HPLC is a widely used tool in peptide and protein purification. One of the challenges in this process is maximizing the lifetime of the bulk media sorbents or prepacked columns, which degrade due to the buildup of material in the column.
There are many causes of performance degradation and backpressure buildup, including incomplete removal of precipitated compounds, strongly retained sample components and dissolution or other degradation of the sorbent.
By nature, peptides and proteins tend to aggregate, making it difficult or impossible to regenerate media under standard wash conditions. Proteins such as insulin are prepared by recombinant techniques and contain nonsoluble materials that need to be washed and eliminated periodically from the purification media to maintain column performance.
Peptides and proteins produced by chemical synthesis contain multiple disulfide bridges. During folding of these compounds, polymerization and scrambling may occur, which can lead to the formation of impurities. These impurities can aggregate during purification under reversed-phase conditions and clog the column inlet frit or bind to the media itself.
Washing techniques are commonly used to regenerate media, typically by treating it with strong basic solutions that remove performance-damaging impurities from the column frit and/or packed media bed.
Columns are typically replaced when washing procedures are no longer able to restore column performance or when backpressure remains high. Most reversed phase silica-based sorbents can only withstand standard wash conditions of acetic acid, methanol and low concentrations of sodium hydroxide. The use of more aggressive wash conditions offers significant improvements in cleaning effectiveness but can only be carried out with media of wide pH stability.
Polymeric sorbents are able to withstand washing with higher concentrations of sodium hydroxide but they do not provide the needed selectivity or efficiency to make them competitive with silica-based media for protein and peptide purification.