JP2016220701A - Methods for producing and purifying factor viii and its derivatives - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for producing proteins having the Factor VIII procoagulant activity by in vitro culture of mammalian cells in a serum free medium where the serum free medium contains an inhibitor against the proteases released from the cultured cells.SOLUTION: In accordance with this invention, the inhibitor can protect the cleavage of a target protein during cultivation and increase homogeneity of a target molecule, wherein the inhibitor can be a dextran sulfate. This invention also relates to a method of purifying the target molecules from the culture medium containing both the target molecules and the selected inhibitor by affinity chromatography.SELECTED DRAWING: Figure 1

Description

  Factor VIII is a plasma glycoprotein associated with blood clotting. Its functional deficiencies or abnormalities lead to a serious hereditary disease called hemophilia A (Eaton, D. et al., 1986, Biochemistry 25: 505-512; Toole, JJ et al., 1984, Nature 312: 342-347; Vehar, GA et al., 1984, Nature 312: 337-342). To date, the only treatment for hemophilia A has been intravenous administration of Factor VIII made from human blood or recombinant sources. For safety reasons, recombinant factor VIII is preferred over plasma-derived factor VIII. However, because the expression level of factor VIII is about 100 to 1000 times lower than other molecules in the same expression system (Lynch CM, 1993, Human Gene Therapy 4: 259-272), production of recombinant factor VIII is in demand. Could not be satisfied.

  Various attempts have achieved improved expression of factor VIII by removing the B-domain known to have no function in the cofactor activity of factor VIII (Eaton et al , 1986, Biochemistry 25: 8343-8347; Burke, RL et al., 1986, J. Biol. Chem., 261: 12574-12578; Toole, JJ et al., 1986, Proc. Natl. Acad. Sci. USA, 83: 5939-5942; Fay et al., 1986, Biochem. Biophys. Acta, 871: 268-278). US Pat. No. 5,661,008 and WO 91/09122 presented a B-domain deleted form of Factor VIII similar to the shortest form of plasma Factor VIII. US Pat. No. 5,112,950 and US Pat. No. 7,041,635 present a single chain form of a B-domain deleted factor VIII molecule.

  As a choice for mammalian cell expression, the Chinese hamster ovary (CHO) cell expression system has been used to produce a number of therapeutic proteins including Factor VIII (Chu, L et al., 2001, Curr. Opin. Biotehnol). 12: 180-187). The characteristics of the CHO cell line have been elucidated. CHO cell lines can be grown in a fixation-dependent or suspension manner that is compatible with serum-containing or serum-free media, particularly supporting post-translational modifications of proteins that are almost identical to human glycosylation patterns (Brooks SA, 2004, Mol. Biotechnol., 28: 241-255; Jenkins, N., et al., 1996, Nat. Biotechnol., 14: 975-981; Chen, Z., et al , 2000, Biotechnol. Lett., 22: 837-941; Mols, J., et al., 2005, 41: 83-91). For the most part, CHO cells that produce therapeutic proteins were cultured in animal-free protein-free media to resolve the safety associated with the transmission of animal-derived viruses or prions and for easier purification (Chu, L., et al , 2001, Curr. Opin. Biotechnol., 12: 180-187). However, removal of serum from the culture medium also removes naturally contained protease inhibitors in serum supplements, making it difficult to maintain cell viability during the production process (Mols, J., et al., 2005, 41: 83-91; Sandberg, H., et al., 2006, Biotechnol Bioeng., 95: 961-971).

  Reduced viability and stressful environment are seen to increase the production of proteases secreted or released from dead cells, which attacks the therapeutic protein and induces heterogeneity Can do. Heterogeneity induced by internal cleavage of therapeutic proteins can be a major problem because the cleaved protein becomes inactive and maintains “lot to lot” identity during the production and purification process It is difficult to do. It is therefore important to maintain a relatively low level of protease during production and prevent protease activity.

  Although some successful efforts have been reported to prevent such proteolysis induced by proteases released from CHO cell lines during culture, all therapeutic proteins produced from CHO cell lines A universal inhibitor that can be applied has not yet been discovered. Satoh M et al. Report the presence of cysteine and serine proteases released from CHO cells. Chotteau et al. (Chotteau, V., et al., 2001, in Animal cell technology: from target to market, Kluwer Academic publishers, pp. 287-292) have identified unidentified extracellular metal-dependent proteases from CHO cell culture media. It was revealed that it contributes to proteolysis of cleaved factor VIII. WO 90/02175 describes that some serine or cysteine proteases from CHO cell cultures are blocked by an inhibitor peptide, and that the inhibitor increases factor VIII productivity. . European Patent Publication No. 0306968 describes that the addition of aprotinin to the culture medium increased factor VIII expression up to three times in the CHO cell medium.

  In US Pat. No. 5,851,800, the inventors disclosed that metalloprotease and chymotrypsin inhibitors can reduce the deleterious effects on factor VIII production in cell culture. Sandberg H et al. Characterized two proteolytic activities released by CHO cells in cell culture. One originated from a metalloprotease and the other originated from a serine protease. Only metalloproteases were found to have a strong adverse effect on factor VIII activity. However, even if inhibitors of metalloproteases such as EDTA and 1,10-o-phenanthroline were able to block factor VIII cleavage as presented by Sangberg H et al. Judging from the experiments, it cannot be added directly to the CHO cell culture medium due to cytotoxic effects.

  Commercially available protease cocktail containing all the protease inhibitors mentioned above and inhibitors against serine, cysteine, aspartic acid and aminopeptidase such as aprotinin, bestatin, leupeptin, E-64 and pepstatin A Were applied to single chain factor VIII derivative (disclosed in US Pat. No. 7,041,635) cultures, all of which were released from cleavage by proteases released from CHO cell cultures during culture from factor VIII It has been discovered that it is not effective in protecting derivatives.

  US Pat. No. 6,300,100 suggests that sulfated polysaccharides such as heparin protect intact tissue factor pathway inhibitor (TFPI) from cleavage by proteases present in the culture medium. In addition, US Pat. No. 5,112,950 presents sulfated dextran that replaces the stabilizing effect of von Willebrand factor on factor VIII in serum-free medium. However, to the best of our knowledge, there are no reports on the inhibitory effect of dextran sulfate on proteases in relation to factor VIII molecules.

  The present invention aims to demonstrate the protective effect of dextran sulfate against the cleavage of Factor VIII or its derivatives from proteases produced during CHO cell culture.

US Pat. No. 5,661,008 International Publication No. 91/09122 US Pat. No. 5,112,950 US Pat. No. 7,041,635 International Publication No. 90/02175 European Patent Publication No. 0306968 US Pat. No. 5,851,800 US Pat. No. 7,041,635 US Pat. No. 6,300,100

Eaton, D. et al., 1986, Biochemistry 25: 505-512; Toole, J.J. et al., 1984, Nature 312: 342-347; Vehar, G.A. et al., 1984, Nature 312: 337-342 Lynch C.M., 1993, Human Gene Therapy 4: 259-272 Eaton et al., 1986, Biochemistry 25: 8343-8347 Burke, R.L. et al., 1986, J. Biol. Chem., 261: 12574-12578 Toole, J.J. et al., 1986, Proc. Natl. Acad. Sci. USA, 83: 5939-5942 Fay et al., 1986, Biochem. Biophys. Acta, 871: 268-278 Chu, L et al., 2001, Curr. Opin. Biotehnol. 12: 180-187 Brooks S.A., 2004, Mol. Biotechnol., 28: 241-255 Jenkins, N., et al., 1996, Nat. Biotechnol., 14: 975-981 Chen, Z., et al., 2000, Biotechnol. Lett., 22: 837-941 Mols, J., et al., 2005, 41: 83-91 Chu, L., et al., 2001, Curr. Opin. Biotechnol., 12: 180-187 Mols, J., et al., 2005, 41: 83-91 Sandberg, H., et al., 2006, Biotechnol Bioeng., 95: 961-971 Chotteau, V., et al., 2001, in Animal cell technology: from target to market, Kluwer Academic publishers, pp. 287-292

In one aspect of the invention, there is provided a method for producing Factor VIII or a derivative thereof in a mammalian host cell line applied to a serum-free medium supplemented with dextran sulfate. When dextran sulfate is added to the culture medium, (a) the factor VIII-cleavage function of some proteases is effectively reduced or blocked, and at the same time the factor VIII molecule produced Homogeneity increases.
In another aspect of the invention, a method is provided for efficiently purifying factor VIII molecules from dextran sulfate-containing media using a monoclonal antibody dependent purification method.

  The present invention provides an effective inhibitor capable of protecting the single chain factor VIII derivatives disclosed in US Pat. No. 7,041,635 from cleavage by proteases released during mammalian host cell culture and the production It relates to increasing the homogeneity of the factor VIII derivatives. The present invention also relates to a method for purifying the factor VIII without being affected by the addition of the protease inhibitor.

  The mammalian host cell can be any animal cell capable of expressing recombinant factor VIII, preferably an animal cell from which a desired transformed cell can be easily isolated, such as a Chinese hamster ovary (CHO) cell, BHK cells or COS cells, more preferably CHO cells.

  A prior patent, US Pat. No. 6,300,100, describes the protective effect of sulfated polysaccharides on target proteins against several proteases, where the target protein is a tissue factor pathway inhibitor ( TFPI). Therefore, it was tested whether the sulfated polysaccharide could protect the target molecule-factor VIII. The present invention has demonstrated that only dextran sulfate has a very strong protective effect against factor VIII cleavage when added to the culture medium during the culture process.

  Dextran sulfate can be obtained from bacterial fermentation or chemical synthesis. The molecular weight of dextran sulfate can vary with the molecular weight of 20 to 5,000 kDa, preferably 50 to 2,000 kDa.

  The sulfur content of dextran sulfate can also vary depending on its source material. Regardless of the sulfur content of dextran sulfate, if the dextran sulfate can protect factor VIII from cleavage by proteases released from the cell culture process, it can be used in the present invention. The sulfur content of dextran sulfate is preferably in the range of 5 to 20% by weight, more preferably over 17% by weight of the sulfated polysaccharide.

  The amount of dextran sulfate added to the growth medium can be controlled by the expression level of Factor VIII and its host cell line, and is not limited to the preferred embodiment of the present invention.

  In a preferred embodiment of the invention, the factor VIII molecule is a factor VIII derivative named dBN (64-53) (hereinafter referred to as I2GdBN) presented in US Pat. No. 7,041,635. One of them. The Factor VIII derivative was designed to have an internal deletion in part of the B-domain and the N-terminal part of A3 and a new N-glycosylation recognition sequence at the fusion site. Like the method disclosed in Example 6 of US Pat. No. 7,041,635, the CHO cell line stably expressing the I2GdBN was produced and applied to a commercially verifiable serum-free medium. . Hereinafter, the clone is indicated as “# 39 clone”, and all the cells mentioned in the examples refer to the CHO cell line (# 39).

  The present invention also relates to a method for purifying Factor VIII or derivatives expressed in mammalian host cells from culture media supplemented with dextran sulfate using affinity chromatography. The affinity chromatography includes an affinity column containing affinity molecules bound to a solid support such as agarose or sepharose. The affinity molecule may be a peptide having high affinity for monoclonal or polyclonal anti-factor VIII.

Figure 2 represents the comparative effect of different sulfated polysaccharides on protecting intact factor VIII cleavage. FIG. 6 represents the molecular weight and concentration effect of dextran sulfate on fragmentation of the factor VIII, I2GdBN, from which the B-domain has been deleted. Fig. 6 represents the effect of sulfate, dextran and dextran sulfate on I2GdBN fragmentation. FIG. 6 represents the effect of dextran sulfate on I2GdBN fragmentation in perfusion culture according to an embodiment of the invention. FIG. 4 represents a Coomassie brilliant blue R250-stained SDS-PAGE gel of elution fractions from immunoaffinity chromatography according to an embodiment of the present invention.

  The invention is further illustrated by the following examples, which can be applied to other Factor VIII molecules and other cell lines, as will be appreciated by those skilled in the art. Accordingly, the following examples should not be construed as limiting the scope of the invention.

Heparin, low molecular weight heparin (-3 kDa and 4-6 kDa), dermatan sulfate, dextran (500 kDa), sodium sulfate, dextrin sulfate (500 kDa, 10 kDa, 8 kDa) were purchased from Sigma. Dextran was derived from Leuconostoc mesenteroides, strain B512. Different molecular weight dextran sulfate was produced by limited hydrolysis and fractionation. The sulfuric acid group was added by esterification with sulfuric acid under mild conditions. The dextran sulfate contained about 17% sulfur.
(Http://www.sigmaaldrich.com/sigmaaldrich/product_information_sheet/d6001pis.pdf)

Smear of # 39 CHO cell line The aforementioned # 39 clone having a DNA fragment encoding I2GdBN was cultured in a serum-free medium (ProCHO5 medium purchased from Cambrex). After thawing, 4 × 10 ⁵ cells were divided into each well of a 6-well plate at the second passage.

Western blot analysis Culture medium containing the expressed factor VIII was applied to a 7.5% SDS-PAGE gel and blotted onto a PVDF membrane. Blotted membranes were probed with an A2 domain-specific antibody designated # 26-1 produced by the inventors of the present invention. Factor VIII-antibody complexes on the blot were visualized using secondary mouse IgG conjugated with horseradish peroxidase.

Example 1
Comparison of the protective effects of various sulfated polysaccharides on I2GdBN fragmentation High molecular weight dextran sulfate (approximately 500 kDa), heparin, two low molecular weight heparins (-3 kDa and 4-6 kDa), and dermatan sulfate from Sigma Purchased, resuspended in water and filter sterilized. Twenty-four hours after the cells were split as previously mentioned, the medium was replaced with fresh one and five sulfated polysaccharides were added to each well at 25 mg / L, 50 mg / L, 100 mg / L, or 200 mg / L, respectively. Added at a final concentration of L. After culturing for 48 hours, the culture supernatant was removed and analyzed through Western blot analysis. As shown in FIG. 1, there was almost no protective effect of the three heparins that effectively protect TFPI described in other patents. However, dextran sulfate efficiently protected factor VIII cleavage in a concentration-dependent manner. Factor VIII in culture medium without additives (41%; Lane 1 in FIG. 1- (D)) and Factor VIII in culture medium with heparin or dermatan sulfate (52% -67%; FIG. 1- ( Compared with lanes 3 to 6) in D), more than 92% of Factor VIII (lane 2 in FIG. 1- (D)) remained completely in the medium containing dextran sulfate. This indicates that not all sulfated polysaccharides can protect all target proteins, and the protective effect of some sulfated polysaccharides is very specific to the target protein.

Example 2
Effect of molecular weight of dextran sulfate on the cleavage of I2GdBN If a lower molecular weight dextran sulfate can be applied to protect the cleavage, a lower molecular weight dextran sulfate is easier from factor VIII based on its size difference Can be separated. Therefore, in order to investigate whether the lower molecular weight dextran sulfate can protect the cleavage of I2GdBN expressed in cell culture, 8 kDa, 10 kDa and having the same content of sulfur and originating from the same source 500 kDa dextran sulfate was added to the medium at various concentrations of 100 mg / L, 200 mg / L, 400 mg / L, and 1000 mg / L. 72 hours after the addition of dextran sulfate, the culture medium was harvested and analyzed by Western blot analysis. As shown in FIG. 2, the amount of low molecular weight dextran sulfate was increased (lane 1 to lane 8 in FIG. 2) and added to the cell culture medium, but an efficient protective effect against I2GdBN cleavage was completely observed. There wasn't. Only 500 kDa dextran sulfate (lane 9 to lane 12) was found to protect the fragmentation of single chain I2GdBN.

Example 3
Only sulfated dextran can protect the cleavage.
In order to examine whether a separate functional group of dextran sulfate has a cleavage inhibitory effect, equimolar amounts of dextran (500 kDa), sodium sulfate and dextran sulfate (500 kDa) were added to the culture medium. Cells were sorted as described above. After 24 hours of stocking, various concentrations of dextran sulfate (500 kDa) and dextran (500 kDa) ranging from 250 mg / L to 1000 mg / L or various concentrations of sodium sulfate ranging from 71 g / L to 384 g / L are added to the medium. It was. Forty-eight hours after addition, medium was collected from each well and analyzed by Western blot analysis. As shown in FIG. 3, only the 500 kDa dextran sulfate (lanes 5 to 7) showed a protective effect against the cleavage of I2GdBN. It was found that dextran alone (lanes 2 to 4) and sodium sulfate (lanes 8 to 10) could not suppress the protease activity of the protease released during CHO culture.
The fragmentation pattern of the culture medium containing only dextran and sodium sulfate was similar to that of the culture medium without the additive (control group, lane 1).

Example 4
Application of dextran sulfate to suspension culture Dextran sulfate (500 kDa) was applied to the perfusion culture system. One vial of cells was thawed and the cell number was increased in a T75 flask and further increased in a T125 flask. Cells in T125 flasks are transferred continuously to 250 mL, 1 L and 3 L spinner flasks and cultured in suspension culture on a magnetic stirrer plate at 37 ° C. under a 5% CO ₂ / air mixture at a rotation speed of 100 rpm. It was. Cells growing exponentially in a 3L spinner flask were collected and inoculated into a 7.5L bioreactor with a 5L range of motion. Dextran sulfate (500 kDa) was added to the serum-free medium in the bioreactor at a concentration of 200 mg / L.
During culture perfusion, cell viability was maintained above 92.7%, and during the culture process, factor VIII fragments were detected at less than 5%, as judged by densitometric analysis of each band on a Western blot. It was done. An exemplary Western blot of culture media collected on days 6, 12, and 18 is shown in FIG.

Example 5
Purification of I2GdBN from Culture Medium by Immunoaffinity Chromatography Due to the strong negative charge feature of dextran sulfate, ion exchange chromatography can be applied to purify factor VIII secreted in culture medium supplemented with dextran sulfate. Can not. Therefore, the culture medium purified in Example 4 was concentrated by tangential flow ultrafiltration and pre-equilibrated with parallel buffer (20 mM Tris-HCl [pH 7.0], 400 mM NaCl, 5 mL CaCl ₂ , 3 mM EDTA). Applied to the immunoaffinity column. An immunoaffinity column was prepared by binding a monoclonal anti-factor VIII that recognizes the A2 site of the factor VIII heavy chain to CNBr-activated sepharose. A factor VIII-conjugated immunoaffinity column was added to 2.5 layers of equilibration buffer and 2.5 layers of wash buffer (20 mM Tris-HCl [pH 7.0], 400 mM NaCl, 5 mL CaCl ₂ , 3 mM Washed with EDTA, 10% ethylene glycol). Elution was performed with step gradient elution using an elution buffer containing ethylene glycol at concentrations ranging from 40-60%. Nine of the 12 elution fractions (elution fractions 1 to 9 corresponding to E1 to E9 in FIG. 5) were sampled and applied to 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue R250 dye. did. As illustrated in FIG. 5, single-stage purification of the culture medium alone provided very pure single-chain factor VIII exceeding 95%.

The present invention may also be the following aspects.
[1] Recombinant factor VIII is produced from mammalian host cells transformed with an expression DNA vector containing cDNA encoding FVIII or FVIII derivative in the culture medium, and the factor VIII is bound to a solid support. A method of purification using a factor VIII-specific affinity molecule, wherein (a) the mammalian host cell is cultured in a culture medium supplemented with dextran sulfate;
(B) concentrating the culture medium containing the factor VIII through ultrafiltration;
(C) A method comprising purifying the Factor VIII from the concentrated culture medium by an immunological method.
[2] The method according to [1], wherein the dextran sulfate has an average molecular weight of 20 to 5,000 kDa.
[3] The method according to [1], wherein the amount of the sulfated polysaccharide in the culture medium is 10 mg / L to 2 g / L.
[4] The method according to [1], wherein the culture medium is a medium containing no animal protein.
[5] The method according to [1], wherein the mammalian host cells are CHO, BHK and COS cells.
[6] The method according to [1], wherein the immunological method is immunoprecipitation or immunoaffinity chromatography.
[7] The chromatography is
(A) a column packed with a solid support comprising agarose and sepharose bound to an anti-factor VIII specific antibody; and (b) a factor VIII molecule bound to the solid support to which the antibody is bound. The method according to [6], comprising an elution buffer containing a buffer, a salt, calcium chloride, a surfactant and ethylene glycol.

Claims (9)

A method for producing recombinant factor VIII or a derivative thereof from a mammalian host cell transformed with an expression DNA vector containing a cDNA encoding factor VIII or a factor VIII derivative in a culture medium, comprising:
Supplementing the culture medium with dextran sulfate having an average molecular weight of 20-5000 kDa to reduce the factor VIII-cleaving activity of the protease released during cell culture, wherein the factor VIII or derivative thereof is dBN (64-53).   The method according to claim 1, wherein the amount of dextran sulfate in the culture medium is 10 mg / L to 2 g / L.   The method according to claim 1, wherein the culture medium is a medium not containing animal protein.   2. The method of claim 1, wherein the mammalian host cell is a CHO, BHK or COS cell. Producing recombinant factor VIII or a derivative thereof from a mammalian host cell transformed with an expression DNA vector containing a cDNA encoding factor VIII or a factor VIII derivative in a culture medium, and said factor VIII or a derivative thereof A factor VIII-specific affinity molecule bound to a solid support, comprising:
(A) culturing the mammalian host cell in a culture medium supplemented with dextran sulfate having an average molecular weight of 20-5000 kDa to reduce the factor VIII-cleaving activity of the protease released during the cell culture;
(B) a step of concentrating the culture medium containing the factor VIII or a derivative thereof through ultrafiltration; and (c) purification of the factor VIII or a derivative thereof from the concentrated culture medium by an immunological method. The process of
Wherein the factor VIII or derivative thereof is dBN (64-53).   The method according to claim 5, wherein the amount of dextran sulfate in the culture medium is 10 mg / L to 2 g / L.   The method according to claim 5, wherein the culture medium is a medium not containing animal protein.   6. The method of claim 5, wherein the mammalian host cell is a CHO, BHK or COS cell.   6. The method of claim 5, wherein the immunological method is immunoprecipitation or immunoaffinity chromatography.

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