A New Purification Tool in Peptide Chemistry

by biopharmacharis in Biopharma Process Systems, BPS News, BPS News Cat 1, BPS Right Sidebar Content, Uncategorized

Peptide chemistry has become a significant player in the therapeutics industry, with many large pharmaceutical companies entering into the interesting world of UNAAs, formic acid and pyridine. Peptides have held their own in the drug discovery world, against rival small molecule and biological therapeutics, overcoming poor pharmacokinetics through utilising unique delivery methods, such as Liposome encapsulating.

Peptide chemistry does have pros and cons, however, the achievement of synthesising your first X-mer is second to none as, one of the more challenging and time-consuming steps in the process is not always the synthesis itself, but the purification.

For most peptide chemists, the instinctive reaction to this is reverse phase HPLC methods, yet there is another way, one paved with less solvent consumption, reduction in analytical time and unique separation sometimes not possible through HPLC; let’s consider instead, SFC.

Supercritical Fluid Chromatography, or SFC for short, is used for the separation of chiral compounds and is also becoming more commonly used for the separation of achiral compounds too. It is a form of normal phase chromatography which utilises CO₂ as the mobile phase, but in its supercritical phase where the CO₂ is neither liquid nor a gas. Typically, CO₂ will be combined with an organic polar co-solvent such as methanol to produce a gradient that will allow the separation of a compound from a column of choice by the end user.

Utilising SFC has proven to be beneficial to many research groups including ChemPartner[1] and Virginia Tech[2] both citing results demonstrating the ability of SFC to separate peptides of up to 40-mers, with Virginia Tech additionally stating that the peptides contained both a variety of acidic and basic residues. Cyclic peptides have also shown to benefit from the use of SFC technology, some of these geometric peptides have also been reported to have separated easier using SFC when compared to multi step separation method through HPLC [3].

In addition to the successful application of SFC in the separation of larger and more niche peptides, it has also been shown to purify peptides that RP-HPLC is not able to. This has been shown in a study at Uppsala University where, when using SFC, the peptides leuprolide and triptorelin were able to be separated when using a XT organo-silane stationary phase (α ≈ 1.19). This was not possible when using conventional reverse phase HPLC [4].

Figure 1: SFC250 can utilise up to 30mm ID columns with 4 solvent modifiers and 8 fraction collection vessels of unlimited volume.

Peptide purification is not the only link in the chain where SFC can be used. Most unique peptides use expensive non-natural amino acids. One way around this is synthesising your own. This too can be possible utilising SFC technology, with promising results showing separation of 5 underivatized amino acids, among which 4 are aromatic, with polysaccharide-based chiral columns in SFC [5].

Previously SFC was met with much hype and popularity when the technology was first introduced. However, it was only recently that it has been able employed in a way to live up to the initial expectations. In today’s market, you can now purchase an SFC systems like the Sepiatec Prep SFC range (shown in figure 1) comprising of bench top non-modular models coupled with an intuitive touch screen display that allows both practiced and inexperienced users the ability to operate to the units full capabilities.

This, combined with a fivefold decrease in analysis time compared to some HPLC systems [6] and a dramatic decrease in solvent due to utilising CO2 as a modifier, makes SFC a powerful tool that any peptide chemist should consider.

SFC is growing in popularity and being adopted by more and more facilities and projects, proving its ongoing credibility as a solution for large-scale peptide purification methods for consideration.

To discover more how Biopharma Group’s range of analytical, prep & semi-prep chromatography equipment solution could meet your project and lab needs, contact our team of peptide purification specialists, who will be happy to assist you in finding a tailored solution.

  1. (https://pubmed.ncbi.nlm.nih.gov/32353707/)
  2. (https://pubmed.ncbi.nlm.nih.gov/16503605/)
  3. (https://www.researchgate.net/publication/47810521_Rapid_peptide_separation_by_supercritical_fluid_chromatography)
  4. https://uu.diva-portal.org/smash/get/diva2:1343046/FULLTEXT01.pdf) pg 37
  5. (https://www.sciencedirect.com/science/article/abs/pii/S0021967318314493)
  6. (https://pubmed.ncbi.nlm.nih.gov/21083112/)