Purification of factor viii using a mixed-mode or multimodal resin

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A method for purifying a recombinant protein using a multimodal or mixed mode resin containing ligands which comprise a hydrophobic part and a negatively charged part is described. The invention is advantageous in that it is a single step chromatographic process which does not require adjustment of pH or conductivity during loading step and results in high yield and potency. The process is used for the purification of recombinant compositions of coagulation factor, particularly recombinant Factor VIII.

Field of the Invention The present invention is related to a method for purifying a recombinant protein using a multimodal or mixed mode resin containing ligands which comprise a hydrophobic part and a negatively charged part. The protein of interest is a coagulation factor, particularly relevant to the purification of compositions of recombinant Factor VIII.

Human Factor VIII, also known as antihaemophilia factor or FVIII:C is a human plasma protein consisting of two polypeptides with light chain molecular weight of 80,000 daltons and a heavy chain molecular weight variable from 90,000 to 220,000. It is considered as one of the key cofactors in the coagulation pathway necessary for the conversion of Factor X into its active form Factor Xa. Factor VIII circulates in plasma as a non-covalent complex with von Willebrand Factor (also known as FVIII : RP). Hemophilia, a bleeding disorder is caused due to abnormal levels of Factor VIII. Factor VIII levels below 20% normal may result in hemophilic condition in humans. A drop in the levels of less than 1% of Factor VIII leads to severe bleeding disorder, with spontaneous joint bleeding being the most common symptom.

The structure and biochemistry of recombinant factor VIII have been described previously.

Traditionally, isolation and purification of Factor VIII has been from a plasma derived source (cryoprecipitate). Purification procedures from plasma-derived sources include those exploring the use of immunoaffinity purification using polyclonal and monoclonal antibodies for the purification of FVIII. However, there may be instances where the Factor VIII effluent contains some residual antibody due to leaching from the support matrix, which may result in antigenicity during ultimate use, i.e when introduced into human or animal system. Purification procedures exploring the use of ion exchange chromatography on e.g. agarose beads have also been used for purification of factor VIII from plasma. These methods, however, often suffer from certain levels of contamination of the resulting FVIII: C. However, purification of Factor VIII from genetically engineered recombinant source has gained importance in the past decade. Protein recovery and concentration of the final product is of utmost importance in the separation of recombinant proteins. The contaminants in recombinantly produced protein may include secreted proteins in the culture medium, media components, cell lysates, unwanted proteins produced by the cells and the nucleic acids.

When purifying a recombinant protein, the aqueous source materials in which the polypeptides of interest are found are furthermore often seen to be contaminated with one or more viruses. Techniques for inactivating viruses in polypeptide mixtures are known in the art, such as e.g. chemical methods, using solvent/detergent solutions, irradiation methods, or thermal methods, but attempts to combine such techniques with known polypeptide purification processes have produced methods with a multiplicity of steps unsuitable for large-volume production. It is also important to exert caution in that the used viral-inactivating agents do not denature the protein or are difficult to separate from the protein of interest. These agents have, however, been either denaturing or difficult to separate from the polypeptide of interest, and have required a special treatment or separation step. Other conventional methods for treating polypeptide-containing preparations for potential viral contamination, such as heat or irradiation, have resulted in either significant denaturation of the polypeptide of interest and/or insufficient inactivation of viruses. Many of the commercially available recombinant Factor VIII products (Advate®, Helixate®, Kogenate FS®, ReFacto®) are made using immunoaffinity chromatography including a detergent for purification and viral inactivation.