The egg white protein avidin is known for its strong binding to biotin. Because of various applications in bioanalytics and biopharmaceutical industries, there is a great interest in the manufacturing of highly purified avidin. For the first time, the distribution behavior of avidin and the major contaminants occurring in egg white, ovalbumin, ovotransferrin (conalbumin), ovomucoid (ovotrypsin-inhibitor) and lysozyme in aqueous two-phase systems (ATPS) are presented. The results revealed a high capability of PEG/phosphate systems for the intermediate purification of the highly valuable protein avidin from chicken egg white solutions. Several PEG/phosphate systems varying in PEG molecular weight, pH and NaCl concentration were investigated in an automated small scale screening performed on a liquid handling station. The high-throughput screening was used to determine binodal curve and tie-lines as well as protein distribution and recovery. Tandem Reversed Phase HPLC was applied for very fast quantification of both impurities and active (biotin binding) avidin within 4.25 min per sample. The main finding was that avidin partitioned in almost all ATPSs to the salt-rich bottom phase while distribution of impurities depended strongly on chosen experimental conditions. Starting from a pre-purified avidin solution containing 11% (c/c) avidin, in the most effective small-scale ATPS, a purification factor of 5.7 was obtained with an avidin yield of 92% in the bottom phase. In this particular system, a removal of ovalbumin of 65%, ovomucoid of 99.4%, lysozyme of 99.7% and ovotransferrin of 95.4% was achieved. The screening results were confirmed in common lab scale experiments representing a scale up by factor 15.
Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using high-throughput techniques
Patrick Diederich, Sven Amrhein, Frank Hämmerling, Jürgen Hubbuch
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Chemical Engineering Science