Evaporation and Lyophilization for Converting RP-HPLC Peptide Fractions into Stable Dry Powders

Here, we describe a method for obtaining research peptides in a stable, dry, and contamination-free form for long-term storage.

Peptides can generate impurities during the synthesis process, which can affect the effectiveness of the target peptide fractions. For example, acidolytic cleavage following SPPS yields crude peptides that contain the desired sequences along with various impurities, such as deletion peptides, truncated peptides, deprotected peptides, modified peptides, scavengers, and by-products derived from cleaved protecting groups. All of these contaminants must be removed to obtain peptides of high purity.

Solid-Phase Peptide Synthesis

Purification of synthetic peptides has been carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) since the late 1970s, and it has become the method of choice for purifying peptides. This technique combines high resolution and recovery with ease and speed of operation, which make it attractive as it is also applicable to a wide range of peptides with diverse physicochemical properties.

Peptides are desorbed from the hydrophobic surface by organic solvents. During the solvent gradient, when the concentration of the organic solvent reaches a specific level unique to each peptide, the peptide desorbs from the hydrophobic surface, travels down the column and elutes. In practice, only a few organic solvents are routinely used in peptide reversed-phase chromatography. The two most widely used are acetonitrile and methanol, although acetonitrile is generally preferred because:

>>It is volatile and easily removed from the sample

>>Has low viscosity, resulting in lower back pressure

>>Is highly transparent at low UV wavelengths

>>Has a long history of successful peptide separations

Fractions should be collected when the desired peptide begins to elute from the preparative column. These fractions should be analysed by analytical HPLC, and all fractions meeting the required purity can be combined. At this stage, the pooled peptide fractions still contain RP-HPLC solvents (water, organic solvents such as acetonitrile, and ion-pairing agents like TFA), which must be removed. RP-HPLC Peptide Fractions are typically dried using a parallel sample evaporator or freeze dryer, and it is important during this process to maintain the quality and integrity of the sample. Utilising a centrifugal evaporator or freeze dryer to dry HPLC fractions offer many benefits, but with a few challenges.

What challenges could you face during the evaporation and lyophilization of RP-HPLC Peptide Fractions?

Freeze drying RP-HPLC peptide fractions containing water and solvents, such as acetonitrile, poses several challenges for lyophilization. Acetonitrile has a low freezing point (-65°C), and if this is not achieved during lyophilization, sample bumping and contamination will result. If acetonitrile accumulates in the ice trap, it can impair the vacuum and prevent lyophilization. In addition to the challengers faced in working with solvents, the lyophilization process itself can be time consuming, sometimes requiring days to achieve the desired sample dryness.

Working with a centrifugal evaporator addresses many of the challenges associated with lyophilization, however evaporation also has obstacles to overcome. Trifluoroacetic acid (TFA) is often added to the reverse phase HPLC mobile phase for its buffering capabilities, to enhance retention, or to suppress certain ionic interactions. At times, centrifugal evaporation alone can be insufficient to remove all of the TFA due to sample interactions, and the residual element could have detrimental effects on analysis. Further, this approach might produce a film-like dried sample on the bottom of the vial, making it difficult to re-suspend and weigh. The solution for overcoming these challenges is the HT-12 centrifugal evaporator developed by Genevac, that utilises LyoSpeed™ technology.

How does the HT-12 and LyoSpeed™ Help?

The challenges faced above led to the development of a combined method, incorporating the speed and efficiency of centrifugal evaporation with the superior drying capability and improved sample integrity achieved with lyophilization. This combined method development has been achieved using the SP Genevac HT Series 3i centrifugal evaporator and Lyospeed™ technology which creates a hybrid drying process combining conventional centrifugal evaporation and lyophilization through these steps:

Stage 1: Removal of Acetonitrile from the solvent mixture at 40’C with a variable Dri-Pure vacuum ramp to prevent solvent bumping, (no heat);

Stage 2: Using controlled vacuum pressure to concentrate the remaining water, (some chamber heating) to a few millilitres.

Stage 3: Applying full vacuum pressure between 5 and 20 hours.

For smaller numbers of HPLC fractions, for example up to twenty-four 20ml vials, or up to forty-eight 16 x 100mm tubes, the latest EZ-2 Elite model utilises the same external scroll pump for a fully automated HPLC Lyo run program.

For many years, this application proved successful and is now widely adopted as the preferred method for drying HPLC reverse phase solvent fractions.

If you are in chemical biology and working with peptide RP-HPLC fractions, you would want dry powder instead of the traditional thin film. This makes your further studies much easier, as compounds can be more easily removed from the tube or vial, or re-dissolved in a suitable solvent. Biopharma Group has the solution. Do not hesitate to contact us to know more!