deltaPhaseTM Purification
The deltaPhase recombinant expression/purification system is based on the transition properties of ELPs and their ability to retain this inverse temperature phase transition when conjugated to other molecules. The phase transition offers a new method of purification for therapeutic proteins and peptides. The process consists of fusing an ELP sequence to the N or C terminus of the polypeptide of interest by recombinant DNA techniques. The DNA coding for the polypeptide or protein tagged with an ELP is introduced into an expression system (e.g. E. coli or mammalian cells) for production (Figure 2).
Figure 2. deltaPhase Expression/Purification
Once the protein ELP-fusion is produced, the cells are lysed (E. Coli expression) or the culture supernatant is collected (mammalian expression), the ELP fusion protein is phase transitioned to form insoluble aggregates which are isolated by centrifugation or filtration. After isolating the aggregated ELP-protein, it is resolubilized by decreasing the temperature. The ELP may then be cleaved enzymatically from the fusion protein. Another cycle of phase transition purification will separate any uncleaved product from the ELP which remains in the insoluble fraction and leaves behind the purified product in the soluble fraction.
This simple three step process provides a number of unique competitive advantages over both traditional recombinant methods and chemical synthesis:
- High Throughput. Since the expression and purification can be accomplished within a day using simple techniques, such as centrifugation, pharmacologic screening of purified proteins can be handled quickly and simply.
- Parallel Processing. The high throughput nature of the process lends itself to parallel production. As many as 96 samples can be processed in a batch in only one week.
- Seamless Scalability. The use of centrifugation or filtration to separate ELP aggregates can be applied with little or no modification to 10mL, 10 L or 100 L volumes.
- High Yields. Recombinant expression is efficient for short peptides because the ELP protects the peptide from proteolysis.
- Improved Solubility. ELP-protein fusions are stored at concentrations of 200mg/mL.
We believe that the deltaPhase technology offers an innovative method for protein/polypeptide purification and avoids costly chromatography. PhaseBio's deltaPhase technology represents the first new method for production and purification of biologics in 20 years and has led to a number of important research collaborations with biotechnology and pharmaceutical partners.