WO2000038743A1 - Removal of biological contaminants - Google Patents
Removal of biological contaminants Download PDFInfo
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- WO2000038743A1 WO2000038743A1 PCT/AU1999/001171 AU9901171W WO0038743A1 WO 2000038743 A1 WO2000038743 A1 WO 2000038743A1 AU 9901171 W AU9901171 W AU 9901171W WO 0038743 A1 WO0038743 A1 WO 0038743A1
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- WIPO (PCT)
- Prior art keywords
- solvent stream
- biomolecule
- membrane
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- biological
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0017—Filtration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/03—Electric current
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
- B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
- B01D61/425—Electro-ultrafiltration
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
- B01D61/425—Electro-ultrafiltration
- B01D61/4251—Electro-ultrafiltration comprising multiple electro-ultrafiltration steps
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- 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
- C07K1/24—Extraction; Separation; Purification by electrochemical means
- C07K1/26—Electrophoresis
Definitions
- the present invention relates to methods for the removal of biological contaminants, particularly removal of biological contaminants from biological preparations.
- Viruses are some of the smallest non-cellular organisms known. These simple parasites are composed of nucleic acid and a protein coat. Viruses are typically very small and range in size from 1.5x10 " m to 5.0x10 " m. Viruses depend on the host cells that they infect to reproduce by inserting their genetic material into the host, often literally taking over the host's function. An infected cell produces more viral protein and genetic material, often instead of its usual products. Some viruses may remain dormant inside host cells. However, when a dormant virus is stimulated, it can enter the lytic phase where new viruses are formed, self-assemble occurs and burst out of the host cell results in killing the cell and releasing new viruses to infect other cells.
- Viruses cause a number of diseases in humans including smallpox, the common cold, chicken pox. influenza, shingles, herpes, polio, rabies. Ebola. hanta fever, and AIDS. Some types of cancer have been linked to viruses.
- Pyrogens are agents which induce fever. Bacteria are a common source for the production of endotoxins which are pyrogenic agents. Furthermore, another detrimental effect of endotoxins is their known adjuvant effect which could potentially intensify immune responses against therapeutic drugs.
- the endotoxin limit set by the Food and Drug Administration (FDA) guidelines for most pharmaceutical products is for a single dose 0.5ng endotoxin per kilogram bodv weight or 25ng endotoxin/dose for a 50kg adult. Due to their size and charge heterogeneity, separation of endotoxins from proteins in solution can often be difficult. Endotoxin inactivation by chemical methods are unsuitable because they are stable under extremes of temperature and pH which would destroy the proteins.
- endotoxins tend to adhere to proteins in a fashion similar to detergents.
- endotoxin activity often clusters with the protein when chromatographic procedures such as ion exchange chromatography or gel filtration are employed.
- chromatographic procedures such as ion exchange chromatography or gel filtration are employed.
- the purification of biomolecules is sometimes a long and cumbersome process especially when purifying blood proteins. The process is made all the more complex by the additional step of ensuring the product is "bug'- free. The costs associated with this task is large and further escalates the purification costs in total.
- the Gradiflow technology rapidly purifies target proteins with high yield. For example, a proteins like fibrinogen (a clotting protein) can be separated in three hours using the Gradiflow while the present industrial separation is 3 days.
- Certain monoclonal antibodies can be purified in 35 minutes compared to present industrial methods which take 35 hours.
- the membrane configuration in the Gradiflow enables the system to be configured so that the purification procedure can also include the separation of bacteria viruses and vectors. It has now been found by the present inventors that appropriate membranes can be used and the cartridge housing the membrane configured to include separate chambers for the isolated bacteria and viruses.
- Gradiflow is a unique preparative electrophoresis technology for macromolecule separation which utilises tangential flow across a polyacrylamide membrane when a charge is applied across the membrane (AU 601040).
- the general design of the Gradiflow system facilitates the purification of proteins and other macromolecules under near native conditions. This results in higher yields and excellent recovery.
- the Gradiflow technology is bundled into a cartridge comprising of three membranes housed in a system of specially engineered grids and gaskets which allow separation of macromolecules by charge and/or molecular weight.
- the system can also concentrate and desalt/dialyse at the same time.
- the multimodal nature of the system allows this technology to be used in a number of other areas especially in the production of biological components for medical use.
- the structure of the membranes may be configured so that bacteria and viruses can be separated at the point of separation - a task which is not currently available in the biotechnology industry and adds to the cost of production through time delays and also because of the complexity of the task. Disclosure of Invention
- the present invention consists in a method of removing a biological contaminant from a mixture containing a biomolecule and the biological contaminant, the method comprising: (a) placing the biomolecule and contaminant mixture in a first solvent stream, the first solvent stream being separated from a second solvent stream by an electrophoretic membrane; (b) selecting a buffer for the first solvent stream having a required pH;
- step (e) maintaining step (c). and optional step (d) if used, until the second solvent stream contains the desired purity of biomolecule.
- the present invention consists in a method of removing a biological contaminant from a mixture containing a biomolecule and the biological contaminant, the method comprising:
- step (e) maintaining step (c). and optional step (d) if used, until the first solvent stream contains the desired purity of biomolecule.
- the biomolecule is selected from the group consisting of blood protein, immunoglobulin. and recombinant protein.
- the biological contaminant can be a virus, bacterium, prion or an unwanted biomolecule such as lipopolysaccharide. toxin or endotoxin.
- the biological contaminant is collected or removed from the first stream.
- the buffer for the first solvent stream has a pH lower than the isoelectric point of biomolecule to be separated.
- the electrophoretic membrane has a molecular mass cut-off close to the apparent molecular mass of biomolecule. It will be appreciated, however, that the membrane may have any required molecular mass cut-off depending on the application. Usually, the electrophoretic membrane has a molecular mass cut-off of between about 3 and lOOOkDa. A number of different membranes may also be used in a desired or useful configuration.
- the electric potential applied during the method is selected to ensure the required movement of the biomolecule. or contaminant if appropriate, through the membrane.
- An electric potential of up to about 300 volts has been found to be suitable. It will be appreciated, however, that greater or lower voltages may be used.
- the benefits of the method according to the first aspect of the present invention are the possibility of scale-up. and the removal of biological contaminants present in the starting material without adversely altering the properties of the purified biomolecule.
- the present invention consists in use of Gradiflow in the purification or separation of biomolecule from a biological contaminant.
- the present invention consists in biomolecule substantially free from biological contaminants purified by the method according to the first aspect of the present invention.
- the present invention consists in use of biomolecule according to the third aspect of the present invention in medical and veterinary applications.
- the present invention consists in a substantially isolated biomolecule substantially free from biological contaminants.
- HSA was purified from endotoxin spiked plasma. Samples were taken from up- and downstream at 30 minute intervals during a 90 minute purification (x-axis). Analysis of the samples using a LAL Chromogenic assay was performed to establish the endotoxin concentration
- FIG. 4 (y-axis) in the samples.
- Figure 4. Four to 25% native gel electrophoresis of samples from an HSA purification from endotoxin spiked plasma. Lane 1 contains molecular weight markers. Lane 2 contains starting plasma sample, Lanes 3-5 contain upstream samples at time 30. 60. and 90 minutes. Lanes 6-9 contain downstream samples at time 0. 30. 60 and 90 minutes, respectively.
- Contamination with virus is a major concern when purifying plasma proteins, such as IgG and human serum albumin (HSA).
- a contaminant virus can potentially infect a patient receiving the contaminated plasma products.
- a virus that infects bacteria is known as a phage, and they are readily detected by examining culture plates for cleared zones in a coating or lawn of bacteria.
- Aim To isolate IgG. HSA. and Fibrinogen from human plasma spiked with virus, using the Gradiflow. with simultaneous removal of the contaminating virus. IgG purification procedure
- IgG is the most abundant of the immunoglobulins. representing almost 70% of the total immunoglobulins in human serum.
- This class of immunoglobulins has a molecular mass of approximately 150kDa and consists of 4 subunits. two of which are light chains and two of which are heavy chains.
- the concentration of IgG in normal serum is approximately lOmg/ml.
- IgGs are conventionally purified using Protein A affinity columns in combination with DEAE-cellulose or DEAE-Sephadex columns.
- the main biological contaminants in IgG isolations are ⁇ -lipoprotein and transferrin.
- the product of conventional protein purification protocols is concentrated using ultrafiltration. Immunoaffinity can also be used to isolate specific IgGs.
- Platelet free plasma was diluted one part in three with Tris- borate. pH 9.0 running buffer and placed in the upstream of Gradiflow and spiked with either Llambda or T7 phage to a concentration of ⁇ 10 8 pfu/ml (plaque forming units/ml).
- a potential of 250V was placed across a separating membrane with a molecular weight cut off of 200kDa (3kDa restriction membranes).
- a membrane of this size restricts IgG migration whilst allowing smaller molecular weight contaminants to pass through the membrane, leaving IgG and other large molecular weight proteins in the upstream.
- a second purification phase was carried out using a GABA/Acetic acid buffer. pH 4.6 with a 500kDa cut off separating membrane (3kDa restriction membranes).
- a potential of 250V reversed polarity was placed across the system resulting in IgG migration through the membrane leaving other high molecular weight contaminants upstream.
- Albumin is the most abundant protein component (50mg/r ⁇ ) in human plasma and functions to maintain blood volume and oncotic pressure. Albumin regulates the transport of protein, fatty acids, hormones and drugs in the body. Clinical uses include blood volume replacement during surgery, shock, serious burns and other medical emergencies. Albumin is 67kDa and has an isoelectric point of approximately 4.9. The protein consists of a single subunit and is globular in shape. About 440 metric tons of albumin is used annually internationally with worldwide sales of US$1.5 billion. Albumin is currently purified using Cohn fractionation and commercial product contains many contaminants in addition to multimers of albumin. The high concentration, globular nature and solubility of albumin make it an ideal candidate for purification from plasma using Gradiflow technology.
- Albumin was isolated from platelet free plasma in a one-phase process using the charge of albumin at a pH above its isoelectric point (pi) and its molecular weight.
- a cartridge with a 75kDa cutoff separation membrane was placed between two 50kDa cutoff restriction membranes.
- the albumin was removed from high molecular weight contaminants by its migration through the separation membrane whilst small molecular weight contaminants dissipated through the 50kDa restriction membrane. Samples were taken at regular intervals throughout a 90 minutes run.
- fibrinogen has a role as fibrin glue, which is used to arrest bleeding and assist in the wound healing process.
- Fibrinogen is an elongated molecule of 340kDa that consists of three non-identical subunit pairs that are linked by a disulphide knot in a coiled coil conformation.
- the isoelectric point of fibrinogen is 5.5 and it is sparingly soluble when compared with other plasma proteins.
- Fibrinogen is conventionally purified from plasma by a series of techniques including ethanol precipitation, affinity columns and traditional electrophoresis. This process takes about 48-72 hours and the harsh physical and chemical stresses placed on fibrinogen are believed to denature the molecule, resulting in activity that is removed from that of fibrinogen in plasma.
- Cryo-precipitation is the first step in the production of factor VIII and involves the loss of most of the fibrinogen in plasma. Processing of this waste fibrinogen is of considerable interest to major plasma processors and provides an opportunity to demonstrate the rapid purification of fibrinogen from cryo-precipitate using the Gradiflow.
- Method Cryo-precipitate 1. produced by thawing frozen plasma at 4 C overnight was removed from plasma by centrifugation at lOOOOxg at 4°C for 5 minutes. The precipitate was re-dissolved in Tris-Borate buffer (pH 9.0) and placed in the upstream of a Gradiflow apparatus.
- the upstream was spiked with either Llambda or T7 phage to a concentration of ⁇ 10 8 pfu/ml.
- a potential of 250V was applied across a 300kDa cutoff cartridge and run for 2 hours.
- the downstream was replaced with fresh buffer at 30 minute intervals.
- a second phase was used to concentrate the fibrinogen through a 500kDa cutoff separation membrane at pH 9.0.
- the downstream was harvested at 60 minutes.
- the product was dialysed against PBS pH 7.2 and analysed for clotting activity by the addition of calcium and thrombin (final concentrations lOmM and 10NIH unit/ml respectively).
- the presence of purified fibrinogen was confirmed by examination on reduced SDS PAGE 4-25% gels.
- the presence of either T7 or Llambda in the time point samples was tested using the previously described method.
- Prion removal during plasma protein purification using Gradiflow technology There is an international concern regarding the contamination of plasma proteins by prion protein.
- Prion is a glycoprotein of 27-33kDa in size which occurs naturally in many human derived materials, including white blood cells, platelets, plasma and plasma proteins preparations, e.g. HSA, IgG. FVIII and fibrinogen. Prion can become folded abnormally and cause neurological disorders such as Creutzfeld-Jacob disease (CJD) and Kuru.
- the samples were incubated for 1-2 hours at room temperature before being dispensed, and the plate washed three times as previously described.
- a solution of prion-specific polyclonal antibody, denoted R029 (Prionics Inc. Switzerland) was diluted at l:1000(v:v) in PBS/T20. and added to the wells of the plate (lOO ⁇ l/well). The mixture was incubated for 1-2 hours at room temperature, before being decanted.
- the plate was washed three times and lOO ⁇ l/well of a horseradish peroxidase conjugated polyclonal anti-rabbit IgG antiserum (purchased from Dakopatts) was added. The conjugate was incubated for 30-60 minutes at room temperature and then removed.
- Endotoxins are a lipopolysaccharide derived from the lipid membrane of gram negative bacteria. The presence of endotoxin in a human blood fraction therapeutic can lead to death of the receiving patients.
- IgG purification procedure Method Platelet free plasma was diluted one part in three with Tris- borate. pH 9.0 running buffer and placed in the upstream of a Gradiflow apparatus and spiked with purified E. coli endotoxin to a concentration of 55ng/ml. A potential of 250V was placed across a separating membrane with a molecular weight cut off of 200kDa (3kDa restriction membranes). A membrane of this size restricts IgG migration whilst allowing smaller molecular weight contaminants to pass through the membrane, leaving IgG and other large molecular weight proteins in the upstream. A second purification phase was carried out using a GAJBA/ Acetic acid buffer. pH 4.6 with a 500kDa cut off separating membrane (3kDa restriction membranes). A potential of 250V reversed polarity was placed across the system resulting in
- the mixture was placed in the upstream of a Gradiflow apparatus.
- Albumin was isolated from platelet free plasma in a one-phase process using the charge of albumin at a pH above its pi and its molecular weight.
- a cartridge with a 75kDa cutoff separation membrane was placed between two 50kDa cutoff restriction membranes.
- the albumin was removed from high molecular weight contaminants by its migration through the separation membrane whilst small molecular weight contaminants dissipated through the 50kDa restriction membrane. Samples were taken at regular intervals throughout a 90 minutes run. The presence of the purified HSA in the downstream was demonstrated by examination by SDS-PAGE.
- Endotoxin was tested for in both up- and down-stream samples using a LAL Chromogenic assay supplied by Cape Cod Associates. All samples were diluted 1 in 10 and the endotoxin assay was performed according to the manufacturer instructions. Results of IgG and HSA purification
- Contamination with bacteria is a major concern when purifying plasma proteins, such as IgG and HSA. Contaminant bacteria can potentially infect a patient receiving the plasma products, or during pasteurisation of the products the bacteria dies releasing dangerous endotoxins. that are harmful to the patient. Bacteria are easily detected by culturing samples on nutrient agar plates. Aim: To isolate IgG. and HSA. from human plasma spiked with bacteria. using the Gradiflow.
- Platelet free plasma was diluted one part in three with Tris- borate. pH 9.0 running buffer and placed in the upstream of Gradiflow and spiked with E. coli to a concentration of 4xl0 8 cells/ml. A potential of 250V was placed across a separating membrane with a molecular weight cut off of
- Prion present in plasma can be moved across a 75kDa separation membrane with albumin, however, unlike albumin, the prion is not retained by the 50kDa restriction membrane.
- albumin can be purified from plasma with simultaneous removal of Prion protein.
Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002356894A CA2356894A1 (en) | 1998-12-23 | 1999-12-23 | Removal of biological contaminants |
JP2000590694A JP2002540912A (en) | 1998-12-23 | 1999-12-23 | Removal of biological contaminants |
AU25266/00A AU769070B2 (en) | 1998-12-23 | 1999-12-23 | Removal of biological contaminants |
EP99968291A EP1140214A4 (en) | 1998-12-23 | 1999-12-23 | Removal of biological contaminants |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPP7906A AUPP790698A0 (en) | 1998-12-23 | 1998-12-23 | Separation of microorganisms |
AUPP7906 | 1999-12-23 |
Publications (1)
Publication Number | Publication Date |
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WO2000038743A1 true WO2000038743A1 (en) | 2000-07-06 |
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ID=3812113
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/AU1999/001171 WO2000038743A1 (en) | 1998-12-23 | 1999-12-23 | Removal of biological contaminants |
Country Status (6)
Country | Link |
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US (2) | US6464851B1 (en) |
EP (1) | EP1140214A4 (en) |
JP (1) | JP2002540912A (en) |
AU (1) | AUPP790698A0 (en) |
CA (1) | CA2356894A1 (en) |
WO (1) | WO2000038743A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2002093168A1 (en) * | 2001-05-15 | 2002-11-21 | Gradipore Limited | Prion diagnostic test |
EP1483377A1 (en) * | 2002-03-12 | 2004-12-08 | Gradipore Limited | Recovery of viruses |
CN111458396A (en) * | 2019-01-18 | 2020-07-28 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
Families Citing this family (7)
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AUPP790698A0 (en) * | 1998-12-23 | 1999-01-28 | Life Therapeutics Limited | Separation of microorganisms |
US20040000482A1 (en) * | 1998-12-23 | 2004-01-01 | Gradipore Limited | Viral removal |
AUPP971399A0 (en) * | 1999-04-12 | 1999-05-06 | Life Therapeutics Limited | Separation of plasma components |
US7077942B1 (en) * | 1999-12-23 | 2006-07-18 | Gradipore Limited | Removal of biological contaminants |
US20030019763A1 (en) * | 1999-12-23 | 2003-01-30 | Brendon Conlan | Apparatus and method for separation of biological contaminants |
EP1786827A4 (en) * | 2004-05-14 | 2008-10-29 | Kirin Holdings Kk | Methods for immunoglobulin purification |
US20080135437A1 (en) * | 2006-12-11 | 2008-06-12 | Leslie Barnett Taneri | Stress relief sounds in consumer goods |
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WO2002093168A1 (en) * | 2001-05-15 | 2002-11-21 | Gradipore Limited | Prion diagnostic test |
US6991715B2 (en) | 2001-05-15 | 2006-01-31 | Gradipore Limited | Prion diagnostic test |
EP1483377A1 (en) * | 2002-03-12 | 2004-12-08 | Gradipore Limited | Recovery of viruses |
EP1483377A4 (en) * | 2002-03-12 | 2005-03-30 | Gradipore Ltd | Recovery of viruses |
CN111458396A (en) * | 2019-01-18 | 2020-07-28 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
CN111458396B (en) * | 2019-01-18 | 2022-07-08 | 成都康弘生物科技有限公司 | Method for detecting charge heterogeneity of protein |
Also Published As
Publication number | Publication date |
---|---|
US6464851B1 (en) | 2002-10-15 |
US20020084187A1 (en) | 2002-07-04 |
EP1140214A1 (en) | 2001-10-10 |
AUPP790698A0 (en) | 1999-01-28 |
JP2002540912A (en) | 2002-12-03 |
CA2356894A1 (en) | 2000-07-06 |
EP1140214A4 (en) | 2003-05-02 |
US7060173B2 (en) | 2006-06-13 |
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