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A new resin for purification of recombinant FVIII

IP.com Disclosure Number: IPCOM000170274D
Publication Date: 2008-May-14
Document File: 3 page(s) / 13K

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Abstract

The invention discloses a new affinity resin for purification of recombinant FVIII. The resin uses a ligand made of single domain heavy chain antibody fragments from Llamas. The ligand can be immobilized to agarose via NHS activation to give a resin with excellent selectivity and is suitable for industrial use. Coupling chemistries such as BrCN, expoxy, allyl, alkylhalo and other techniques for attachment of a protein ligand can also be used. The ligand can be immobilized on other sugar polymers as well as synthetic or ceramic beads. In an example the ligand includes a 13 kD recombinant protein produced in S.cerevisiae.

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A new resin for purification of recombinant FVIII

The purification of FVIII is of great interest due to the increasing market of biopharmaceuticals consisting of recombinant produced anti-coagulation factors such as FVIII. Purification of FVIII can be done using conventional ion exchange and/or hydrophobic interaction chromatography as well as affinity chromatography. These multi-step processes usually give a low yield of FVIII since it is a difficult protein to purify. The number of purification steps should be kept as few as possible to minimize inactivation of FVIII. It is crucial to maintain the specific activity of factor VIII during its purification for the quality of the final product.

To overcome problems in recombinant FVIII purification, an affinity resin has been developed. The resin uses a ligand made of single domain heavy chain antibody fragments from Llamas. The ligand can be immobilized to agarose via NHS activation to give a resin with excellent selectivity and is suitable for industrial use. Coupling chemistries such as BrCN, expoxy, allyl, alkylhalo and other techniques for attachment of a protein ligand can also be used. The ligand can be immobilized on other sugar polymers as well as synthetic or ceramic beads.

The ligand, which is a 13 kDa recombinant protein produced in S.cerevisiae, has showed high affinity towards recombinant FVIII.  This makes the resin an excellent choice for purification of recombinant FVIII. Due to multiple binding points between ligand and base matrix the leakage should be minimized. A single point attachment would be more sensitive to leakage but would, on the other hand, probably result in higher capacity. Possible leakage from the resin is likely to be decreased with extensive washing at either high or low pH or with increased amounts of salt and other additives.

Recombinant FVIII can be applied directly from clarified cell lysates or supernatants. One typical example on equilibration and washing buffer is 100 mM ammonium acetate buffer pH 6,8; 400 mM NaCl; 5mM CaCl2; 1 % Triton X-100 and 0,3% tri-n-butyl phosphate used on a peptid based FVIII affinity ligand resin (Brian D. Kelly et al; Journal of Chromatography, Vol. 1038, 121-130, 2004). The equilibration and washing buffer is not limited the stated pH, concentrations, type of buffer, salt for ionic strength, detergent and phosphate. Washing may also be done in the presence of organic solvents.

 Buffers should always contain Ca2+ ions in order to promote the formation of the active conformation of FVIII. Usually NaCl is needed to increase the ionic strength in order to stabilize the molecule, however we have indications that it is possible to use low ionic strength with another additive present. A surfactant (Tween, Triton or other detergents) is usually needed to inhibit surface induced denaturation (Wei Wang et al; International journal of pharmaceutics (Review), 259, 1-15, 2003).

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