CA2035965A1 - Process for producing highly purified monoclonal antibodies and methods for purification of viruses by monoclonal antibody affinity column - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Highly purified monoclonal antibodies are produced by cultivating monoclonal antibody-producing hybridoma in high dense by the method of perfusion system using serum-free medium, collecting the said monoclonal antibodies and purifing by cation-exchange column chromatography. The method for purifying viruses by monoclonal antibody affinity column is also provided.

Description

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PROCESS FOR PRODUCING HIGHLY PURIFIED MONOCLONAL
ANTIBODIES AND METHODS FOR PURIFICATION OF VIRUSES BY
MONOCLONAL ANTIBODY AFFINITY COLUMN

Backqround of the Invention (1) Field of the Invention The present invention relates to process for producing highly purified monoclonal antibodies and methods for purification of infectiouspancreatic necrosis virus (IPNV), which characterizes to use affinity column coated with anti-IPNV monoclonal antibody.

(2) Description of the Prior Art Recently, biologically active substances are progressively produced using animal cells and these products are used for pharmacy, food, and chemical industrial fields. Especially, interferon, virus, vaccine from human lymphocytes or fibroblast, monoclonal antibodies from murine hybridomas, and erythropoietin or tissue plasminogen activators from recombinant CHO
cells, can be industrially produced by large-scale of animal cells. However, as we usually often use media with fetal bovine serum to c~tivate such as animal cells, different lot of serum sometimes causes damage to cells and non-reproducible results. Therefore, nowadays such a serum containing medium is replaced by 2~

serum-free medium. Serum is highly co~ted and often results in troubles to purify because of highly containing proteins when produced and purified large amount of monoclonal antibodies from the supernatant of hybridomas. Therefore, it is required their profitability and convenience to purify largely-produced monoclonal antibodies. Thus, efficient production methods for monoclonal antibodies by serum-free media are strongly needed.
And then viruses require their host cells to grow because they are different from other microorganisms and grow only in the cells. For prevention to these viral infectlon, up to date, vaccination is carried out using vaccines, but purified vaccines are required to promote more efficient immune responseagainst the viruses in vivo. Therefore, it is more preferable that virus particles should be purified to apply the virus particles to vaccines.
When virus cultures are purified, the viruses must be inoculated to the host cells and then fractionated from the infected cell cultures.
So far, we have used ultracentrifugation with density gradient using sucrose or CsCl, ion-exchange chromatography, and gel filtration to purify the viruses.
However, these methods are time consuming, and not 2~3~

efficient because of sophisticated methods.
Recently, virus purification are sometimes carried out by affinity chromatography using gel of cellulose sulfate, but even if used this method, it is hard for some species of viruses to purify. Thus, it is needed to be established the methods for virus purifi-cation in the more convenient, rapid, and higher purity.
Summary of the Invention The present invention established the methods to produce largely and highly purified monoclonal anti-bodies; firstcultivate monoclonal antibody-producing hybridoma in high dense by the method of perfusion system using serum-free medium which comprises insulin, transferrin, ethanolamine, and selenium composed, secondary purify produced monoclonal antibodies by cation-exchange column chromatography.
This invention provides the methods which character-izes tocultivate monoclonal antibody-producing hybridoma in high dense by perfusion method using serum-free medium comprised of insulin, transferrin, ethanol amine, and selenite and to purify these produced monoclonal antibodies by cation-exchange column chromatography.
And then, the inventors established the purification methods for viruses in high purity using affinity 2~3~6$

column coated with monoclonal antibody. This invention was completed by attempting to apply some monoclonal antibody to affinity chromatography to purify the viruses.
This invention provides the methods for purifying infectious pancreatic necrosis viruses (IPNV), which characterizes to purify using affinity column coated with anti-IPNV monoclonal antibody.
Brief description of the Drawings Fig. 1 shows growth of hybridoma 4PG-3N in high density culture system and productivity of monoclonal antibody.
Fig. 2 shows purification of monoclonal antibody by cation-exchange chromatography.
Fig. 3 shows neutralizing activity of purified anti-IPNV monoclonal antibody to IPNV.
Fig. 4 shows purification of infectiouspancreatic necrosis virus (IPNV) from the culture supernatant of IPNV by anti-IPNV monoclonal antibody affinity column chromatography.
Fig. 5 shows SDS-polyacrylamide gel electro-phoretogram of infectious pancreatic necrosis virus (IPNV) purified by anti-IPNV monoclonal antibody affinity column chromatography.

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Detailed Descriptionofthe Preferred Embodiments This invention does not particularly define mono-clonal antibodies producing hybridomas, but preferably use hybridomas which produce monoclonal antibodies to fish viruses and require to make highly efficient and lowly production cost. Concretely, hibridomas which produce monoclonal antibodies to infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, and Hirame rhabdovirus, and more concretely can be used hybridoma 4PG-3N (FERM BP 3068) and 4PG-2N, which produce monoclonal antibody with neutralizing activity to infectious pancreatic necrosis virus.
The medium in this invention can be serum-free medium such as E-RDF (Kyokuto), RPMI1640 (Nissui), and MEM (Nissui) with insulin, transferrin, ethanolamine, and selenium compound. These additives can be used in the concentrations of 2 ~ 10 ~g/ml (insulin), 10 ~ 50 g/ml (transferrin), 5 ~ 30 ~M (ethanolamine), and 1 ~ 3 xlO M (solenite). This medium may sometimes contain 25 ~ 80 ~g/ml of egg yolk lipoprotein, which makes much better growth of hybridomas. Especially, more preferable medium can be E-RDF with insulin, transferrin, ethanolamine, selenite, and egg yolk lipoprotein.
As the selenium compound, there can be used a 2 ~

selenium compound utilizable by hybridomas for example, selenic acid, sodium selenate, potassium selenate, selenious acid, sodium selenite, potassium selenite, selenocysteine~ selenocystine, salts thereof and so on.
Perfusion system mentioned above is called as culture fluid exchanging system, which incubate continu-ously hybridoma by exchanging exhaused medium with same volume of fresh medium after the hybridoma grow upto set cell density. In the present invention, the hybridoma can becultivated until the cell density reach to about 1 ~ 5 x 105 cells/ml and then continuously cultivated in high dense by perfusion system. In this case, concentration of dissolved oxigen and pH should be 0.5 ~ 5.0 ppm and 6.5 ~ 7.3, respectively~ To perform this high density culturing, we can use SHC-l (Shimadu) as one of the high density culture systems.
Culture fluid obtained by the methods mentioned above, can be centrifuged and filtered to be separated the hybridoma cells, and then directly purified the monoclonal antibodies from other protein fractions by using protein A or G affinity chromatography after precipitated protein fraction- containing monoclonal antibodies by ammonium sulfate, resuspended, and dialysed in buffer solution. However, this invention may be more superior to above description with respect 2~3~

to easy purification of these antibodies because our methods using cation-exchange column chromatography can purify the antibody directly from culture fluid of hybridoma after the fluid is directly applied into the column without any proceeding like precipitating protein fractions with ammonium sulfate. Furthermore, our method in this invention should be suitable to purify the antibodies because recovery of the antibody was 20~ higher than that using protein A or G affinity chromatography when the inventors compared both methods.
The columns used for such a cation-exchange column chromatography, for example, should be SP-650 (Tohso), Mono SHR (Pharmacia), CM Biogel A (Bio-rad), and so on.
Purified monoclonal antibodies are prepared to give a concentration of 5 ~ 15 mg/ml with 100 mM
citrate buffer (pH 5.0) and NaCNBH3 is dissolved into the solution to give a concentration of 10 ~ 20 mg/ml.
One ml of the mixture is fixed into aldehyde-column by repeatedly injecting using syringes. After fixation, the column is rinsed orderly with citrate and phosphate buffer (pH 7.2). To purify the virus, first virus culture supernatant is loaded onto the column at 1 ml/min and then washed out unadsorbed substances in the column with 650 mM NaCl in 10 mM phosphate buffer (pH 7.2). By this washing procedure, 2 ~ J

only virus particles should be adsorbed in the column.
Adsorbed virus particles can be fractionated and purified from the column by eluting with 100 mM
glycine-HCI buffer (pH 2.8) at about 2 ml/min.
The present invention will now be described in detail with reference to the following examples that by no means limit the scope of the invention.
Example 1 Procedures for large-scale production and purifi-cation of neutralizing monoclonal antibodies to infectious pancreatic necrosis virus are described as follows.
Hybridoma 4PG-3N (FERM BP-3068) which produces anti-IPNV monoclonal antibody and stocked in freeze pack (Xawasumi) at -80C was resuspended in 500 ml serum-free medium, ITES-YLP-E-RDF (E-RDF medium which was supple-mented with 5 ~g/ml insulin, 20 ~g/ml transferrin, 20 ~M ethanolamine, 2.5 x 10 8 M sodium selenite, and 30 ~g/ml egg yolk lipoprotein) and incubated continuously in high density culture apparatus (SHC-l, Shimadu) at 37C. During the incubation period, fresh medium was prepared to have pH7.0 and 4.0 ppm dissolved oxygen, and incubation was conducted stirring at 45 rpm except for 30 rpm in first day of incubation. Average perfusion rate was set up in 16.4 ml/h during 24-day-incubation period. Eventually, 9,850 ml of culture fluidwas obtained. Out of this culture fluid, 10 ml super-natant was subjected to cation-exchange column chromatography using the column (SP-650, Tohso) which run at 1 ml/min and equilibrated with 20 l~M Tris-HCl buffer (pH 6.0), after adjusted to pH 6.0 with 1 M
Tris-HCl buffer. After adsorbed the proteins in the column, unadsorbed protein fractions were washed out from the column at 1 ml/min with 20 mM
Tris-HCl buffer (pH 6.0) and the rest adsorbed monoclonal antibody fractions were eluted with 1 M NaCl in phosphate buffer (pH 7.5) at 1 ml/min.
Anti-IPNV monoclonal antibody fraction was deter-mined by measuring the absorvance at 280 mn and ELISA.
Another hybridoma 4PG-2N which also produce anti-IPNV
monoclonal antibody also was tested and the similar result as above 4PG-3N was obtained.
Example 2 Hybridoma 4PG-3N (FERM BP-306~) with neutralizing activity to IPNV was suspended in serum-free E-RDF medium with ITES and YLP to give 3 x 105 cells/ml, cultivated continuously at 37C for 24 days using high density culturing apparatus (SHC-l, Shimadu), and then the culture supernatant was recovered. Two hybridomas 4PG-3N and 4PG-2N in this invention showed similar results 2 ~ 3 ~ P$ ~ ~a for the productivities of monoclonal antibody and their recovery was almost same after purification.
Therefore, the inventors describehereespecially about hybrodoma 4PG-3N as the representative.
(a) Cell density of hybridoma 4PG-3N in the culture Hybridoma 4PG-3N was suspended in 500 ml of serum-free E-RDF medium with ITES-YLP to give 3 x 10 cells/ml and incubated in high density culturing apparatus (SHC-l, Shimadu). The results are indicated in Fig. 1.
Until 8 days after incubated, the hybridoma grew logarithmically upto 1.6 x 107 cells/ml. During 24-day-incubation period, 4PG-3N could becultivated continu-ously using high density culture system, showing well growth, though the filters were chagned five times because disrupted cells pluged them.
(b) Viable cell number of hybridoma 4PG-3N
Viable cells of hybridoma 4PG-3N during the incubation period, were counted by trypan blue staining method using final concentration of 0.1% trypan blue.
This result is also shown in Fig. 1. The viable cells were 80% among the total cells 8 days after incubation, and then the rate decreased sharply to about 50%.
(c) Amount of monoclonal antibody Amount of anti-IPNV MAb produced in culture during the incubation period was measured by ELISA. The result 2~3~6~

is also shown in Fig. 1. 4PG-3N produced only 5 ~ 10 ~g/ml of monoclonal antibody by static culture with Petri dishes, but the monoclonal antibody was produced continuously more 20 ~ 40 fold than the dish culture when high density culture system was used the cell density to raise upto 107 cells/ml.
(d) Purification of anti-IPNV monoclonal antibody by cation-exchange column chromatography Monoclonal antibody largely produced as mentioned above was purified by cation-exchange column chromato-graphy. Out of 9,850 ml culture fluid obtained by the high density culture system, 10 ml supernatent was applied to cation-exchange column (SP-650, Tohso) which equilibrated to pH 6.0 with 20 mM Tris-HCl buffer after adjusted pH to 6.0 with 1 M HCl. Monoclonal antibody adsorbed in the eolumn was eluted with 1 M
NaCl in 50 mM phosphate buffer (pH 7.5) after nonadsorbed protein fractions were washed out with 20 mM tris-HCl buffer (pH 6.0). This result is indicated in Fig. 2.
Antibody could be recovered in fr.action No. 6 shown as one sharply peak and the coneentration was 283 ~g/ml. The recovery of antibody by such a method is shown in Table 1.

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E~ Q1~4 It proved that high density culture system by 2 ~ 6 using seerum-free medium made significantly large amount of monoclonal antibody, showing 2,350 ~g/ml of total protein. 1,000 ~g of total amount of antibody, and 43%
of ratio of antibody/protein. Furthermore, 85% of purified antibody was recovered from the culture fluid by cation-exchange column chromatography and the purity was 80%, which was higher than that of pre-purification.
(e) Neutralizing activity of purified anti-IPNV monoclonal antibody Purified anti-IPNV monoclonal antibody was diluted with E-RDF medium to give serial 10-fold concentration from 100 ~ 0.01 ~g/ml and 100~1 of filtered-sterile monoclonal antibody was reacted with same volume of IPNV
stock suspension (1.0 x 107 PFU/ml) at 15C for 1 hour.
After reaction, the neutralizing activity was measured by plaque reduction assay. The result is shown in Fig.

3. Anti-IPNV monoclonal antibody neutralized about of IPNV at 1 ~g/ml and more than 105 at 100 ~g/ml, showing higher neutralizing activity when the concen-tration more increased.
Example 3 Hybridoma 4PG-3N (FERM BP-3068) which produces monoclonal antibody with neutralizing activity to IPNV was suspended in 500 ml serum-free medium. ITES-YLP-E-RDF (E-RDF medium (kyokuto) which was supplemented with 5 ~g/ml insulin, 20 ~g/ml transferrin, 20 ~M
ethanolamine, 2 ~ 5 x 10 8 M sodium selenite, and 30 ~g/ml egg yolk lipoprotein) to make 3 x 105 cells/ml and incubated continuously in high density culture apparatus 2~3~

(SHC-l, Shimadn) at 37~C. After incubation, monoclonal antibody was purified directly from the culture supernatant by using Cation-exchange column chromatography described above. The purified monoclona-l antibody was prepared to give a concentration of 13 mg/ml in 100 mM citrate buffer (pH
5.0) and added with 20 mg of NaCNBH3. The mixture was loaded into aldehyde column (Chromatop aldehyde column, 4.6 mm ~410 cm, Nippon Gaishi) for 20 min using two syringes to fix monclonal antibody into this column.
After fixation,an adsorbed ligand was washed out from the column with 100 mM citrate buffer (pH 5.0), 10 mM
citrate buffer (pH 3.3), 10 mM phosphate buffer (pH 7.0), 10 mM carbonate buf er (pH 7.4), and lM NaCl in 10 mM
phosphate buffer (pH 7.0), for each 5 min at 5 ml/min.
Fixed antibody was calculated indirectly by measuring the amount of inadsorbed antibody by ELISA. Eventually, 12.4 mg of antibody could be fixed into column since the amount of unadsorbed antibody was 0.6 mg.
Example 4 Purification of IPNV was attempted by using mono-clonal antibody affinity chromatography made inExample 3 Stock IPNV (VR 299 strain) was inaculated to CHSE-214 cells derivedfrom c ~ oo~ salmon in MEM 10-Tris medium (MEM medium (Nissui) contained 10% fetal bovin serum, 1.6~ of 1 M Tris-buffer, and 0.075% NaHCO3) and incubated at 15C for about 1 week.

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After incubation, the virus culture was centrifuged to eliminate the cells and the supernatant was filtered with 0.22 ~m pore-sized filter. The filtered virus culture was directly applied to the antibody fixed affinity column chromatography which described in Example 3 and equilibrated with 150 mM NaCl in 10 mM phosphate buffer (pH 7.2), loading at 1 ml/min. After loading, unadsorbed proteins like fetal bovine serum were washed out with 650 mM NaCl in 10 mM phosphate buffer (pH 7.2). The virus particles adsorbed in the columns were eluted with 100 mM glycine-HCl buffer (pH 2.8) at 2 ml/min and the column was washed out with 3.48 M
acetate buffer (pH 2.8). This eluate was immediately neutralized with 6N NaOH and the viral infectivity was determined by plaquing assay. The concentration of viral proteins both before and after purification was measured by Bio-Rad protein assay. The purity of the virus was certlfied by SPS-polyacrylamide gel electrophoresis.
Eventually, the inventors obtained one sharply peak of 1 PNV fraction in eluate of 100 mM glycine-HCl buffer. The result is illustrated in Fig. 4. The concentration of the viral protein obtained was233~g in total and the recovery was 87~ when determined by plague assay. These results are shown in Table 2.

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The protein of purified IPNV certified by SDS-polyacrylamide gel electrophoresis is shown in Fig. 5, indicating almost completely purified IPNV, since IPNV proteins, ~ and ~ , were shown clear in lane 4 which contains no other contaminated proteins different from lanes 2 (culture supernatant of IPNV) and 3 (unadsorbed fraction of IPNV) which contains proteins of fetal bovine serum.

Claims (6)

1. Process for producing highly purified monoclonal antibodies, which comprises cultivating monoclonal antibody-producing hybridoma in high dense by the method of perfusion system using serum-free medium comprised insulin, transferrin, ethanolamine and selenium compound, collecting the produced monoclonal antibodies, purifying the said monoclonal antibodies by cation-exchange column chromatography.

2. The process according to claim 1, wherein said serum-free medium contains eggyolk lipoprotein.

3. The process according to claim 1, wherein said the monoclonal antibody has neutralizing activity to infectious pancreatic necrosis virus.

4. The process according to claim 1, wherein said the monoclonal antibody-producing hybridoma derived from fusion between the lymphocyte immunized with infectious pancreatic necrosis virus and the myeloma cell.

5. The process according to claim 1, wherein said the hybridoma is hybridoma 4PG-3N(FERM BP-3068).

6. A method for purification of infectious pancreatic necrosis virus which comprises using affinity column coated with anti-infectious pancreatic necrosis virus monoclonal antibody.