(239b) Heme Extraction and Separation for the Preparation of Apohemoglobin

Background: In this study we outline the development of a procedure to extract and separate the iron containing heme group from the blood protein hemoglobin (Hb) to produce apohemoglobin (apoHb) using a tangential flow filtration (TFF) extraction and purification procedure. ApoHb is a protein derived biomolecule currently being investigated as a delivery vehicle for hydrophobic molecules targeting the CD163 clearance pathway in monocytes and macrophages. Previous strategies to prepare this biomolecule use either an acidified-acetone precipitation or a methyl ethyl ketone liquid-liquid extraction. Unfortunately, these procedures require large volumes of highly flammable, chemically incompatible solvents that necessitate the use of expensive separations equipment.

Methodology: To facilitate scale-up of apoHb production, we developed a procedure for apoHb extraction that combines extraction with purification within a single liquid phase. In addition, during the development of this process, we also developed methods to analyze these materials with an assay to target the functionality of the hydrophobic heme-binding pocket of Hb. Here, an acidified ethanol-based solution was used to protonate the proximal histidine residue, partially unfold the Hb protein, and extract the heme group. The resulting material then underwent diafiltration to continue extraction by separating the apoHb from the heme solution. After separation, total protein and active protein yields were quantified via the molar extinction coefficient of apoHb and a heme-binding pocket activity assay, respectively. Analysis of the Soret peak was used to determine residual heme content. Mass spectrometry and SDS-PAGE were used to confirm the chemical identity and purity of the apoHb. The storage stability of the apoHb product was analyzed at varying temperature. Size exclusion chromatography was used to verify haptoglobin binding was similar to native Hb. Finally, heme was reincorporated into apoHb to produce reconstituted Hb (rHb) for biophysical characterization of the O₂ equilibrium curve, O₂ dissociation and CO association kinetics of the rHb.

Results and Conclusions: The new apoHb-TFF process resulted in total protein yields of more than 95% and active protein yields of approximately 75%. The apoHb had less than 1% residual heme and SDS-PAGE indicated more than 99% purity of apoHb with an identical globin chemical profile to Hb, except for the absence of heme. Storage studies demonstrated no loss of heme-binding activity in solutions at 4 °C, -80 °C, and in lyophilized form over extended periods of time. Size exclusion chromatography confirmed that apoHb bound to haptoglobin. Furthermore, the mass of the α and β subunits match the theoretical mass of the α and β subunits of human Hb. Biophysical properties of rHb were approximately the same as native Hb. Overall, this study describes a novel and improved method to produce apoHb. This scalable and effective method for purification can prepare sufficient materials for much preclinical and clinical studies that aim to use apoHb as a drug encapsulation and delivery biomolecule.