TY - JOUR
T1 - Purification of hemoglobin by ion exchange chromatography in flow-through mode with PEG as an escort
AU - Lu, Xiuling
AU - Zhao, Dongxu
AU - Su, Zhiguo
PY - 2004
Y1 - 2004
N2 - Development of hemoglobin-based blood substitutes requires production of highly purified hemoglobin. Process of hemoglobin purification by ion exchange chromatography in flow-through mode was researched and optimized. Three kinds of media including, QMA Spherosil LS (Biosepra, France) and Q Sepharose Big Beads (Amersham Bioscience, Sweden), and an anion exchange membrane column, Mustang Q (PALL, USA) were investigated and compared. Adding polyethylene glycol (PEG) as an escort in ion exchange chromatography improved the purity and recovery, and the recovery in the chromatography was increased from 75 to 95%. The mechanism of PEG effects on chromatography was discussed. The optimal chromatography step, in combination with hypotonic dilution hemolyzing and membrane separation, formed an integrated hemoglobin purification process. The total recovery in the process was 87.6%. The activity of hemoglobin was well preserved: P50 23.2 mmHg, and Hill coefficient 2.31. The product appeared as a single band in SDS-PAGE, and GF-HPLC showed only one peak. The purity of the prepared hemoglobin was more than 99.9%. The optimized process is time saving and suitable for large-scale preparation of hemoglobin to provide materials for further preparation of blood substitutes.
AB - Development of hemoglobin-based blood substitutes requires production of highly purified hemoglobin. Process of hemoglobin purification by ion exchange chromatography in flow-through mode was researched and optimized. Three kinds of media including, QMA Spherosil LS (Biosepra, France) and Q Sepharose Big Beads (Amersham Bioscience, Sweden), and an anion exchange membrane column, Mustang Q (PALL, USA) were investigated and compared. Adding polyethylene glycol (PEG) as an escort in ion exchange chromatography improved the purity and recovery, and the recovery in the chromatography was increased from 75 to 95%. The mechanism of PEG effects on chromatography was discussed. The optimal chromatography step, in combination with hypotonic dilution hemolyzing and membrane separation, formed an integrated hemoglobin purification process. The total recovery in the process was 87.6%. The activity of hemoglobin was well preserved: P50 23.2 mmHg, and Hill coefficient 2.31. The product appeared as a single band in SDS-PAGE, and GF-HPLC showed only one peak. The purity of the prepared hemoglobin was more than 99.9%. The optimized process is time saving and suitable for large-scale preparation of hemoglobin to provide materials for further preparation of blood substitutes.
KW - Blood substitutes
KW - Hemoglobin
KW - Ion exchange chromatography
KW - Process integration
KW - Protein purification
UR - http://www.scopus.com/inward/record.url?scp=3042551057&partnerID=8YFLogxK
U2 - 10.1081/BIO-120037828
DO - 10.1081/BIO-120037828
M3 - Article
C2 - 15274429
AN - SCOPUS:3042551057
SN - 1073-1199
VL - 32
SP - 209
EP - 227
JO - Artificial Cells, Blood Substitutes, and Immobilization Biotechnology
JF - Artificial Cells, Blood Substitutes, and Immobilization Biotechnology
IS - 2
ER -