KR20060038210A - Monoclonal anti factor viii antibody for purification of coagulation factor viii - Google Patents

Abstract

The present invention relates to a mouse monoclonal antibody for purification of coagulation factor 8, and more specifically, to coagulation with human blood coagulation factor 8 (Factor VIII) in a specific and high affinity, only high concentration of coagulation factor 8 The present invention relates to a mouse monoclonal antibody which can be isolated and purified purely, a gene encoding a heavy or light chain of the antibody, and a hybridoma producing the antibody.

Coagulation factor 8, tablets, monoclonal antibody, hybridoma

Description

Monoclonal Antibodies for Purification of Human Blood Coagulation Factor 8             

FIG. 1 shows the result of electrophoresis on SDS-polyacrylamide gel after purification of recombinant coagulation factor 8 (FVIII) for the production of antibodies specifically binding to human blood coagulation factor 8 (FVIII).

Figure 2 shows the results of Coomassie staining (a) and Western blot by electrophoresis of recombinant FVII (refecto) on SDS-polyacrylamide gel to determine the recognition site of FVIII using the monoclonal antibody of the present invention. (b) is shown.

Figure 3a is a schematic diagram showing the location and size of the five fragments of FVIII to determine the recognition site of FVIII using the selected monoclonal antibody.

Figure 3b is the result of Western blot the site recognized by the antibody selected using five fragments of FVII.

Figure 4 shows the result of ELISA using sequential dilution to determine the reactivity of the selected monoclonal antibody to the antigen.

Figure 5 shows the nucleotide sequence encoding the variable region of the heavy chain portion of the mouse monoclonal antibody and the amino acid sequence translated therefrom.

Figure 6 shows the nucleotide sequence encoding the variable region of the light chain portion of the mouse monoclonal antibody and the amino acid sequence translated therefrom.

The present invention relates to a monoclonal antibody for the purification of human coagulation factor 8, more specifically, to coagulation factor 8 (Factor Ⅷ (F Ⅷ)) in a specific and high affinity to bind only the coagulation factor 8 high concentration A mouse monoclonal antibody that can be isolated and purified purely, a gene encoding a heavy or light chain of the antibody, and a hybridoma producing the antibody.

There are 13 known factors related to blood coagulation. Blood coagulation factor 8 (FⅧ) is a coenzyme that helps factor 9 promote the activity of factor 10, fibrinogen. It is converted into fibrin and helps to form a stable fibrin network. FVIII circulates in vivo in complex with the von Willebrand Factor (vWF) factor, the portion of the complex having the coagulation activity of the factor VIII coagulation protein, FV coagulation activity or simply FVIII: The other part that is not C coagulation function is called factor Ⅷ related antigen, F Ⅷ: Ag, F Ⅷ: RP factor, vWF. Hemophilia A, which accounts for the largest proportion of hemophilia diseases, is caused by a deficiency of FⅧ: C. Until now, treatment has been continued by injecting FⅧ: C continuously throughout life.

For this reason, the whole plasma was initially concentrated to obtain FⅧ: C for the purpose of treating hemophilia. Early FⅧ: C concentrates improved the situation in patients with hemophilia, prolonged life, and enabled some normal daily activities. However, plasma-derived FⅧ: C concentrates pose a major problem of viral infection. Chromatographic methods have been developed to obtain high-purity FV: C, but still cannot be free from infection with AIDS or hepatitis virus. In addition, a large amount of plasma must be supplied to obtain a large amount of FV: C, but other methods of producing FV: C are required due to the limitation of the supply amount.

As such, there has been an attempt to synthesize FⅧ: C based on genetic engineering methods to overcome the impurity problem in plasma-derived FⅧ: C and the limitation of supply, and in another aspect, to obtain high purity and high concentration of FⅧ: C through purification. An attempt was made. The commonly used method was ion exchange chromatography, but this method has a problem that it is difficult to elute the protein from charged ionic materials without affecting the activity of the protein or to elute the protein with the same activity. .

To solve this problem, many researchers have attempted to develop antibodies that specifically bind to FⅧ: C to obtain only high-purity FⅧ: C. Initially, studies have been made to purify FVIII: C by making an antibody against von Willibrand factor (vWF) that binds FVIII: C in vivo. Hoyer and Trabold report a method for purifying FV: C as an immunosorbent chromatography with rabbit polyvalent antibodies against vWF [J. Lab. Clin. Med., 97: 50-64, 1981. This method creates an immunoaffinity column with a ligand of the polyvalent antibody against vWF, injects plasma containing FV: C / vWF into the column, adsorbs both FV: C / vWF, washes impurities, and then removes vWF and Bound FVII: C was eluted with calcium ions. Although it is improved in the purity and efficiency of the eluted F Ⅷ: C, due to the use of a multivalent antibody has a disadvantage that a large amount of impurities and vWF is mixed and eluted.

In addition, Zimmerman et al. Reported a method of making an immunosorbent column by making monoclonal antibodies of vWF-specific mice to separate FV: C with an ion exchange resin column (US Pat. No. 4,361,509). That is, an immunoaffinity column was constructed by binding a monoclonal antibody specific for vWF to agarose beads. Pass the solution containing FⅧ: C / vWF through this column to adsorb FⅧ: C / vWF first, then wash with buffer to remove unadsorbed protein from the column, and combine with vWF with a solution containing calcium Only FⅧ: C which was made was eluted. However, since the immunoaffinity column using an antibody having a binding ability to vWF is a method of specifically binding FV: C after binding to vWF bound to FV: C, FV: C that does not bind vWF is an antibody. There is a problem that a large amount of FⅧ: C is lost by simply passing through the column without combining with.

Amphlett et al. Reported that the purification of FV: C using antibodies against vWF was not suitable as an efficient purification method because the amount of FV: C not bound to vWF was 50% or more. [US Pat. No. 4,508,709].

The methods such as Heuer and Zimmerman have been improved in terms of purity and efficiency compared to the conventional methods, but still have not overcome the disadvantages of low concentration of the final purified product and containing a large amount of impurities.

Therefore, there is a need for a technology that can specifically purify FⅧ: C with high concentration and high purity by specifically binding to FⅧ: C rather than an antibody to vWF complexed with FV: C.

Thus, the present inventors specifically bind to FⅧ: C except for vWF complexed with FⅧ: C, while solving the problems of low efficiency, low purity, and low concentration when F 정제: C is purified using an antibody against vWF. The present invention was completed by the first development of a monoclonal antibody capable of purifying only FV: C with high concentration and high purity.

Accordingly, the present invention provides a hybridoma F171-8 [KCLRF-BP-00106] and a monoclonal antibody produced therefrom which produce a monoclonal antibody for effectively isolating and purifying only the coagulation factor 8 only. have.

Another object of the present invention is to provide a method for purifying coagulation factor 8 using the monoclonal antibody.

The present invention provides a hybridoma F171-8 [KCLRF-BP-00106] and a monoclonal antibody produced therefrom for the production of mouse monoclonal antibody that specifically binds to the coagulation factor 8 except for von Willebrand Factor (vWF). It is characterized by.

In addition, the present invention includes a method of purifying only the coagulation factor 8 with high concentration and high purity using the monoclonal antibody.

The present invention will be described in more detail as follows.

The present invention relates to a mouse monoclonal antibody which binds to human blood coagulation factor 8 (Factor VII) with specific and high affinity, and is capable of purely separating and purifying only blood coagulation factor 8 at high concentration, and heavy and light chains of the antibody. It relates to a gene encoding and a hybridoma producing the antibody.

First, cDNA encoding human-derived recombinant FVIII was prepared by RT-PCR, and this cDNA was inserted into pcDNA 3.1 to produce a recombinant expression vector, which was transformed into a human kidney-derived fibroblast line to prepare recombinant FVIII.

The recombinant FVIII antigen was used as an antigen and injected into mice to obtain splenocytes. Among the hybridomas obtained by the cell fusion method, splenocytes were used to select hybridoma F171-8 for producing monoclonal antibody having specificity for FVIII. It was.

Selected hybridoma F171-8 was deposited with the Korea Cell Line Research Foundation on October 18, 2004 and received accession number KCLRF-BP-00106.

The mouse monoclonal antibody produced from the hybridoma F171-8 includes the sequence of FIG. 5 (SEQ ID NOs: 25, 26) encoding the heavy chain and the sequence of FIG. 6 (SEQ ID NOs: 27, 28) encoding the light chain. .

In addition, the antibody also includes a variant having at least 50% homology with the heavy or light chain sequence.

Using the monoclonal antibody as a ligand, F ^: C (the active part of coagulation factor 8 and coagulation factor 8 except vWF) can be purified from frozen plasma precipitate or cell culture by injecting it into an immuno-affinity column. .

FⅧ: C obtained by this purification method showed a high purity of 10562.50 ~ 22392.83 IU / mg with a high purification yield of 66.42% in the cell culture medium.

Accordingly, the mouse monoclonal antibody according to the present invention specifically binds only to the coagulation factor 8 (FⅧ: C) except vWF, thereby effectively acting on the purification of FⅧ, minimizing contamination of other proteins, and concentrating high FⅧ: C concentrations. Provide only high purity. This purified FⅧ: C can be usefully used to treat hemophilia.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1: Construction of human derived recombinant FVII: C for immunity

CDNA encoding human derived recombinant FVIII was constructed using RT-PCR method.

Specifically, FⅧ cDNA was obtained by RT-PCR method using human liver mRNA to secure FⅧ cDNA. Reverse transcription is performed using gene specific primer (primer A: 5'-AGCACAAAGGTAGAAGGCAAGC-3 ', SEQ ID NO: 1) having complementarity with 7237-7258 portion of human Factor VIII cDNA sequence (GenBank accession number: NM 0013). It was. Conditions for RT were 9.5 total, including 50 μg human liver mRNA, 1 μl 10X reverse transcription buffer, 1 μM A primer, 4 mM dNTPs, 1 unit of RNase inhibitor and 10 units of reverse transcriptase. It was carried out in a volume of μl. The RT reaction was carried out at 42 ° C. for 90 minutes, and the FⅧcDNA was amplified with three pairs of primers capable of amplifying the pfu polymerase and the FⅧ cDNA into three parts using the synthesized cDNA. The primer pairs used are as follows.

B primer Forward direction 5'-CCTTTTGCTTCTCCAGTTGAAC-3 '(SEQ ID NO: 2) Amplify nucleotides 133-1997 of Fâ ‚‚ cDNA (NM 00132) Reverse 5'-TTCTCTGTGAGGTACCAGCTTC-3 '(SEQ ID NO: 3) C primer Forward direction 5'-TGCCTGACCCGCTATTACTCTA-3 '(SEQ ID NO: 4)  Amplifies F10 cDNA (NM 00132) nucleotides 1810-4295 Reverse 5'-TCTATCTGTGTGAGGGTGCTCG-3 '(SEQ ID NO: 5) D primer Forward direction 5'-GGAGGAAGAAAACTTGGAAGGC-3 '(SEQ ID NO: 6) Amplifies F44 cDNA (NM 00132) nucleotides 4044-7320 Reverse 5'-AGCACAAAGGTAGAAGGCAAGC-3 '(SEQ ID NO: 7)

PCR conditions were the first 1 cycle of DNA denaturation at 95 ℃ for 1 minute 30 seconds, then amplified for 30 minutes at 95 ℃, 30 seconds at 56 ℃, 6 minutes at 68 ℃ for 45 cycles, the extension (extension) The reaction was carried out for 10 minutes at 68 ℃ for 1 cycle. The amplified FVIII cDNA fragments were each subcloned into pCR2.1 TOPO vectors. Three fragments of FⅧ cDNA were linked using KpnI and NsiI, one-time restriction enzyme cleavage sites, to obtain full-length FⅧ cDNA. Total sequencing was confirmed by total DNA sequencing. In order to prepare FVIII cDNA from which a part of B-domain and A3 domain was removed for the purpose of increasing the amount of expression, sequences 2783-4804 were first cut by EcoNI, and blunt end (using DNA polymerase I Klenow fragment) was used. The blunt-end was formed and then ligation was performed. Using the vector as a template, F ′ cDNA was removed from primers 765 through 1652 of the F ′ amino acid sequence using primer E (5′-CACCACAATTCCAGAAAATACTACTCTTCAGTCAGTC-3 ′, SEQ ID NO: 8) using a loop out mutagenesis method. Whether the desired sequence was obtained was confirmed by full sequencing.

The c-DNA from which the B-domain was removed was separated into XhoI and Kpn1, and then linked to pcDNA3.1 incised with the same restriction enzyme to confirm its orientation by restriction enzyme and sequencing. This was transformed by transfection into a human kidney-derived fibroblast line (HEK293). Transformed cell lines were cultured in serum medium to express human derived recombinant FVIII.

The expressed culture supernatant was passed through a DEAE Sepharose column (DEAE Sepharose, Pharmacia, Sweden) and eluted with recombinant FVIII with sodium chloride. The eluate was injected into an immuno-affinity column containing the anti-FV antibody as a ligand and only the recombinant FV was eluted. The anti-FV antibody used at this time is ESH-8 (American Diagnostics, USA). The eluate eluted from the immunoaffinity column was again added to a Q Sepharose column (Q Sepharose, Pharmacia, Sweden) and eluted with sodium chloride. It was confirmed that the eluate eluted in Q Sepharose was purified by Coomassie blue after developing on SDS-PAG, and the results are shown in FIG. 1.

Example 2 Preparation of Hybridomas for the Production of Anti-Human Recombinant FVII Mouse Monoclonal Antibodies

Preparation of the mouse monoclonal antibody, such as quiller and Milstein [Nature. Vol. 256, p. 495-497, 1975] and the corresponding method.

1) Preparation of hybridoma for antibody production

First, the antigen prepared in Example 1 was mixed 1: 1 with Freund's complete adjuvant (Qiagen, USA) in an emulsified solution to female Balb / c mice 8 weeks old to immunize FVII: C. The preparations were administered intraperitoneally with a recombinant FVIII protein of about 5 μg / mouse. Then, 4 to 15 times at intervals of 2 weeks, a 1: 1 mixture of the antigen solution for intraperitoneal immunization and Freund's complete adjuvant (Qiagen, USA) was prepared such that the recombinant FVIII was 5 µg / mouse. Intraperitoneal administration. Blood was collected 3 days after the last immunization, and antibody titers in serum were measured by ELISA.

The antibody titer in serum was measured using the ELISA method, and spleen cells were isolated from mice with elevated antibody titers to recombinant FVIII from the result, thereby preparing splenocytes in a conventional manner.

Parent cells for cell fusion were previously selected as a medium containing 20 μg / ml of 8-azaguanine and Bal b / c mice, strains deficient in hypoxanthine, guanine and phosphoribosyl transferase (HGPRT) Derived transformed cells SP2 / 0-Ag14 were used. For SP2 / 0-Ag14 cells 2.0 × 10 ⁷ cells and splenocytes 1.0 to 1.3 × 10 ⁸ cells, HAT (100 μM hypoxanthine, 400 nM aminopterin, 16 μM thymidine, 10% fetal bovine serum, 2.5 μg / ml Cell fusion, HAT selection, and cloning were performed using DMEM) medium containing gentamicin.

Screening of the cloned hybridoma culture supernatant was carried out on a plate to which 1 µg / ml of a recombinant FVII: C protein solution prepared in the same manner as the antigen for intraperitoneal administration was combined, and the ELISA was performed with 50 µl of the hybridoma culture supernatant. Was performed. In these ELISA results, hybridomas that showed an immune response to the recombinant FVIII-well were selected.

Thus, by screening the culture supernatant of HAT-selected hybridomas by ELISA method, clone F171-8 having stable antibody production capacity and proliferative capacity and specificity to recombinant FVIII was obtained.

2) Preparation and Purification of Mouse Monoclonal Antibodies

For 10-week-old female Balb / c mice, 0.5 ml / pristan (pri-stane, Sigma, USA) was administered intraperitoneally and 15 days later, hybridoma cells F171-8 obtained by cloning above. Was injected intraperitoneally at 5 × 10 ⁶ to 1.0 × 10 ⁷ cell counts / 1 ml / horse. Six days later, the abdominal hypertrophy of the mouse was observed and ascites was collected. The collected ascites was centrifuged at 3,000 rpm for 20 minutes at 4 ° C, and the supernatant and the precipitate were separated. Monoclonal antibody F171-8 was purified from the obtained supernatant using affinity column protein A (Pharmacia, Sweden). The absorbance was measured at 280 nm with respect to the obtained antibody solution, and antibody concentration was calculated from the molecular absorbance coefficient of mouse IgG.

Example 3 Characterization of Selected Monoclonal Antibodies 171-8

0.5 ml of the culture supernatant of hybridoma cell F171-8 obtained in Example 2-1) was diluted in 4.5 ml of TBS-T, and mouse monoclonal antibody isotyping kit (Mouse) was used for 3 ml in the dilution. Monoclonal antibody isotyping kit (Pierce, USA) was used to compare isotypes. As a result, the isotype of heavy chain was IgG1, and the isotype of light chain was kappa type.

Refecto (fVII: SQ), a currently commercially available FVIII agent, was used to identify the recognition site of FVIII: C for the monoclonal antibody F171-8 selected in Example 2-2). Refecto was expressed in animal cells by cleaving a portion of the B-domain that was found to have no effect on FVIII function to increase the expression level and conjugating the 743th amino acid Serine and the 1638 glutamine amino acid [ WO 91/09122]. Refecto shows an expression form in which the heavy chain (90 kDa) and the light chain (80 kDa) are cleaved and secreted out of the cell during the expression process. The present inventors obtained the refecto gene by PCR as in Example 1, inserted into a pcDNA 3.1 (Invitrogen, USA) vector, and transfected into a human kidney-derived fibroblast line (HEK293) to transform it. Refecto was purified from the expressed cell culture using an ion exchange resin column and an immuno-affinity column as in Example 1 [FIG. 2A]. Thus purified Refecto was developed on SDS-polyacrylamide gel and Western blot was performed. As a result, monoclonal antibody F171-8 recognized the heavy chain region of FVIII: C, and the result is shown in FIG. 2B.

Five fragments of FVIII were constructed to identify more precise recognition sites for monoclonal antibody F171-8. The primers for making the five fragments are as follows.

One Forward primer 5'-ATGGATCCTAACATGGCTTCCCATCC-3 '(SEQ ID NO: 9) Reverse primer 5'-GGTCTCGAGTTATACTTTGACATAAGCTTC-3 '(SEQ ID NO: 10) 2 Forward primer 5'-ATGGATCCTCACCAACATGATGGCATG-3 '(SEQ ID NO: 11) Reverse primer 5'-GGTCTCGAGTTAATCCTTCAAATGTTTTAC-3 '(SEQ ID NO: 12) 3 Forward primer 5'-ATGGATCCTGATCCAGAGTTCCAAG-3 '(SEQ ID NO: 13) Reverse primer 5'-GGTCTCGAGTTAGGTAAATACCATGTCCC-3 '(SEQ ID NO: 14) 4 Forward primer 5'-ATGGATCCTGGCCTGATTGGACCCC-3 '(SEQ ID NO: 15) Reverse primer 5'-GGTCTCGAGTTATGGATAGAGATTGTACAG-3 '(SEQ ID NO: 16) 5 Forward primer 5'-ATGGATCCTCACGGCATCAAGACCC-3 '(SEQ ID NO: 17) Reverse primer 5'-GGTCTCGAGTTATAGAGAGTTCACCACAGG-3 '(SEQ ID NO: 18)

Of the five pairs of primers, the forward primers were constructed to have restriction enzyme BamH I cleavage sites and reverse primers to restriction enzyme Xho I cleavage sites and STOP codons. The restriction enzymes BamH I and Xho I were cut and inserted into the pRSET vector (Invitrogen, USA). After confirming the gene sequence of each fragment inserted into the pRSET vector, it was introduced into E. coli and developed on SDS-polyacrylamide gel to confirm expression. The location and size of the five fragments is shown in Figure 3a.

The five fragments expressed as above were developed on an SDS-polyacrylamide gel, and then Western blot was performed with the monoclonal antibody F171-8 selected in Example 2-2). Western blot results indicate that monoclonal antibody F171-8 recognizes the A2 site in the heavy chain site of FVIII: C, and the results are shown in FIG. 3B.

Example 4 Identification of Gene Sequences of Variable Regions of Selected Mouse Monoclonal Antibodies and Identification of Complementarity-determining Regions (CDRs)

In order to elucidate the gene sequence of the variable region of the antibody expressed in the hybridoma F171-8 [Accession No .: KCLRF-BP-00106] which binds specifically to the recombinant FVIII obtained above, the hybridoma F171-8 was replaced with T. Cultured in a -75 (Falcon, USA) flask. Total RNA was isolated from the cultured hybridoma cells according to the method of Chomczynski (1993), and mRNA was isolated by the method of QIAGEN. Using the isolated mRNA as a template, the variable region genes of the heavy and light chains of mouse IgG were amplified by RT-PCR using the following primers. Using the isolated mRNA as a template, the variable region genes of the heavy and light chains of mouse IgG were amplified by RT-PCR using the following primers. Complementary DNA synthesis primers isolated and complementary (complementary) DNA synthesis put together and heated at 70 ℃, and then quenched to bind the mRNA and the primer. After adding buffer, DTT and dNTP to the solution and heating at 42 ° C. for 2 minutes, the enzyme was added to Promega's enzyme and incubated at 42 ° C. for 50 minutes. In order to inactivate the enzyme, it was heated at 70 ℃ for 15 minutes, and then quenched. To remove the remaining mRNA, QIAGEN enzyme was added and incubated at 37 ° C. for 30 minutes.

The polymerase chain reaction was performed to amplify the variable region gene with the complementary DNA obtained above. Complementary DNA, buffer, dNTP, forward primer, reverse primer and Solgent's enzyme (pfu-Taq polymerase) were added, 94 ° C.-5 min / 94 ° C.-1 min; 60 ° C.-1 min; A chain reaction of 72 ° C.-2 min (30 repetitions) / 72 ° C.-7 min / 4 ° C.-∞ minutes was performed.

* Primers for complementary DNA synthesis

① Heavy chain primer: 5'-CTC AAG TTT CTT GTC CAC CTT GGT GC-3 '(SEQ ID NO: 19)

② Light chain primer: 5'-CTC ATT CCT GTT GAA GCT CTT GAC ATT-3 '(SEQ ID NO: 20)

* Primer for variable region gene amplification

① Heavy primer forward primer: 5'-AGG TCC AGC TGC AGG AGT CTG G-3 '(SEQ ID NO: 21)

② Reverse primer for heavy chain: 5'-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC -3 '(SEQ ID NO: 22)

③ Forward primer for light chain: 5'-GAC ATT GAG CTC ACC CAG TCT CCA-3 '(SEQ ID NO: 23)

④ reverse primer for light chain: 5'-GTT TTA TCT CGA GCT TGG TCC C-3 '(SEQ ID NO: 24)

The amplified gene was cloned by inserting into a pGEM-T vector (Promega, USA) and analyzed by sequencing using an automatic gene analyzer (ABI Prism 310, Applied Biosystem Co.). The nucleotide sequence of the analyzed gene was confirmed by comparison with the previously reported nucleotide sequence, and the sequence of the complementarity-determining sites (CDR H1, H2, H3 / CDR L1, L2, L3) was artificially translated. It was used for identification. Complementarity-determination sites were identified using Kabat's database (http://www.bioinf.org.uk/abs/). As a result of determining the complementarity-determining site for F171-8, the amino acid sequence of the CDR has a mismatched CDR amino acid sequence compared to other antibodies known to date, and the results are shown in FIGS. 5 and 6 (SEQ ID NOs 25 to 28). ).

Example 5: Confirmation of Responsiveness to Antigens of Selected Antibodies

ELISA was performed by sequentially diluting the culture medium of the hybridoma cells obtained in 2-1) to confirm the reactivity of the selected F171-8 mouse monoclonal to FVIII: C. Recombinant FVIII: C purified at all times in Example 1 was added to the microwell plate 1 ㎍ each well for 1 hour at 37 ℃ and blocked with PBS buffer containing 3% BSA for 1 hour at 37 ℃. After diluting the F171-8 hybridoma culture medium to 1/2 to 1/1024 times and adding 50 μl per well, the reaction was performed at 37 ° C. for 1 hour and 30 minutes. After washing with PBS buffer containing 0.1% of Tween-20, HRP-conjugated anti mouse antibody was diluted 6000-fold, and 50 µl was added per well, followed by reaction at 37 ° C. for 1 hour. After washing three times with PBS buffer containing 0.1% Tween-20, HRP was added by adding a substrate. By measuring the color development at absorbance 450 nm, it was confirmed that the F171-8 reacts with FⅧ: C according to the dilution ratio, the results are shown in FIG.

Example 6: FVII: C Purification Using Mouse Monoclonal Antibodies

The mouse monoclonal antibody produced in Example 2-2) was dialyzed in carbonate buffer (pH 8.3) containing 0.5M sodium chloride, and the medium CNBr-Activated sepharose 4B (CNBr-Activated sepharose 4B, Pharmacia, Sweden) An immunoaffinity column was constructed by reacting with the gel to allow the anti-FVIII antibody to bind at 1 mg per ml of solid medium gel.

1) Purification of FⅧ: C from Frozen Plasma Cryoprecipitate

100 g of human frozen plasma precipitate was stirred with 20 mM Tris buffer solution for 1 hour to completely dissolve. The solution was added with 0.25% aluminum hydroxide, stirred for 20 minutes, and centrifuged at 8,500 rpm for 10 minutes. Only the supernatant was taken, and glycine was added to 2.2 M, stirred at 37 ° C. for 30 minutes, and centrifuged at 8,500 rpm for 10 minutes to remove fibrinogen. Again, only the supernatant was taken up to 15% sodium chloride, completely dissolved and left for 1 hour. The solution was centrifuged at 8,500 rpm for 10 minutes to obtain only precipitates with concentrated FV: C. This precipitate was completely dissolved in 20 mM Tris buffer to give a FⅧ: C concentrate.

The FⅧ: C concentrate obtained from the frozen plasma precipitate was injected into the immunoaffinity column prepared as described above, and washed with Tris buffer containing 50 mM calcium and 150 mM potassium chloride. Again, only FC: C was eluted with Tris buffer containing 50 mM calcium and 60% ethylene glycol to obtain high-purity FⅧ: C with a recovery of 23.39%.

Purification of FⅧ: C from Human Frozen Plasma Precipitates Purification steps Active (IU / ml) Volume (ml) Total activity (IU) Yield (%) FⅧ: C Concentrate 3.04 20 60.8 100 Column non-adsorbent 1.743 20 34.68 57.33 Column wash 0.891 10 8.91 14.65 Column eluate 2.37 6 14.22 23.39

2) Purification of FVIII: C in Animal Cell Line Cultures Expressing Recombinant FVIII: C

Animal cell lines expressing recombinant F ′: C were cultured and the culture was centrifuged at 8,000 rpm, 4 ° C. for 10 minutes to remove cells or suspended matter in the culture. To remove some impurities in the supernatant and concentrate the recombinant FⅧ, the culture supernatant was passed through a DEAE Sepharose column (DEAE Sepharose, Pharmacia, Sweden) at a rate of 10 ml per minute, followed by Tris buffer containing 1M sodium chloride. Recombinant FVIII was eluted.

The eluted recombinant FVIII concentrate was injected into Sepharose 4B column with anti-FVIII antibody bound to ligand. After washing with Tris buffer solution containing 50 mM calcium, recombinant FVIII was eluted with 60% ethylene glycol containing 50 mM calcium.

Finally, a Q Sepharose column was passed through to remove the small amount of impurities remaining in the eluate. After eluting with an elution solution containing 1M sodium chloride, a high-purity recombinant FV: C was obtained, and the recovery was about 66.42%.

Purification of Recombinant FVII: C from Cell Cultures Purification steps Active (IU / ml) Volume (ml) Total activity (IU) Yield (%) FⅧ: C Concentrate 37 246 9102 100 Column non-adsorbent 2.815 290 816.35 8.97 Column wash 0.69 175 120.75 1.33 Column eluate 58.12 104 6045.2 66.42

As described above, the mouse monoclonal antibody according to the present invention specifically binds only to FⅧ: C, thereby effectively acting on the purification of FⅧ, thereby minimizing contamination of other proteins and providing only high concentration of FⅧ: C with high purity. Can be. Thus, the FVIII: C factor thus obtained can be usefully used for the treatment of hemophilia.

<110> IN2GEN CO., LTD. <120> Monoclonal Anti Factor VIII antibody for purification of          coagulation Factor VIII <160> 28 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> gene specific primer <400> 1 agcacaaagg tagaaggcaa gc 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 ccttttgctt ctccagttga ac 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 ttctctgtga ggtaccagct tc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 tgcctgaccc gctattactc ta 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 tctatctgtg tgagggtgct cg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 6 ggaggaagaa aacttggaag gc 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 7 agcacaaagg tagaaggcaa gc 22 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer E <400> 8 caccacaatt ccagaaaata ctactcttca gtcagtc 37 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 atggatccta acatggcttc ccatcc 26 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 ggtctcgagt tatactttga cataagcttc 30 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 atggatcctc accaacatga tggcatg 27 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 ggtctcgagt taatccttca aatgttttac 30 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 atggatcctg atccagagtt ccaag 25 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 14 ggtctcgagt taggtaaata ccatgtccc 29 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 15 atggatcctg gcctgattgg acccc 25 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 16 ggtctcgagt tatggataga gattgtacag 30 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 atggatcctc acggcatcaa gaccc 25 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 ggtctcgagt tatagagagt tcaccacagg 30 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> heavy chain primer <400> 19 ctcaagtttc ttgtccacct tggtgc 26 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> light chain primer <400> 20 ctcattcctg ttgaagctct tgacatt 27 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer of heavy chain <400> 21 aggtccagct gcaggagtct gg 22 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of heavy chain <400> 22 tgaggagacg gtgaccgtgg tcccttggcc cc 32 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer of light chain <400> 23 gacattgagc tcacccagtc tcca 24 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of light chain <400> 24 gttttatctc gagcttggtc cc 22 <210> 25 <211> 344 <212> DNA <213> Artificial Sequence <220> <223> heavy chain of monoclonal antbody 171-8 <400> 25 aggtccagct gcaggagtct ggaggaggct tggtgcaacc tggaggatcc atgaaactct 60 cctgtgttgc ctctggattc actttcagta acttctggct gaactgggtc cgccagtccc 120 cagaaaaggg gcttgagtgg gttgctgaaa ttagattgaa atctaataat tatgcaacac 180 attatgcgga gtctgtgaaa gggaggttca ccatctcaag agatgattcc aaaagtagtg 240 tctacctgca aatgaacaac ttaagagctg aagacactgg catttatttc tgtactacta 300 cggctgctta ctggggccaa gggaccacgg tcaccgtctc ctca 344 <210> 26 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> heavy chain of monoclonal antbody 171-8 <400> 26 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser   1 5 10 15 Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Phe Trp              20 25 30 Leu Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala          35 40 45 Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser      50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Val  65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Phe                  85 90 95 Cys Thr Thr Thr Ala Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val             100 105 110 Ser Ser         <210> 27 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> light chain of monoclonal antbody 171-8 <400> 27 gacattgagc tcacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300 tcggaggggg gaccaagctc gagataaaac 330 <210> 28 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> light chain of monoclonal antbody 171-8 <400> 28 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser              20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro          35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala      50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His  65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg                  85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Ser Arg *** Asn             100 105 110  

Claims (5)

Hybridoma F171-8 for production of mouse monoclonal antibody that specifically binds to coagulation factor 8 (FVII: C) except for von Willebrand Factor (vWF) [KCLRF-BP-00106]. Monoclonal antibody against coagulation Factor 8 (FVII: C) produced from hybridoma F171-8 [KCLRF-BP-00106] of claim 1. The antibody according to claim 2, which has a heavy chain coding nucleotide sequence represented by SEQ ID NO: 25 and a light chain coding nucleotide sequence represented by SEQ ID NO: 27. The antibody of claim 3, wherein the antibody has at least 50% homology with the heavy or light chain sequence. A method for purifying only coagulation factor 8 from frozen plasma precipitate or cell culture by injecting an antibody selected from claims 2 to 4 into a immunoaffinity column using a ligand.

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