Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Definitions

• the invention relates to the removal of viruses aqueous solutions, as a rule protein solutions, by ultrafiltration.
• This entails the viruses to be removed being increased in size by incubation with high molecular weight ligands binding thereto, preferably specific antibodies, so that, on the one hand, the separation effect is improved and, on the other hand, a larger pore diameter which can now be chosen for the filters used also makes it possible for smaller viruses to be separated from larger protein molecules present in protein solutions, and, where appropriate, the filtration rate is increased.
• Proteins purified and concentrated from human plasma are used for therapy and prophylaxis of human diseases. These products are prepared from plasma pools consisting of about 10,000 individual donations. Since some of these donations may potentially be contaminated with human pathogenic viruses such as HIV-1 ⁇ 2, hepatitis B virus, hepatitis C virus and other viruses, there is the possibility of infection being caused by administration of the plasma proteins. In order to minimize this contamination hazard, donations are obtained only from healthy donors who are additionally tested for infection markers (antibodies against HIV 1 and HIV 2, UBsAg, antibodies against HCV and elevated liver function test results (ALT)); positive donations are rejected and not used for obtaining plasma proteins.
• the purification and concentration steps used in the industrial preparation of plasma proteins and, in particular, steps specifically introduced into the production to eliminate and/or inactivate viruses lead to plasma proteins with a very high safety standard.
• Filter units for eliminating viruses are produced by various companies (DiLeo, A. J. et al. Biologicals 21, 275-286 (1993); DiLeo A. J. et al. Biologicals 21, 287-296 (1993); Burnout, T. et al., Vox Sang. 67, 132-138 (1994)).
• Asahi Chemical Industry Co. Ltd. Tokyo, Japan, produces filter units stating a defined (average) pore size, while, for example, Millipore Corp., Bedford, Mass., USA, produces filter units stating a nominal molecular weight cut off
• viruses are held back at different rates by filters with different pore sizes, depending on the diameter of said viruses (HIV: 80-100 nm: HCV: 40-60 nm; HBV: 40-45 nm; picornaviruses: 24-30 nm; parvoviruses: 18-25 am): (I) filters with an average pore diameter of 75 nm essentially retain HIV, while the other specified viruses are found in the filtrate; (II) filters with an average pore diameter of 35 nm retain HIV completely and HCV and HBV to a large extent, while, for example, picornaviruses and parvoviruses are found in the filtrate; (III), filters with an average pore diameter of 15 nm retain HIV, HCV and HBV and, to a large extent, for example picornaviruses and parvoviruses.
• Filtration through a filter with an average pore diameter of 15 nm leads to a general increase in the virus safety of plasma proteins.
• most plasma proteins have such a high molecular weight that they cannot be filtered through a 15 nm filter, i.e. are likewise held back, only filters with an average pore diameter of 35 nm (or a nominal molecular weight cut off of 70,000, D to 100,000 D) are suitable for filtering most plasma proteins, but these do not remove to an adequate extent at least picornaviruses (such as, for example, hepatitis A virus) and parvoviruses (such as the human pathogenic parvovirus B 19) from plasma proteins.
• picornaviruses such as, for example, hepatitis A virus
• parvoviruses such as the human pathogenic parvovirus B 19
• the object therefore was to achieve an adequate, i.e. complete, retention even of small viruses by filtration, and to make filtration methods also applicable to those proteins which resemble simply in terms of their size a small virus.
• it was intended to increase the filtration rate as far as possible.
• the object is achieved by the present invention un that the viruses to be removed are increased in size by binding to high molecular weight ligands, preferably specific antibodies, particularly preferably monoclonal antibodies, in principle of all subclasses, but preferably subclass IgG or IgM or parts thereof still capable of binding, which are, where appropriate, modified or enlarged by genetic engineering, to such an extent that they can be held back by filtration.
• the increase in size can also be achieved by aggregate formation. It is in fact possible with this method to separate relatively large proteins such as factor VIII or von Willebrand factor from such viruses of increased size by filtration, it now being possible to choose the pore size such that the proteins pass through and the viruses of increased size are held back.
• the present invention makes it possible to increase the size of any constituents of an aqueous solution by binding to high molecular weight ligands to such an extent that separation is then possible from the now smaller constituents in a filtration step.
• Antibodies possibly modified HIV CD4 receptor HIV Sialic acid ( ⁇ derivatives, for Influenzaviruses example sialooligosaccharides) Heparan sulfate HSV C3d complement receptor/complement EBV receptor 2 (CR2) Acetylcholine receptor Rabiesviruses ICAM-1 (intracellular adhesion Rhinoviruses molecule-1) Gangliosides Paramyxoviruses IgA receptor HBV Epidermal Growth Factor Receptor Vaccinia Beta adrenergic receptor Reovirus Serotype 3 Immunoglobulins superfamily protein Poliovirus H-2 antigens Semliki Forest Virus
• Human pathogenic viruses such as, for example, HBV, HCV, HIV, picornaviruses and parvoviruses may, despite selection of donors, be present in a plasma pool. These viruses bind to antibodies present in the protein solution, in particular either to antibodies present in the plasma pool during incubation of the low-cryo plasma or of the resuspended cryoprecipitate at 2° C. to 37° C. for 15 minutes to 36 hours, preferably at 2° C. to 8° C. for a period of 2 to 36 hours, in particular 4 to 18 hours, or at 10° C. to 25° C.
• the virus-antibody complexes formed in this way can be removed from the plasma protein solution by filtration, for example dead-end filtration or, preferably, tangential flow filtration.
• Bovine parvovirus (BPV; ATCC VR-767), as model virus for the human pathogenic parvovirus B 19, was grown in diploid fetal bovine lung cells in EME medium containing 5% FCS and then separated from cells and cell debris by low-speed centrifugation (2000 g, 15 minutes, 4° C.); the virus-containing supernatant was divided into aliquots and stored at ⁇ 70° C. until investigated.
• Porcine parvovirus PPV; ATCC VR-742
• PPV was grown and isolated like BPV but in a permanent porcine kidney cell line (IB-RS-2 D10; ATCC CRL 1835).
• test mixtures were mixed, incubated at 20° C. for 1 hour and then filtered through BMM process filter PLANOVATM 35 (from Asahi Chemical Industry Co. Ltd., Tokyo, Japan) in accordance with the instructions of the manufacturing company.
• the infectiosity titer (CCID 50 : cell culture infectious dose 50%) was determined in the starting material and in the filtrate after filtration.
• Virus BPV BPV BPV PPV PPV material (30 ml) Antibody- PBS Human Human Human Human Human con- (no anti- serum serum serum serum taining bodies) (B19- (B19- (B19- (B19- solution positive/ negative/ positive/ negative/ (30 ml) ELISA) ELISA) ELISA) ELISA) ELISA) Infection 10 5.2 10 4.2 10 5.1 10 6.4 10 6.2 titer before filtra- tion (CCID 50 ) Infection 10 4.9 ⁇ 10 0.5 10 4.3 10 6.0 10 5.9 titer after filtra- tion (CCID 50 )
• a licensed poliovirus vaccine for oral immunization (Oral-Virelon®; live attenuated vaccine) was suspended in a protein solution comprising 10% fetal calf serum in DMEM; purified immunoglobulin (Beriglobin®) was added to part of this virus suspension, and the same volume of PBS was added to the other part. After incubation at 15° C.
• the resuspended samples were initially diluted 1:2 in tris/glycine buffer pH 8.3 and then further diluted in a 1:3 dilution series; 100 ⁇ l portions of each dilution were applied to a nitrocellulose filter (pore size 0.4 ⁇ m), the membrane was blocked with skimmed milk powder (3%), incubated with antiserum against polioviruses (from rabbit) at 37° C. for 1 hour and then incubated further with POD-labeled anti-rabbit antibodies. The bound antibodies were visualized with 4-chloro-1-naphthol/H 2 O 2 (dot-blot procedure as described by Cardosa, M J & Tio, P. H., Bull.
• the poliovirus-antibody complexes cannot, in contrast to uncomplexed antigens, be filtered; removal from the protein solution can therefore be achieved by adding immunoglobulins.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Analytical Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Water Supply & Treatment (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Separation Using Semi-Permeable Membranes (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 1995
  • 1995-02-09 DE DE19504211A patent/DE19504211A1/de not_active Withdrawn
• 1996
  • 1996-01-10 DE DE59610319T patent/DE59610319D1/de not_active Revoked
  • 1996-01-10 AT AT96100268T patent/ATE236659T1/de not_active IP Right Cessation
  • 1996-01-10 ES ES96100268T patent/ES2193210T3/es not_active Expired - Lifetime
  • 1996-01-10 EP EP96100268A patent/EP0727226B1/de not_active Revoked
  • 1996-02-07 AU AU44401/96A patent/AU708757B2/en not_active Ceased
  • 1996-02-07 US US08/598,264 patent/US20020068368A1/en active Granted
  • 1996-02-07 US US08/598,264 patent/US6391657B1/en not_active Expired - Fee Related
  • 1996-02-07 KR KR1019960002859A patent/KR100421763B1/ko not_active IP Right Cessation
  • 1996-02-08 JP JP8022195A patent/JPH08242849A/ja active Pending
  • 1996-02-08 CA CA002169122A patent/CA2169122A1/en not_active Abandoned

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