Purification of coagulation factor VIII by immobilized metal affinity chromatography

Abstract

Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.

Biosynthesis and purification of histidine‐tagged human soluble catechol‐O‐methyltransferase

“…Immobilized‐metal affinity chromatography (IMAC) has emerged over the last decade as one of the most widely used methods at the laboratory scale to isolate proteins from fermentation broths, mammalian cell culture supernatants or other biological sources . Over the years, several proteins with different topologies had been successfully purified using IMAC as the main chromatography step such as the human growth hormone, immunoglobulins, the coagulation factor VIII or membrane‐bound proteins such as the dehydrogenase/reductase SDR family member 7 . The IMAC is a separation technique in which metal ions entrapped on solid chromatographic supports serve as affinity ligands for several proteins, usually histidine‐tagged fusion proteins .…”

Covalent immobilization of coagulation factor VIII on magnetic nanoparticles for aptamer development

“…FVIII is a valuable therapeutic protein that has been purified from plasma or recombinant sources by different chromatography processes, especially affinity chromatography. 52,53 In this regard, Fe 3 O 4 MNPs was first synthesized by a co-precipitation method, and then a silica coating was applied to stabilize the iron-based magnetic core. Then, a second layer of functional silica was coated onto the MNPs to improve their reactivity to the NH 2 or OH groups of proteins.…”

Polysaccharide-based chromatographic adsorbents for virus purification and viral clearance

“…IMAC and HIC have been seldom applied to the purification of viral/subviral particles and VLPs (see sections 2.1.5 and 2.1.6, respectively). IMAC has also been used occasionally to purify plasma coagulation factors: factor VIII [241] and, more particularly, factor IX [231,242]. Roberts [231,242] assessed the virus elimination efficiency of the chromatographic process during the manufacture of Replenine®-VF (Bio Products Laboratory), a human plasma-derived concentrate of coagulation factor IX [243].…”