide possible for researchers to use. There is a minimum of acceptable purity levels that are welcomed and used for different research purposes. Take an in vitro study, for example. They have a need for a high standard of purity (>95%). ELISA, on the other hand, has a minimal acceptability of >70% for measuring titers of antibodies. Therefore the minimum level is essential to attain.

It is also important to understand and know the types of impurities that can come forth along with their characteristics. This way, researchers know what purification method would be most effective as each situation is different.

Specific impurities during peptide synthesis can happen; These consist of:
1. Hydrolysis products of labile amide bonds
2. Deletion sequences generated in solid-phase peptide synthesis (SPPS)
3. Diastereomers
4. Insertion peptides and by-products formed during removal of protection groups

Other than impurities, polymeric forms of peptides may also occur. This transpires due to a byproduct resulting from a formation of cyclic peptides which have disulphide bonds. Therefore, the purification process method that is chosen must make certain to isolate the selected peptide while in a multifaceted mixture of compounds and impurities.

At BiotechPeptides, we adhere to the most stringent synthesis and purification practices in the industry. Through our dedication to these standards, our company is able to provide peptides that exceed 99% purity and are fit for any research study or application.