WO2014075205A1 - Method for purifying blood coagulation factor viii and the variants thereof - Google Patents

Abstract

A method for purifying blood coagulation factor VIII by using antibody fragments. The antibody fragments may be single-chain variable fragments and can be produced by bacterial expression.

Description

 Method for purifying coagulation factor and its variants

 The present invention discloses a method for purifying coagulation factor by antibody fragment. Back ft technology Coagulation factor 8, FVIII is a protein that plays an important role in blood coagulation. Defects in coagulation factor eight cause hemophilia A (Nature 312: 342-347, 1984; Nature 312: 337-342, 1984). Hemophilia is a serious genetic disease. From the birth of the patient, bleeding will occur repeatedly. This type of bleeding, not only in the case of trauma or surgery, but also spontaneously occurs in the joints, muscles, etc. Over time, it can cause chronic arthritis and eventually lead to disability. If intracranial hemorrhage or laryngeal bleeding occurs, it will directly endanger life.

 The only treatment for hemophilia A is intravenous injection of eight factors. The coagulation factor can be purified from plasma or can be produced by mammalian cell culture by genetic engineering. Recombinant coagulation factor VIII is better because plasma materials are potentially infectious. However, since the coagulation factor VIII is large and difficult to express, the coagulation factor of the cell synthesis is difficult to secrete extracellularly, and the coagulation factor released into the extracellular culture medium is easily degraded, and the yield of genetically engineered coagulation factor VIII is higher than that. The yield of other molecules is several hundred to several thousand times lower (Human Gene Therapy 4: 259-272, 1993; Blood Cells, Molecules, and Diseases. 28: 234-248, 2002; Blood. 103: 3412-3419, 2004). Therefore, genetic engineering recombinant coagulation factor VIII production costs are rampant and expensive. In the past, there have been many attempts to increase the coagulation factor production. There are two main strategies for increasing the production of coagulation factor VIII. The first is to increase the yield of coagulation factor in mammals upstream, and the second is to increase the purification efficiency downstream.

 One of the main strategies for increasing the yield of coagulation factor VIII is to co-express coagulation factor VIII with vWF (von Willebrand factor) (J. Biol. Chem. 263, 6352-6362, 1988; Mol. Cell. Biol. 9, 1233- 1242, 1989; J. Biol. Chem. 266, 21948-21955, 1991). Coagulation factor can also be expressed in combination with other molecules to increase expression. For example, Fontes et al. combined the expression of coagulation factor VIII with the P140K gene to increase the expression of coagulation factor VIII in 293T cells (Genet Mol Res. 11 (1): 775-89, 2012).

 The B domain of coagulation factor VIII does not play a role in clotting activity, and the coagulation octa-factor variant of B domain is removed, and the molecule is smaller than the full-length molecule, and the expression rate is high (US Patent No. 5, 661, 008, W0- A-91/09122, U.S. Patent No. 5,112,950, U.S. Patent No. 7,041,635. Adding certain components to the culture medium, increasing the expression of coagulation factor or reducing the degradation of coagulation factor VIII is another major strategy for increasing the yield of octafactors. The addition of exogenous vWF factor to the culture medium can reduce the coagulation factor VIII degradation in combination with coagulation factor (J. Biol. Chem. 263:6352-6362, 1988). In the medium, ortho-phospho-L-serine (0PLS) was added to reduce the binding of coagulation factor to the cell membrane to increase expression (J. Biotechnol. 151: 357-62, 2011). The addition of high molecular weight dextran sulfate to the culture medium also reduces the secretion of coagulated eight factor degradation (U.S. Patent Application No. US20100112641). In terms of improving the purification yield, the use of immobilized antibodies to purify the antigen coagulation factor to improve purification recovery is a widely used strategy (US Patent No. 5, 470, 954, US Patent No. RE32011), and an ion exchange layer. Pure

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Confirmation Compared with the chemical method, the recovery rate of affinity chromatography is higher, and the purity of the obtained protein is higher. All of these antibody affinity chromatography methods employ full length antibody molecules. Full length antibody molecules need to be obtained by hybridoma cell culture or other mammalian cell culture. The cost of preparing full length antibody molecules by mammalian cell culture methods is relatively high, which accounts for a significant proportion of the total cost of coagulation factor production. In the present invention, a coagulation factor VIII or a vWF factor antibody fragment molecule, which is usually immobilized by a single chain antibody fragment (scFv) molecule, is used for separation and purification of coagulation factor VIII. SUMMARY OF THE INVENTION Immunoaffinity purification of "coagulation eight factors" using antibody fragments instead of intact antibody molecules.

 Say

 Antibody Fragments scFvs are usually prepared by expression production using bacteria. The full length of the antibody is usually prepared by mammalian cell culture for expression production. Compared to mammalian cell culture, production with bacteria can significantly reduce production costs. Book

 Many full length antibody molecules have varying degrees of antigen neutralization activity that reduce or eliminate antigen activity. Many scFvs do not have the function of neutralizing antigens, but only have the function of binding antigens. Purification of the "coagulation factor" by scFv without antigen neutralization can maximize the activity of the "coagulation factor" purification process, thereby improving the activity of purifying the "coagulation eight factor" drug and improving the drug quality accordingly. The scFv has no Fc portion in the full-length molecule of the antibody, which can reduce the non-specific adsorption of Fc, thereby improving the purity of the "coagulation factor" and correspondingly improving the quality of the drug. The molecular weight of scFv is generally about 30KD. The full length antibody has a molecular weight of approximately 150 kD. The genetic engineering "coagulation factor" molecular weight is approximately 300KD. In the "coagulation factor" affinity purification process, whether it is immobilized full-length antibody molecule or scFv, a certain shedding occurs during use. These exfoliated antibodies or scFv are impurities in the drug and are removed in subsequent molecular sieve chromatography. Molecular sieve chromatography is isolated and purified according to the size difference between molecules. Due to the small size of the scFv molecule, the difference in size from the "coagulation factor" molecule can be more effectively removed in molecular sieve chromatography, thereby improving the purity of the "coagulation eight factor" and correspondingly improving the quality of the drug. The implementation of the technology of the present invention can greatly reduce the production cost of the "coagulation eight factor" drug and significantly improve the quality of the drug. DRAWINGS

No specific implementation

Monoclonal antibody preparation

The antibody preparation method may be a mouse hybridoma method, or a phage display or the like. The antigen prepared by the monoclonal antibody may be a purified plasma protein, a purified full-length recombinant protein or variant, or a synthetic polypeptide. Instruction manual

 2. Antibody fragment preparation of scFv

 Antibody Fragments The commonly used structure of scFv is the heavy chain variable region (VH) - Linker - Light Chain Variable Region (VL). Commonly used in the junction area (Gly4Ser) 3. The light chain variable region and heavy chain variable region sequences of the antibody can be obtained by PCR. When an antibody sequence is known, antibody fragments can be obtained synthetically. Antibody fragments can be expressed in bacterial, yeast, and mammalian cells. It is commonly expressed in E. coli. Purification of the antibody fragment can be affinity-purified with protein A or protein G or protein L, or affinity-purified with antigen coagulation factor VIII, or purified by ion exchange or the like.

3. Antibody fragment scFv tandem solid medium

 The solid substrate can be agarose, Sepharose, or other matrix for ligand coupling, such as size controlled glass, silicon, and the like. The activation method of the solid substrate may be CNBr activation, NHS activation, epoxy activation, and other activation methods.

4. Immobilized antibody fragment scFv affinity purification process

 The immobilized antibody fragment was loaded onto a chromatography column. Raw materials containing coagulation factor VIII, such as plasma or recombinant cell line culture medium, are diluted or adjusted for pH and applied to the column. The antibody fragment chromatography column was washed with a buffer. Salts, detergents, etc. may be added to the buffer. Coagulation factor VIII can be eluted with buffer.

Claims

Claim 1. A method for separating and purifying coagulation factor VIII and its variants using antibody fragments.  2. According to claim 1, the coagulation factor can be a full-length coagulation factor, a B domain deletion variant, a point mutation variant, or other variant.  3. According to claim 1, antibody fragments can be derived from antibodies against anticoagulant octafactors and variants thereof.  4. According to claim 1, in the case of vWF co-expression or vWF in the raw material, the antibody fragment may be derived from antibodies to vWF and its variants.  5. According to claim 1, the antibody fragment can be an scFv, or other antibody fragment molecule.  6. According to claim 1, antibody fragments can be produced in bacteria, yeast, and mammalian cells, and are more suitable for production with bacteria, and are most suitable for production with E. coli.  7. According to claim 1, antibody fragments can be derived from humans, mice, rabbits, and sheep.  8. According to claim 1, the material containing coagulation factor VIII may be a blood paddle or a recombinant coagulation octa factor cell culture medium.  9. According to claim 1, when the raw material containing coagulation factor is cell culture solution, the cells may be CH0, BHK or other animal or human cells, and the culture solution may be serum-containing or serum-free.  10. According to claim 1, the immobilized antibody fragment can be used in place of the immobilized antibody or as a complementary process to further purify the coagulation factor.