Tackling the complex area of peptides

16th December 2015

Posted By Paul Boughton

Chromatographic results for a challenging peptide mixture

The challenges of peptide analysis and purification. By Cecilia Mazza

As the industry and academia continue to expand their work into the promising area of peptides, with the focus of bringing more effective therapeutic drugs to the market, there is an increased need for the analysis and purification of peptide mixtures.

Traditionally, peptide molecules are separated and purified using reversed phase chromatography with silica based stationary phases at low pH conditions. However, as the studies of peptide structures develop and some of those innovative structures become more basic in nature, not all peptide mixtures can be isolated to satisfaction with the standard conditions of low pH, representing a significant challenge to the scientific community.

Furthermore, while many of the therapeutic peptides are initially analysed and purified at a small scale within drug discovery departments, there is the expectation that some of the peptides will exhibit positive results to the point that scale-up and manufacturing will occur. As many of these substances are expensive to produce and their impurities need to be removed to very low concentrations, HPLC is the ideal technique to use in the purification of these drugs also at a large scale. There is therefore a need to determine successful preparative methods early on that can be transferred from discovery to development and then to production.

At the same time as the field of therapeutic peptides grows rapidly, state-of-the-art stationary phases are being developed based on organosilane enforced silica to expand the range of pH scientists can operate at, maintaining the chemical stability of traditional silicas but at high pH. These innovative materials provide scientists with a new tool to separate and purify substances, including peptides.

The example discussed here is one of a challenging peptide mixture where the main substance is a small peptide that needs to be purified. The chromatographic results are depicted in Fig. 1. As seen in the figure, the purification at pH 8 is carried out in the same manner as with a classic silica material meanwhile the purification at pH 9.5 is normally used with a reinforced silica material. The purity and yield results are also shown in the figure, indicating that the work carried out at higher pH is more effective. In fact the results at higher pH are significant because under overloaded conditions, the main compound and its impurities travel at a different pace through the column producing purer fractions. When these types of results are found at the discovery phase, there is an aspiration to continue using the same stationary phase, mobile phase and run conditions in the scale up to development and production because of the time savings this represents to a given project where companies are striving to come first to market.

The example shown here is relevant as results are impressive and the purification is already carried out with a 10µm material considered to be a traditional particle size in larger scale chromatography, where the scientist is already future-proofing the development and manufacturing steps if the substance is deemed of value for the drug company.

Consequently, there is more stress on using stationary phase materials that can be scaled with the needs of the pharmaceutical or biotechnology company.

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Cecilia Mazza is with AkzoNobel





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