Albumin and albumin-related proteins such as albumin fusion proteins represent an increasingly important platform for production of therapeutic proteins with extended in vivo half-lives. Affinity ligands directed to the albumin portion of the molecule are ideally suited to development of a purification platform for albumin containing proteins. In addition, significant reductions in manufacturing costs are possible, especially if processing times can be shortened through use of higher flow rates.
Introduction
Mimetic Blue® SA ligand is selective for human serum albumin and commonly used for the purification of human albumin and albumin fusion proteins. Optimization of the PuraBead® base matrix (6% beaded agarose) cross-linking chemistry and Mimetic Blue® SA ligand coupling has led to the development of an improved adsorbent called Mimetic Blue® SA HL P6HF. This new SAHLP6HF product retains the excellent selectivity and high binding capacity (up to 30 g albumin/L) provided by Mimetic Blue® SA HL P6XL product but has significantly improved pressure/flow properties.
Materials and Methods
The pressure-flow rate relationship of Mimetic Blue® SA adsorbents was measured using a 10-cm diameter column (20 cm bed height) with 50 mM sodium phosphate buffer containing 25 mM NaCl, pH 7.0. Albumin fusion protein was purified from yeast lysate using loading conditions at pH 7.0 and eluting with 30 mM caprylate.
Results
Figure 1 summarizes the pressure versus flow relationship for Mimetic Blue® SA HL P6XL (blue) and the new Mimetic Blue® SAHLP6HF (red) adsorbents. The results demonstrate that at 15 psi the linear flow rate for the new HF product is threefold higher than XL product, and at 30 psi there is a fivefold difference.
Figure 2 is the nonreduced SDS-PAGE of the chromatography samples, which indicates that nontarget proteins and impurities flow through the columns, and the elution fractions contain concentrated, purified albumin fusion protein. The dynamic binding capacity and elution recovery data indicates that the XL and HF products perform comparably, both exhibiting a dynamic binding capacity and elution recovery of approximately 10 g/L. Experiments conducted with different albumin containing feeds demonstrate dynamic binding capacities up to 30 g/L are possible.
Conclusions
Mimetic Blue® SA HL P6HF can sustain a linear flow rate of 1,000 cm/h at 30 psi (10 cm column diameter×20cm bed depth), fivefold higher than Mimetic Blue® SA HL P6XL. Both adsorbents perform comparably for the purification of albumin fusion protein from yeast lysate.
Author Details
Cory J. Pigeon (cpigeon@prometicbiosciences.com) and Mark Jackson (mjackson@prometicbiosciences.com) are the commercial directors at ProMetic BioSciences Ltd. 211 Cambridge Science Park, Milton Road, Cambridge CB4 0WA UK; www.prometicbiosciences.com. ProMetic BioSciences Ltd and the PBL logo are trademarks of ProMetic BioSciences Ltd. Mimetic Blue® and PuraBead® are registered with the U.S. Patent and Trademark Office.