ANTIVIRAL POLYCLONAL ANTIBODIES AGAINST EBOLA VIRUS AND THE USES THEREOF

Abstract

The present invention relates to polyclonal antibodies for use in the prevention and/or treatment of the Ebola virus disease. In one embodiment, the polyclonal antibodies are specific for a truncated Ebola virus GP glycoprotein. Said polyclonal antibodies are preferably non-human antibodies and are provided in a serum free pharmaceutical composition. The present invention also relates to a method of prevention and/or treatment of Ebola virus disease in a subject in need thereof, comprising administering to said subject polyclonal antibodies specific for the Ebola virus.

Claims

1-18. (canceled)

19. A method for the prevention or treatment of an Ebola virus disease in a subject in need thereof, comprising administering to said subject a non-human polyclonal antibody specific for the Ebola virus.

20. The method claim 19, wherein said polyclonal antibody is a rabbit polyclonal antibody.

21. The method of claim 19, wherein said polyclonal antibody comprises immunoglobulins which bind to, or have been obtained by immunization of a non-human mammal with, an antigen which comprises or consists of a sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.

22. The method of claim 19, wherein said antibody is obtained or obtainable by immunization of a non-human mammal with a composition comprising an Ebola virus GP glycoprotein antigen in trimeric form.

23. The method of claim 22, wherein said antibody is obtained or obtainable by immunization of a non-human mammal with a composition comprising at least 30% Ebola virus GP glycoprotein antigen in trimeric form and at least 20% Ebola virus GP glycoprotein antigen in monomeric form.

24. The method of claim 22, said composition comprising at least 30% Ebola virus GP glycoprotein in trimeric form.

25. The method of claim 23, wherein said Ebola virus GP glycoprotein antigen comprises or consists of a sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.

26. The method of claim 22, wherein said Ebola virus GP glycoprotein antigen is a recombinant antigen produced in insect cells.

27. The method of claim 19, wherein said polyclonal antibody has a titer of at least 10000 and/or a neutralization effect of at least 400.

28. The method of claim 19, wherein said polyclonal antibody is provided in a pharmaceutical composition.

29. The method of claim 28, wherein said pharmaceutical composition is suitable for subcutaneous, intradermal, intramuscular, intraperitoneal and/or intravenous administration.

30. The method of claim 28, wherein said pharmaceutical composition comprises from 50 mg to 500 mg of said polyclonal antibody.

31. The method of claim 19, wherein said polyclonal antibody is used in combination with at least another anti-Ebola virus drug and/or at least another anti-Ebola virus vaccine.

32. A polyclonal antibody specific for the Ebola virus, obtained or obtainable from at least one biological sample of a non-human animal immunized with (i) at least one antigen comprising or consisting of a sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; (ii) at least one nucleic acid comprising or consisting of a sequence encoding said antigen; and/or (iii) at least one vector comprising at least one nucleic acid comprising or consisting of a sequence encoding said antigen.

33. An antigen comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9 or a sequence at least 80% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

34. A composition comprising an Ebola virus GP glycoprotein having a sequence at least 80% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO. 14, wherein at least 30% of said glycoprotein in said composition is in trimeric form.

35. A nucleic acid comprising or consisting of a sequence encoding an antigen according to claim 33.

36. A vector comprising a nucleic acid according to claim 35.

37. A method for producing polyclonal antibodies, wherein said method comprises: providing at least one biological sample from an animal immunized with (i) at least one antigen comprising or consisting of a sequence at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; and/or (ii) at least one nucleic acid comprising or consisting of a sequence encoding said antigen; and/or (iii) at least one vector comprising a nucleic acid comprising or consisting of a sequence encoding said antigen, and collecting or purifying the polyclonal antibodies from said biological sample.

Description

LEGEND TO THE FIGURES

[0331] FIG. 1: CBB stained SDS PAGE analysis of an Ebola virus antigen of the invention. Lanes A and 15: SeeBlue plus2 prestained marker kDa (MES): 188, 98, 62, 49, 38, 28, 17, 14, 6, 3; lane B: ABTP-02 Streptag AC peak pooled fractions before SEC; Lanes 1-9: Fractions B10-B2 of SEC peak 1; Lanes 10-14: Fractions B1-C4 of SEC peak 2; Lane 16: SEC peak 1 fr B10-B2 concentrated 0.88 mg/ml; Lane 17: SEC peak 2 fr B1-C7 concentrated 0.80 mg/ml.

[0332] FIG. 2: Size Exclusion profile of an Ebola virus antigen of the invention.

[0333] FIG. 3: HPLC analysis of SEC purified trimers of the invention.

[0334] FIG. 4: Immunoblot analysis of an Ebola virus antigen of the invention.

[0335] FIG. 5: anti-GP specific ELISA of D60 (5A) and D68 (5B) polyclonal antibodies.

[0336] FIG. 6: Titer of D60 (6A) and D68 (6B) polyclonal antibodies.

[0337] FIG. 7: Ebola neutralization of D60 (7A) and D68 (7B) polyclonal antibodies.

[0338] FIG. 8: Ebola neutralization of D60 (7A) and D68 (7B) polyclonal antibodies.

[0339] FIG. 9: Cross neutralization of different filoviruses.

EXAMPLES

Example I: Production of Polyclonal Antibodies for Use in the Prevention and/or Treatment of Ebola Virus Disease

[0340] The recombinant antigens of sequence SEQ ID NO: 5 and SEQ ID NO: 7 are produced as follows. Two pMTpuro vectors comprising either sequence SEQ ID NO: 4 or SEQ ID NO: 6 are used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 g/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO.sub.4 for 4 days. The recombinant antigens are then purified on Strep-Tactin resin (for the recombinant antigen carrying a Strep tag) or on a Ni resin (for the recombinant antigen carrying a His tag). The recombinant antigens are further purified by Superdex 200 size-exclusion chromatography in 10 mM Tris and 150 mM NaCl (pH 7,5) (1TBS). If needed, the tag is then removed using an enterokinase acting on the cleavage site present between the truncated GP and the tag. The purified recombinant antigens comprise sequence SEQ ID NO: 11.

[0341] Rabbits are then immunized by injecting 0.1 mg of purified recombinant antigen per injection. Four injections are performed at day 10, 38, 66 and 87. Serum is collected between the injections to monitor the immunization process. The rabbits are sacrificed on day 90 and the serum is collected.

[0342] The polyclonal antibodies are purified from the serum by precipitation in 50% ammonium sulphate followed by purification on protein A-sepharose column, purification ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.

[0343] The specific binding of the polyclonal antibodies to the recombinant antigen comprising sequence SEQ ID NO: 11 is then assessed by ELISA and the neutralizing capacity of the polyclonal antibodies is assessed in infected human cells.

Example II: Production of Highly Immunogenic Ebola Virus GP Antigen

[0344] The recombinant Ebola virus antigen of sequence SEQ ID NO: 5 is produced in insect cells. Briefly, a pMTpuro vector comprising sequence SEQ ID NO: 4 is used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 g/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO.sub.4 for 4 days. The recombinant antigen is then purified on Strep-Tactin resin.

[0345] As shown in CBB stained SDS PAGE analysis of FIG. 1, the antigen under denaturing conditions is composed of GP1 and GP2 subunits.

Fractions before (lane B) and after one of the SEC runs (lanes 1-14); P1 and P2 indicate peak 1 and 2 respectively. After pooling and concentration of the peak fractions: lane 16 (peak 1) and lane 17 (peak2). Lower panel: Loading pattern of gel.

[0346] Furthermore, as shown in FIG. 2 by size-exclusion chromatography, the antigen is produced in two conformations: peak 1 contains predominantly a trimeric form of the antigen, while peak 2 is a mix of monomers and trimers. The different masses were measured by SEC-MALLS. The derived mass data estimates a trimer mass of 178.2 kDa and the monomer estimated mass of 60.1 kDa.

[0347] Peak 1 was further purified by SEC chromatography and analyzed by HPLC. The resulting composition comprises more than 90% antigen in trimeric form. The profile of this composition is shown in FIG. 3, confirming high purification level and essentially trimeric form.

[0348] Samples of antigen produced as above were separated on 12% SDS PAGE and blotted to nitrocellulose. The membrane was then probed with human anti-EBOLA monoclonal antibody (mAb KZ52) and revealed with mouse anti-IgGFc HRP (Thermofisher cat no A10648). The results presented in FIG. 4 confirm the antigen is very well recognized by human antibodies, thus confirming its biological relevance.

Example III: Production of a Potent Neutralizing Anti-EBOLA Polyclonal Antibody

[0349] Rabbits were immunized by injecting 0.3 mg of purified recombinant antigen prepared as disclosed in example II, per injection. In this example, three im injections were performed at day 0, 22, and 50. The rabbits are sacrificed on day 60 and the serum is collected.

[0350] The polyclonal antibodies are purified from the serum by precipitation in 50% ammonium sulphate followed by purification on protein A-sepharose column, purification ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.

[0351] The specific binding of the polyclonal antibody to the recombinant antigen comprising sequence SEQ ID NO: 5 was then assessed by ELISA, and the neutralizing capacity of the polyclonal antibody was determined in infected human cells. The results are presented in FIG. 5A, FIG. 6A, FIG. 7A and FIG. 8A.

[0352] As shown on these figures, polyclonal antibody has a high titer of anti-Ebola immunoglobulins, and can effectively neutralize Ebola virus entry into human cells.

[0353] The specific anti-GP IgG are detected by ELISA. Recombinant GP is coated onto the well and sera are titrated by serial dilution. Binding of polyclonal antibody is measured by colorimetric assay. The titer of specific IgG is calculated by defining the inverse of the dilution presenting 50% of the positive signal. Following three administrations, polyclonal antibody presents a titer of 133572181 (meanSE).

[0354] Neutralization activity of the polyclonal antibody D60 is evaluated by sero-neutralization assay. Recombinant VSV-GP virus is mixed with different dilutions of polyclonal antibody and added to cultured cells. Protection is measured by counting the number of infected cells. The titer of sero-neutralization is determined by the inverse of the dilution where 50% of the cells are protected from the recombinant virus. Following three administrations, polyclonal antibody presents a SN50% of 564154 (meanSE).

Example IV: Production of a Potent Neutralizing Anti-EBOLA Polyclonal Antibody

[0355] Rabbits were immunized by injecting 0.3 mg of purified recombinant antigen prepared as disclosed in example II, per injection. In this example, four im injections were performed at day 0, 22, 36, and 50. The rabbits are sacrificed on day 68 and the serum is collected.

[0356] The polyclonal antibodies are purified from the serum by precipitation in 50% ammonium sulphate followed by purification on protein A-sepharose column, purification ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.

[0357] The specific binding of the polyclonal antibody to the recombinant antigen comprising sequence SEQ ID NO: 5 was then assessed by ELISA, and the neutralizing capacity of the polyclonal antibody was determined in infected human cells. The results are presented in FIG. 5B, FIG. 6B, FIG. 7B and FIG. 8B.

[0358] As shown on these figures, polyclonal antibody has a high titer of anti-Ebola immunoglobulins, and can effectively neutralize Ebola virus entry into human cells The specific anti-GP IgG are detected by ELISA. Recombinant GP is coated onto the well and sera are titrated by serial dilution. Binding of polyclonal antibody is measured by colorimetric assay. The titer of specific IgG is calculated by defining the inverse of the dilution presenting 50% of the positive signal. Following four administrations, polyclonal antibody presents a titer of 171774914 (meanSE).

[0359] Neutralization activity of the polyclonal antibody D68 is evaluated by sero-neutralization assay. Recombinant VSV-GP virus is mixed with different dilutions of polyclonal antibody and added to cultured cells. Protection is measured by counting the number of infected cells. The titer of sero-neutralization is determined by the inverse of the dilution where 50% of the cells are protected from the recombinant virus. Following four administrations, polyclonal antibody presents a SN50% of 860225 (meanSE)

Example V: Large Spectrum of Action of Polyclonal Antibodies of the Invention

[0360] Cross neutralization effect of polyclonals of the invention was tested against several serotypes of Ebola virus, as well as against non-Ebola viruses. Cross neutralization activity of the polyclonal antibody is evaluated by sero-neutralization assay. Several recombinant VSV-GP virus are produced harboring different GP from EBOLA virus (Mayinga, Makona, Tai Forrest and Bundibugyo), or from Marburg or Lloviu. Recombinant virus is mixed with different dilutions of polyclonal antibody and added to cultured cells. Protection is measured by counting the number of infected cells.

[0361] The results are depicted on FIG. 9.

[0362] As shown on FIG. 9, polyclonal antibody D60 is able to neutralize several serotypes of Ebola virus, while it has no side effect against Marburg or Lloviu viruses. This graph illustrates that polyclonal antibody produced following EBOV recombinant GP immunization can neutralized efficiently virus belonging to Zaire strain but also other strains of EBOLA like Tai Forrest and Bundibugyo. No neutralization of distinct filovirus like Marburg and Lloviu is observed, confirming the specificity of the polyclonals of the invention.

Example VI: Production of Anti-Ebola Sudan Virus Polyclonal Antibody Preparations

[0363] The present example discloses the production of an anti-Ebola polyclonal antibody preparations directed against Sudan Ebola virus.

[0364] The GP glycoprotein has been selected as antigen to produce polyclonal antibodies. The antigen has been modified to improve the immunogenicity. More specifically, such modified antigen comprises a consensus sequence. By aligning the sequences of several sequences of the Sudan Ebola virus GP glycoprotein, the inventors have selected most conserved positions and defined a consensus sequence which is referred to as sequence SEQ ID NO: 12.

TABLE-US-00002 SEQIDNO:12: MGGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGVVTNSTLEVTEIDQ LVCKDHLASTDQLKSVGLNLEGSGVSTDIPSATKRWGFRSGVPPKVVSYE AGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHKAQGTGPCPGD YAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIR EAVNYTENTSSYYATSYLEYEIENFGAQHSTTLFKIDNNTFVRLDRPHTP QFLFQLNDTIHLHQQLSNTTGRLIWTLDANINADIGEWAFWENKKNLSEQ LRGEELSFEALSLNETEDDDAASSRITKGRISDRATRKYSDLVPKNSPGM VPLHIPEGETTLPSQNSTEGRRVVNTQETITETAATIIGTNGNHMQISTI GIRPSSSQIPSSSPTMAPSPEAQTPTTHTSGPSVMATEEPTTPPGSSPGS TTEAPTLTTPENITTAVKTVLPQESTSNGLITSTVTGILGSLGLRKRSRR QTNTKATGKCNPNLHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQ NALVCGLRQLANETTQALQLFLRATTELRTYTILNRKAIDFLLRRWGGTC RILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPNQDNDDNWWTGWRQW IPAGIGITGIIIAIIALLCVCKLLC

[0365] In a particular embodiment, the Sudan Ebola virus antigen for use in the invention comprises or consists of a sequence which is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 12. The antigen preferably comprises or consists of sequence SEQ ID NO: 12 or an epitope-containing fragment thereof.

[0366] In a further preferred embodiment, the modified antigen is devoid of a functional mucin-like domain (bold and emphasized on the sequence above).

[0367] In a further preferred embodiment, the modified antigen is devoid of a functional signal peptide (bold on the sequence above).

[0368] In a further preferred embodiment, the modified antigen is devoid of a functional transmembrane domain (emphasized on the sequence above).

[0369] In this respect, a further particular Ebova virus antigen of the invention comprises or consists of a sequence which is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 14. The antigen preferably comprises or consists of sequence SEQ ID NO: 14 or an epitope-containing fragment thereof.

TABLE-US-00003 SEQIDNO:14 MPLGVVTNSTLEVTEIDQLVCKDHLASTDQLKSVGLNLEGSGVSTDIPSA TKRWGFRSGVPPKVVSYEAGEWAENCYNLEIKKPDGSECLPPPPDGVRGF PRCRYVHKAQGTGPCPGDYAFHKDGAFFLYDRLASTVIYRGVNFAEGVIA FLILAKPKETFLQSPPIREAVNYTENTSSYYATSYLEYEIENFGAQHSTT LFKIDNNTFVRLDRPHTPQFLFQLNDTIHLHQQLSNTTGRLIWTLDANIN ADIGEWAFWENKKNLSEQLRGETGILGSLGLRKRSRRQTNTKATGKCNPN LHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQNALVCGLRQLANE TTQALQLFLRATTELRTYTILNRKAIDFLLRRWGGTCRILGPDCCIEPHD WTKNITDKINQIIHDFIDNPLPNQDNDDNWWTGWRQW

[0370] In a further particular embodiment, the antigen further comprises a tag and, optionally, a spacer.

[0371] The recombinant antigen comprising sequence SEQ ID NO: 12 or 14 is produced as follows. Two pMTpuro vectors comprising the respective coding sequences are used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 g/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO.sub.4 for 4 days. The recombinant antigen is then purified on Strep-Tactin resin (for the recombinant antigen carrying a Strep tag) or on a Ni resin (for the recombinant antigen carrying a His tag). The recombinant antigen is also/further purified by Superdex 200 size-exclusion chromatography in 10 mM Tris and 150 mM NaCl (pH 7,5) (1TBS).

[0372] A polyclonal antibody preparation is obtained by administering to a rabbit the antigen as defined above. More specifically, rabbits are immunized by injecting between 0.1 and 1 mg of purified recombinant antigen per injection. Several (3 to 6) injections are performed. Serum is collected between the injections to monitor the immunization process. The rabbits are sacrificed when antibody titer is high and the serum is collected. The polyclonal antibodies are purified from the serum by precipitation in 50% caprylic acid followed by purification on protein A-sepharose column, ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.

Example VI: Production of Anti-Ebola Bundibugyo Virus Polyclonal Antibody Preparations

[0373] The present example discloses the production of an anti-Ebola polyclonal antibody preparations directed against Bundibugyo Ebolavirus.

[0374] The GP glycoprotein has been selected as antigen to produce polyclonal antibodies. The antigen has been modified to improve the immunogenicity. More specifically, such modified antigen comprises a consensus sequence. By aligning the sequences of several sequences of the Bundibugyo Ebolavirus GP glycoprotein, the inventors have selected most conserved positions and defined a consensus sequence which is referred to as sequence SEQ ID NO: 13.

TABLE-US-00004 SEQIDNO:13: MVTSGILQLPRERFRKTSFFVWVIILFHKVFPIPLGVVHN NTLQVSDIDKLVCRDKLSSTSQLKSVGLNLEGNGVATDVP TATKRWGFRAGVPPKVVNYEAGEWAENCYNLDIKKADGSE CLPEAPEGVRGFPRCRYVHKVSGTGPCPEGFAFHKEGAFF LYDRLASTIIYRSTTFSEGVVAFLILPETKKDFFQSPPLH EPANMTTDPSSYYHTVTLNYVADNFGTNMTNFLFQVDHLT YVQLEPRFTPQFLVQLNETIYTNGRRSNTTGTLIWKVNPT VDTGVGEWAFWENKKNFTKTLSSEELSVIFVPRAQDPGSN QKTKVTPTSFANNQTSKNHEDLVPEDPASVVQVRDLQREN TVPTPPPNTVPTTLIPDTMEEQTTSHYEPPNISRNHQERN NTAHPETLANNPPDNTTPSTPPQDGERTSSHTTPSPRPVP TSTIHPTTRETHIPTTMTTSHDTDSNRPNPIDISESTEPG PLTNTTRGAANLLTGSRRTRREITLRTQAK CNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGIMHN QNGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAI DFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDF IDKPLPDQTDNDNWWTGWRQWVPAGIGITG VIIAVIALLCICKFLL

[0375] In a particular embodiment, the Bundibugyo Ebolavirus antigen for use in the invention comprises or consists of a sequence which is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 13. The antigen preferably comprises or consists of sequence SEQ ID NO: 13 or an epitope-containing fragment thereof.

[0376] In a further preferred embodiment, the modified antigen is devoid of a functional mucin-like domain.

[0377] In a further preferred embodiment, the modified antigen is devoid of a functional signal peptide.

[0378] In a further preferred embodiment, the modified antigen is devoid of a functional transmembrane domain.

[0379] In a further particular embodiment, the antigen further comprises a tag and, optionally, a spacer.

[0380] The recombinant antigen comprising sequence SEQ ID NO: 13 is produced as follows. Two pMTpuro vectors comprising the respective coding sequences are used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 g/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO.sub.4 for 4 days. The recombinant antigen is then purified on Strep-Tactin resin (for the recombinant antigen carrying a Strep tag) or on a Ni resin (for the recombinant antigen carrying a His tag). The recombinant antigen is also/further purified by Superdex 200 size-exclusion chromatography in 10 mM Tris and 150 mM NaCl (pH 7,5) (1TBS).

[0381] A polyclonal antibody preparation is obtained by administering to a rabbit the antigen as defined above. More specifically, rabbits are immunized by injecting between 0.1 and 1 mg of purified recombinant antigen per injection. Several (3 to 6) injections are performed. Serum is collected between the injections to monitor the immunization process. The rabbits are sacrificed when antibody titer is high and the serum is collected. The polyclonal antibodies are purified from the serum by precipitation in 50% caprylic acid followed by purification on protein A-sepharose column, ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.