Polyethylene glycol increases purification and recovery, alters retention behavior in flow-through chromatography of hemoglobin

Flow-through chromatography was a method for purification of hemoglobin (Hb) from red cell lysate. The presence of 0-5% polyethylene glycol (PEG) increased the retention time of Hb peak from 15 min to 20 min in flow-through ion-exchange chromatography (IEC) but decreased the retention time from 88 min to 62 min in the hydrophobic interaction chromatography (HIC). However, the purification and the recovery were both increased. For IEC the recovery of hemoglobin increased from 75% to more than 90%, and the purified Hb showed single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and one peak in size-exclusion HPLC. For HIC, the recovery of hemoglobin was improved significantly from 20% to 85% and the removal of lipids was 100%. The bioactivity of hemoglobin was well preserved in these two chromatographic processes. The mechanism for the effect of PEG in these two flow-through chromatographic processes was discussed.