CA2356894A1 - Removal of biological contaminants - Google Patents
Removal of biological contaminants Download PDFInfo
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- CA2356894A1 CA2356894A1 CA002356894A CA2356894A CA2356894A1 CA 2356894 A1 CA2356894 A1 CA 2356894A1 CA 002356894 A CA002356894 A CA 002356894A CA 2356894 A CA2356894 A CA 2356894A CA 2356894 A1 CA2356894 A1 CA 2356894A1
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- solvent stream
- biomolecule
- membrane
- stream
- solvent
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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
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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
Abstract
A method of removing a biological contaminant from a mixture containing a biomolecule and the biological contaminant, the method comprising: (a) placi ng 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 strea m having a required pH; (c) applying an electric potential between the two solvent streams causing movement of the biomolecule through the membrane int o the second solvent stream while the biological contaminant is substantially retained in the first sample stream, or if entering the membrane, being substantially prevented from entering the second solvent stream; (d) optionally, periodically stopping and reversing the electric potential to cause movement of any biological contaminants having entered the membrane to move back into the first solvent stream, wherein substantially not causing a ny biomolecules that have entered the second solvent stream to re-enter first solvent stream; and (e) maintaining step (c), and optional step (d) if used, until the second solvent stream contains the desired purity of biomolecule.< /SDOAB>
Description
L
a Removal of Biological Contaminants Technical Field The present invention relates to methods for the removal of biological contaminants. particularly removal of biological contaminants from s biological preparatiozis.
Backeround Art The modern biotechnology industry is faced with a number of problems especiallv concerning the processing of complex biological solutions which ordinarily include proteins. :nucleic acid molecules and 1o complex sugars and which are contaminated with unwanted biological materials. Contaminants include microorganisms such as bacteria and viruses or biomolecules derived from microorganisms or the processing procedure. The demand is. therefore, for a high purity, scalable separation, which can be confidently used both in product development and production, z5 which in one step gin.=ill both purify macromolE:cules and separate these biological contaminants.
Viruses are some of the smallest non-ce;Ilular organisms known. These simple parasites are composed of nucleic acid and a protein coat. Viruses are typically very small and range in size from I.;ix30'8 m to 5.0x10'5 m. Viruses 2o 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 zn.ay remain dormant inside host cells. However. when a dormant virus is stimulated, it can enter the lvtic 2s phase where new viruses are formed. self assemble occurs and burst out of the host cell results in k1111I1g the cell and releasing new viruses to infect other cells. Viruses cause a number of diseases izz humans including Siliallp0\. the common cold. chicken pox. influenza, shingles, herpes, polio.
rabies. Ebola. hanta fever. and AIDS. Some types of cancer have been linked 30 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 intezZSifv immune responses against therapeutic drugs. The 35 endotoxin limit set by the Food and Drug Administration (FDA) guidelines for most pharmaceutical products is for a single dose 0.5ng endotoxin per a , ..
~ R
a Removal of Biological Contaminants Technical Field The present invention relates to methods for the removal of biological contaminants. particularly removal of biological contaminants from s biological preparatiozis.
Backeround Art The modern biotechnology industry is faced with a number of problems especiallv concerning the processing of complex biological solutions which ordinarily include proteins. :nucleic acid molecules and 1o complex sugars and which are contaminated with unwanted biological materials. Contaminants include microorganisms such as bacteria and viruses or biomolecules derived from microorganisms or the processing procedure. The demand is. therefore, for a high purity, scalable separation, which can be confidently used both in product development and production, z5 which in one step gin.=ill both purify macromolE:cules and separate these biological contaminants.
Viruses are some of the smallest non-ce;Ilular organisms known. These simple parasites are composed of nucleic acid and a protein coat. Viruses are typically very small and range in size from I.;ix30'8 m to 5.0x10'5 m. Viruses 2o 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 zn.ay remain dormant inside host cells. However. when a dormant virus is stimulated, it can enter the lvtic 2s phase where new viruses are formed. self assemble occurs and burst out of the host cell results in k1111I1g the cell and releasing new viruses to infect other cells. Viruses cause a number of diseases izz humans including Siliallp0\. the common cold. chicken pox. influenza, shingles, herpes, polio.
rabies. Ebola. hanta fever. and AIDS. Some types of cancer have been linked 30 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 intezZSifv immune responses against therapeutic drugs. The 35 endotoxin limit set by the Food and Drug Administration (FDA) guidelines for most pharmaceutical products is for a single dose 0.5ng endotoxin per a , ..
~ R
kilogrannbody weight or 25ng endotoxil~/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. Furthermore, due to their arnphipathic nature. endotoxins tend to adhere to proteins in a fashion similar to detergents. In such cases. endotoxin activity often clusters with the protein when chromatographic' procedures such as ion exchange chromatography or geI filtration are employed.
1o Presently. 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 25 purifies target proteins with high yield. For example, a proteins like fibrinogen (a clotting protein) can be separatecL in three hours using the Gradiflocv while the present industrial separatiion is 3 days. Certain monoclonal antibodies can be purified in 35 minutes compared to present industrial methods which take 35 hours.
20 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 faund by the present inventors that appropriate membranes can be used and the cartridge housing the membrane configured to include separate chapnbers for the isolated 25 bacteria and viruses.
The Gradiflow Technology Gradiflocv 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 30 (AU 6010:10). The general design of the Gradiflow system facilitates the purification of proteins and other macromolecules under near native COIldlti0llS. This results in higher wields and excellent recovery.
In essence the GradifIow technology is bundled into a cartridge COIIlp1'ISlIlg Of three 112eII1bI'alleS housed in a system of specially engineered 35 'rids and gaskets ~rhich allow separation of macromolecules by charge and/or molecular tveight. The system can also conceni:rate and desalt/dialvse at the ~ ..
which would destroy the proteins. Furthermore, due to their arnphipathic nature. endotoxins tend to adhere to proteins in a fashion similar to detergents. In such cases. endotoxin activity often clusters with the protein when chromatographic' procedures such as ion exchange chromatography or geI filtration are employed.
1o Presently. 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 25 purifies target proteins with high yield. For example, a proteins like fibrinogen (a clotting protein) can be separatecL in three hours using the Gradiflocv while the present industrial separatiion is 3 days. Certain monoclonal antibodies can be purified in 35 minutes compared to present industrial methods which take 35 hours.
20 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 faund by the present inventors that appropriate membranes can be used and the cartridge housing the membrane configured to include separate chapnbers for the isolated 25 bacteria and viruses.
The Gradiflow Technology Gradiflocv 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 30 (AU 6010:10). The general design of the Gradiflow system facilitates the purification of proteins and other macromolecules under near native COIldlti0llS. This results in higher wields and excellent recovery.
In essence the GradifIow technology is bundled into a cartridge COIIlp1'ISlIlg Of three 112eII1bI'alleS housed in a system of specially engineered 35 'rids and gaskets ~rhich allow separation of macromolecules by charge and/or molecular tveight. The system can also conceni:rate and desalt/dialvse at the ~ ..
same time. The nlultimodaI Mature of the systf:m allows this technology to be used in a number of other areas especially i:n the production of biological COIIIpOIIeIItS for nledical use. The structure of the membranes may be configured so that bacteria arid viruses can be separated at the point of separation - a task which is not currently avaiI;able 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 In a first aspect. the present invention consists in a method of i0 removing a biological COIItaIIlIIlaIlt frOIIl a InlXfLlre COntalnlng a biomolecule and the biological c0IltaIIllilaIlt, the method comprising:
(a) placing the biolnolecule 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;
(c) applying an electric potential between the two solvent streams causing movement of the bionlolecule through the mem.bralle into the second solvent stream ~~~hile the biological contaminant is substantially .retained in the first sample stream. or if elltering the membrane. beiing substantially prevented 2o from entering the second solvent stream;
(d) optionally, periodically stopping and revE:rsing the electric potential to cause movement of ally biological contaminants having entered the membraMe to move back into the first solvent st:reanl. wherein substantially not causing any biomolecules that have entered the second solvent stream to i'e-enter fil'St SOIVeIlt StreaIll: and (e) maintaining step (c), and optional step (d;~ if used, until the second solvent stream contains the desired purity of bionlolecule.
In a second aspect. the present invention consists in a method of Ten10V1I1g a biological COIltaI11111aI1t fTOnl a I111.~CtU.re COIItalIllllg a biomolecule aIld the biological COIItaI111llallt, the method COII'lprlSlllg:
(a) placing the biolnolecule and contaminant mixture in a first solvent stream. the first solvent stream being separated from a second solvent stream b~~ an electrophoretic membrane:
(b) selecting a, buffer for the first solvent strea.ln having a required pH;
(c) applying an electric potential betmeen the two solvent streams causing movement of the biological contaminant through the membrane into the $ r WO.00138743 PCT/AU99/OI171 second solvent stream c~rhile the biomolecule is substantiall~f retained in the first sample stream.. or if entering the membrane, being substantially prevented from entering the second solvent stream: .w (d) optionally, periodically stopping and reversing the electric potential to cause movement of any biomolecule having entered the membrane to move back into the first solvent stream: «, herein substantially not causing any biological COIltaIIl111a11tS that have entered the second solvent stream to re-enter first solvent stream: and . (e) maintaining step (c), and optional step (d) if used, until the first solvent stream contains the desired purity of biomolecule.
In the first and second aspects of the pr,ssent.invention, preferably tile biomoIecule is selected from the group consisting of blood protein, lIIIEIluilOglObuI111. alld reCOIIlblnallt protein. .
The biological cOIltaII1112aI1t can be a virus, bacterium. prior or an v.
s5 unwanted biomolecule such as lipopolysaccharide. toxin or endotoxin.
Preferably, the biological contaminant is collected or removed from the first stream.
Preferably, the buffer for the first solvent stream has a pH lower than the isoelectric point of biomolecule to be separated.
, In a further preferred embodiment of the; first aspect of the present invention. the electrophoretic membrane has a molecular mass cut-off close to the apparent molecular mass of biomolecule;. It will be appreciated.
hov~rever. 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 1000kDa. t'1 number of differezlt W embranes znay 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 biolnolecule, or c:ontanunant if appropriate, through the membrane. ran electric potential of up to about 300 volts has 3o beezl found to be suitable. It ~.vill be appreciated. however. that greater or lom~er voltages may be used.
The benefits of the method according to the first aspect of the presenfi invention are the possibility of scale-up. and the removal of biological contaminants present in the starting material tuithout adversely altering the properties of the purified biomolecule.
~ .r w WO 00/38?43 PCT/AU99/011?1 In a third aspect. the present invention consists in use of Gradiflow in ~ the purification or separation of biomolecule from a biological contaminant.
In a fourth aspect, the present invention consists in biomolecule substantially free from biological contaminants purified by the method 5 according to the first aspect of the present inverition.
In a fifth aspect. the present invention consists in use of biomolecule according to the third aspect of the present invention in medical and veterinary applications.
In a sixth aspect. the present invention consists in a substantially 2o isolated biomolecule substantially free from biological contaminants.
Throughout this specification, unless the context requires otherwise, the word "comprise". or variations such as "cornprises" or "comprising", will be understood t0 llllply the IIlClus10I1 Of a stated element: integer or step, or group of elements. integers or steps, but not the exclusion of any other I5 element. integer or step, or group of elements, integers or steps.
In order that the present invention may be more clearly understood a preferred forms will be described with reference to the accompanying drawings.
Brief Description of Drawings 2o Figure 1. Samples from up and downstream were taken at time intervals (x-axis) during the isolation of albumin from plasma. Albumin was measured in the samples by II11x1I1g with BCG reagent and reading the absorbance of 8311111. The cOIlC811trat10I1 Of albumin in each sample was calculated from the standard curve. and multiplied by the volume of the up-25 or docwnstream to obtain the Total HSA in the up- and downstream (y-axis).
All samples were assayed for prior using a sandwich ELISA. and recording the absorbance values at .t50nm (second y-axis)'.
Figure 2. Samples fl'OIII the second phasf~ of an IgG separation were taken fFOIIl both up- and downstreams (U/S and D/S respectively) at 30 30 minute intervals. The samples were assayed for endotoxin using a LAL
Chromogenic assat= (Cape Cod Assoc.) Figure 3. HSA cv=as purified from endoto~;in spiked plasma. Samples were taken from up- and downstream at 3Q minute intervals during a 90 minute purification (x-a.Yis). Analysis of the samples using a LAL
35 Chromogenic assay mas performed to establish 'the endotoxin concentration (~T-axis) in the samples.
Disclosure of invention In a first aspect. the present invention consists in a method of i0 removing a biological COIItaIIlIIlaIlt frOIIl a InlXfLlre COntalnlng a biomolecule and the biological c0IltaIIllilaIlt, the method comprising:
(a) placing the biolnolecule 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;
(c) applying an electric potential between the two solvent streams causing movement of the bionlolecule through the mem.bralle into the second solvent stream ~~~hile the biological contaminant is substantially .retained in the first sample stream. or if elltering the membrane. beiing substantially prevented 2o from entering the second solvent stream;
(d) optionally, periodically stopping and revE:rsing the electric potential to cause movement of ally biological contaminants having entered the membraMe to move back into the first solvent st:reanl. wherein substantially not causing any biomolecules that have entered the second solvent stream to i'e-enter fil'St SOIVeIlt StreaIll: and (e) maintaining step (c), and optional step (d;~ if used, until the second solvent stream contains the desired purity of bionlolecule.
In a second aspect. the present invention consists in a method of Ten10V1I1g a biological COIltaI11111aI1t fTOnl a I111.~CtU.re COIItalIllllg a biomolecule aIld the biological COIItaI111llallt, the method COII'lprlSlllg:
(a) placing the biolnolecule and contaminant mixture in a first solvent stream. the first solvent stream being separated from a second solvent stream b~~ an electrophoretic membrane:
(b) selecting a, buffer for the first solvent strea.ln having a required pH;
(c) applying an electric potential betmeen the two solvent streams causing movement of the biological contaminant through the membrane into the $ r WO.00138743 PCT/AU99/OI171 second solvent stream c~rhile the biomolecule is substantiall~f retained in the first sample stream.. or if entering the membrane, being substantially prevented from entering the second solvent stream: .w (d) optionally, periodically stopping and reversing the electric potential to cause movement of any biomolecule having entered the membrane to move back into the first solvent stream: «, herein substantially not causing any biological COIltaIIl111a11tS that have entered the second solvent stream to re-enter first solvent stream: and . (e) maintaining step (c), and optional step (d) if used, until the first solvent stream contains the desired purity of biomolecule.
In the first and second aspects of the pr,ssent.invention, preferably tile biomoIecule is selected from the group consisting of blood protein, lIIIEIluilOglObuI111. alld reCOIIlblnallt protein. .
The biological cOIltaII1112aI1t can be a virus, bacterium. prior or an v.
s5 unwanted biomolecule such as lipopolysaccharide. toxin or endotoxin.
Preferably, the biological contaminant is collected or removed from the first stream.
Preferably, the buffer for the first solvent stream has a pH lower than the isoelectric point of biomolecule to be separated.
, In a further preferred embodiment of the; first aspect of the present invention. the electrophoretic membrane has a molecular mass cut-off close to the apparent molecular mass of biomolecule;. It will be appreciated.
hov~rever. 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 1000kDa. t'1 number of differezlt W embranes znay 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 biolnolecule, or c:ontanunant if appropriate, through the membrane. ran electric potential of up to about 300 volts has 3o beezl found to be suitable. It ~.vill be appreciated. however. that greater or lom~er voltages may be used.
The benefits of the method according to the first aspect of the presenfi invention are the possibility of scale-up. and the removal of biological contaminants present in the starting material tuithout adversely altering the properties of the purified biomolecule.
~ .r w WO 00/38?43 PCT/AU99/011?1 In a third aspect. the present invention consists in use of Gradiflow in ~ the purification or separation of biomolecule from a biological contaminant.
In a fourth aspect, the present invention consists in biomolecule substantially free from biological contaminants purified by the method 5 according to the first aspect of the present inverition.
In a fifth aspect. the present invention consists in use of biomolecule according to the third aspect of the present invention in medical and veterinary applications.
In a sixth aspect. the present invention consists in a substantially 2o isolated biomolecule substantially free from biological contaminants.
Throughout this specification, unless the context requires otherwise, the word "comprise". or variations such as "cornprises" or "comprising", will be understood t0 llllply the IIlClus10I1 Of a stated element: integer or step, or group of elements. integers or steps, but not the exclusion of any other I5 element. integer or step, or group of elements, integers or steps.
In order that the present invention may be more clearly understood a preferred forms will be described with reference to the accompanying drawings.
Brief Description of Drawings 2o Figure 1. Samples from up and downstream were taken at time intervals (x-axis) during the isolation of albumin from plasma. Albumin was measured in the samples by II11x1I1g with BCG reagent and reading the absorbance of 8311111. The cOIlC811trat10I1 Of albumin in each sample was calculated from the standard curve. and multiplied by the volume of the up-25 or docwnstream to obtain the Total HSA in the up- and downstream (y-axis).
All samples were assayed for prior using a sandwich ELISA. and recording the absorbance values at .t50nm (second y-axis)'.
Figure 2. Samples fl'OIII the second phasf~ of an IgG separation were taken fFOIIl both up- and downstreams (U/S and D/S respectively) at 30 30 minute intervals. The samples were assayed for endotoxin using a LAL
Chromogenic assat= (Cape Cod Assoc.) Figure 3. HSA cv=as purified from endoto~;in spiked plasma. Samples were taken from up- and downstream at 3Q minute intervals during a 90 minute purification (x-a.Yis). Analysis of the samples using a LAL
35 Chromogenic assay mas performed to establish 'the endotoxin concentration (~T-axis) in the samples.
Figure ~. 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 iI1i111ateS. Lanes 6-9 contain downstream samples at time 0. 30. 60 and 90 :minutes. respectively.
l.~Iodes for Carrvina Out the Invention Virus removal during plasma protein purification using Gradiflow technology Contamination with virus is a major con,cerii 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 knoww as a phage, and they are readily detected by examining culture plates for cleared zones in a coating or Lawn of bacteria.
25 .-iizrz: 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 immunol;lobuIins. representing almost 70% of the total immunoglobulins in human serum. This class of immunogIobulins has a molecular mass of approximately 150kDa and consists of .t subunits. ttvo of cvhich are light chains and two of which are heavy chains. The concentration of IgG 111 IlOT171a1 serum is approximately l0mghnl.
--'S IgGs are conventionally purified using Protein A affinity colulnils in conlbillation with DErIE-cellulose or DEAErSep;Eladex columns. The main biological COIItalIi111a11tS 111 IgG isolations are f~-lipoprotein and transferrin.
The product of conventional protein purification protocols is concentrated using ultrafiltration. I111111uIlOafflnltv can also b~e used to isolate specific IgGs.
.l.Iethod: Platelet free plasma was diluted one part in three with Tris-borate, pH 9.0 ruIlIllIlg buffer and placed in the upstream of Gradiflow and spiked ~a~ith either Llambda or T7 phage to a concentration of ~-108pfu/ml (plaque forming units/nll). A potential of 250V mas placed across a separating membrane ~~>ith a molecular w>eight cot off of 200kDa (3kDa restriction membranes). A membrane of this size restricts IgG migration . ' i ' «°hilst allots~ing smaller molecular weight contaminants to pass through the membrane. leaving IgG and other large molecular weight proteins in the upstream. r1 second purification phase was carried out using a GABA/Acetic acid buffer. pH ~.6 with a 500kDa cut off separating membrane (3kDa restr1Ct10I1 nleII2bTaIles). A potential of 250V reversed polarity was placed across the system resulting in IgG migration through the membrane leaving other high molecular weight contaminants ups~treazn.
Examination of samples taken at 30 minutes intervals was made on reduced SDS-PAGE 4-25% gels:
VIruS tE'StllI~.P' One hundred and fifty ul taken at each time point sample was mixed t~~ith 100u1 of appropriate Escherichia coh culture (Strain HB201 was used for T7 and strain jhI101 for Llambda). The mixtures were incubated for 15 minutes at 37"C, before each was added to 2.5rn1 of freshly prepared molten soft agar. and vortexed. The mixtures were poured over culture plates of Luria Agar. and incubated at 37°C overnight. T'he plates were inspected for the presence of virus colonies (plaques) in the lawn of E. coli. The number of plaques was recorded or if the virus had infected the entire E. coli population the result was recorded as confluent lysis.
HSA purification procedure Albumin is the most abundant protein component (50mg/m) in human plasma and functions to znaintaimblood volume; and oncotic pressure.
Albumin regulates the transport of protein. fatty acids. hormones and drugs in the bode. Clinical uses include blood.volume replacement during surgery, shock. serious bums and other medical emergencies. Albumin is 67kDa and has an isoelectric point of approximately ~.9. The protein consists of a single subunit and is globular in shape. About .t~0 metric tons of albumin is used annualln internationally with worldwide sales of US$1.5 billion. Albumin is currenti~- purified using Cohn fractionation and commercial product contains InaIly COIItaIIllIlaIltS lI1 addlt10I2 t0 I11u1t1IneTS Of albuIllln. The high COIlC8I2trat10I1. globular nature and solubility of .albumin make it an ideal candidate for purification froze plasma using Gradiflow technology.
alethod: Pooled normal plasma was diluted one in three with Tris-Borate (TB) running buffer. pH 9.0 and spiked vfith --~ 208pfu/ml of Llambda or T7 phage. The mixture was placed in the upstream of a Gradiflow apparatus.
:~lbuznin mas isolated from platelet free plasma i;n a one-phase process using n r WO 00/38743 PCTlAU99/01171 the charge of albumin at a pH above its isoele<aric point (pI) and its ' molecular weight. Thus a cartridge with a 751~a cutoff separation membrane was placed bet<veen 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. Virus was detected as so previously described above.
Fibrinogen purification procedure:
Commercially. 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 3:tOkDa that consists of three non-identical subunit ~5 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 2o 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 3aroduction of factor VIII and 25 involves the loss of IIlOSt 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 Gradiflou~.
Method: Cryo-precipitate 1, produced by thawing frozen plasma at 4°C
30 o~~ernight ~~uas removed from plasma by centrifugation at 10000xg at 4°C far 5 minutes. The precipitate «~as re-dissolved in Tris-Borate buffer (pH 9.0) and placed in the upstreaim of a Gradiflow apparatus.. The upstream was spiked t~uith either Llambda or T7 phage to a concentration of ---108pfuJml. A
potential of 250V .vas applied across a 300kDa cutoff cartridge and run for 2 35 hours. The donvnstream tras replaced Gvith fresh buffer at 30 minute inten~als. A second phase teas used to concentrate the fibrinogen through a ° , "
l.~Iodes for Carrvina Out the Invention Virus removal during plasma protein purification using Gradiflow technology Contamination with virus is a major con,cerii 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 knoww as a phage, and they are readily detected by examining culture plates for cleared zones in a coating or Lawn of bacteria.
25 .-iizrz: 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 immunol;lobuIins. representing almost 70% of the total immunoglobulins in human serum. This class of immunogIobulins has a molecular mass of approximately 150kDa and consists of .t subunits. ttvo of cvhich are light chains and two of which are heavy chains. The concentration of IgG 111 IlOT171a1 serum is approximately l0mghnl.
--'S IgGs are conventionally purified using Protein A affinity colulnils in conlbillation with DErIE-cellulose or DEAErSep;Eladex columns. The main biological COIItalIi111a11tS 111 IgG isolations are f~-lipoprotein and transferrin.
The product of conventional protein purification protocols is concentrated using ultrafiltration. I111111uIlOafflnltv can also b~e used to isolate specific IgGs.
.l.Iethod: Platelet free plasma was diluted one part in three with Tris-borate, pH 9.0 ruIlIllIlg buffer and placed in the upstream of Gradiflow and spiked ~a~ith either Llambda or T7 phage to a concentration of ~-108pfu/ml (plaque forming units/nll). A potential of 250V mas placed across a separating membrane ~~>ith a molecular w>eight cot off of 200kDa (3kDa restriction membranes). A membrane of this size restricts IgG migration . ' i ' «°hilst allots~ing smaller molecular weight contaminants to pass through the membrane. leaving IgG and other large molecular weight proteins in the upstream. r1 second purification phase was carried out using a GABA/Acetic acid buffer. pH ~.6 with a 500kDa cut off separating membrane (3kDa restr1Ct10I1 nleII2bTaIles). A potential of 250V reversed polarity was placed across the system resulting in IgG migration through the membrane leaving other high molecular weight contaminants ups~treazn.
Examination of samples taken at 30 minutes intervals was made on reduced SDS-PAGE 4-25% gels:
VIruS tE'StllI~.P' One hundred and fifty ul taken at each time point sample was mixed t~~ith 100u1 of appropriate Escherichia coh culture (Strain HB201 was used for T7 and strain jhI101 for Llambda). The mixtures were incubated for 15 minutes at 37"C, before each was added to 2.5rn1 of freshly prepared molten soft agar. and vortexed. The mixtures were poured over culture plates of Luria Agar. and incubated at 37°C overnight. T'he plates were inspected for the presence of virus colonies (plaques) in the lawn of E. coli. The number of plaques was recorded or if the virus had infected the entire E. coli population the result was recorded as confluent lysis.
HSA purification procedure Albumin is the most abundant protein component (50mg/m) in human plasma and functions to znaintaimblood volume; and oncotic pressure.
Albumin regulates the transport of protein. fatty acids. hormones and drugs in the bode. Clinical uses include blood.volume replacement during surgery, shock. serious bums and other medical emergencies. Albumin is 67kDa and has an isoelectric point of approximately ~.9. The protein consists of a single subunit and is globular in shape. About .t~0 metric tons of albumin is used annualln internationally with worldwide sales of US$1.5 billion. Albumin is currenti~- purified using Cohn fractionation and commercial product contains InaIly COIItaIIllIlaIltS lI1 addlt10I2 t0 I11u1t1IneTS Of albuIllln. The high COIlC8I2trat10I1. globular nature and solubility of .albumin make it an ideal candidate for purification froze plasma using Gradiflow technology.
alethod: Pooled normal plasma was diluted one in three with Tris-Borate (TB) running buffer. pH 9.0 and spiked vfith --~ 208pfu/ml of Llambda or T7 phage. The mixture was placed in the upstream of a Gradiflow apparatus.
:~lbuznin mas isolated from platelet free plasma i;n a one-phase process using n r WO 00/38743 PCTlAU99/01171 the charge of albumin at a pH above its isoele<aric point (pI) and its ' molecular weight. Thus a cartridge with a 751~a cutoff separation membrane was placed bet<veen 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. Virus was detected as so previously described above.
Fibrinogen purification procedure:
Commercially. 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 3:tOkDa that consists of three non-identical subunit ~5 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 2o 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 3aroduction of factor VIII and 25 involves the loss of IIlOSt 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 Gradiflou~.
Method: Cryo-precipitate 1, produced by thawing frozen plasma at 4°C
30 o~~ernight ~~uas removed from plasma by centrifugation at 10000xg at 4°C far 5 minutes. The precipitate «~as re-dissolved in Tris-Borate buffer (pH 9.0) and placed in the upstreaim of a Gradiflow apparatus.. The upstream was spiked t~uith either Llambda or T7 phage to a concentration of ---108pfuJml. A
potential of 250V .vas applied across a 300kDa cutoff cartridge and run for 2 35 hours. The donvnstream tras replaced Gvith fresh buffer at 30 minute inten~als. A second phase teas used to concentrate the fibrinogen through a ° , "
500kDa cutoff separation membrane at pH 9Ø 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 lOmIvI and 10NIH unit/n~l respectively).
The presence of purified fibrinogen wa:; confirmed by examination on reduced SDS PAGE ~-26% gels. The presence of either T7 or Llambda in the tlIlle point samples was tested using the previously described method.
Results of IgG, HSA arid fibrinogen purification.' The procedures described successfully 1?urified IgG, albumin and fibrinogen as judged by electrophoresis. Neither T7 nor Liambda phage were detected in the downstream products. but were present in the upstream samples.
Prion removal during plasma protein purific~~tion 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.
Currently, there is much concern regarding the transmission of these diseases via transfusion and plasma protein fractions administered ciinicallv.
slim: To isolate HSr'~ from human plasma using the Gradifiow, with simultaneous removal of prion.
Method: Pooled platelet rich plasma was diluted one in two with Tris-Borate (TB) running buffer. pH 9.0 and was placed in the upstream of a Gradiflow apparatus. Albumin was isolated from platelet free plasma using the charge of albumin at a pH above its pI and ias molecular weight. Thus a cartridge e~~ith a 75kDa cutoff separation membrane was placed between two 50kDa cutoff restriction membranes. The albmnin was removed from high ' molecular tweight COIItaIIllIlaIltS b~T its nugration through the separation membrane nvhilst small molecular weight contaminants dissipated through the 50kDa restriction membrane. Samples were taken at 20 minute intervals throughout a 2.~0 minute run. The buffer streai:o and cartridge were replaced after the initial tw°o hours, tvith identical solutions and cartridge.
r The presence of the 'purified HSA in th.e downstream was demonstrated by examination by SDS-PAGE, and was measured using a Bromocresol Green Assay (purchase from Trace Scientific. Prior was tested for in both up- and dovvn-stream samples using a sandwich ELISA comprised s of prior specific antibodies obtained from Pri:oriics Inc (Switzerland).
.-~Ibumin qucuttitation Fifty ul of each time point sample was diluted with 50.1 of PBS buffer.
A 20u1 aliquot of each diluted sample was placed in a microplate well. A
standard curve of the kit calibrator from a maximum concentration of 10 ~Omg/ml vvas prepared using PBS as the diluent. The standard curve dilutions were also placed iwthe microplate (.2~.1 plasmalwell). The bromocresol green reagent was added to alI th.e wells (200~llwell) and the absorbance at 630nm was read using a Versamax microplate reader. The standard curve was drawn on a liner scale and the concentration of albumin 25 in the up and downstream samples were read from the curve: The volume in the appropriate stream at the time of sampling; was multiplied by the concentration of each sample. Thus providinf; a value for the total HSA
present in each stream.
Prior detection 2o A solution of 5~g/ml monoclonal antibody denoted 6H4 (Priories Inc.
Switzerland) in a lOIIlIVI carbonate buffer was added to the wells of a microplate (IOO~I/vvell). and incubated overnight at :I"C. The antibody was later decanted and the wells washed three times with 250~1/well of a PBS
solution containing 0.1%(v/v) Tween 20.~ The plate wells were blocked by 25 lllCUbatIIlg at room temperature for 30 minutes with 200~1/well of PBS/T20 containing 1% albumin. The plate was again washed three times with 250ulhvell of PBSlT20 before the up- and dowr.E-stream time point samples tvere added (100~1/~-yell). The samples were incubated for 1-2 hours at room temperature before being dispensed, and the plate a=ashed three times as 3o previousl~~ described. A solution of prior-specific polyclonal antibody, denoted 8029 (Priories Inc. Stvitzerland) ~-vas cliluted at 2:1000(v:v) izz PBS/T20. and added to the wells of the plate (100~1/well). The mixture was incubated for 1-2 hours at room temperature. before being decanted. The plate was tvashed three tlIIleS aIld 100iz1/well of a horseradish peroxidase 3s conjugated polyclonal anti-rabbit IgG antiserum (purchased from Dakopatts) was added. The conjugate ivas incubated for 3t7-fi0 minutes at room WO 00/38743 PC'T/AU99/01171 .
The presence of purified fibrinogen wa:; confirmed by examination on reduced SDS PAGE ~-26% gels. The presence of either T7 or Llambda in the tlIlle point samples was tested using the previously described method.
Results of IgG, HSA arid fibrinogen purification.' The procedures described successfully 1?urified IgG, albumin and fibrinogen as judged by electrophoresis. Neither T7 nor Liambda phage were detected in the downstream products. but were present in the upstream samples.
Prion removal during plasma protein purific~~tion 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.
Currently, there is much concern regarding the transmission of these diseases via transfusion and plasma protein fractions administered ciinicallv.
slim: To isolate HSr'~ from human plasma using the Gradifiow, with simultaneous removal of prion.
Method: Pooled platelet rich plasma was diluted one in two with Tris-Borate (TB) running buffer. pH 9.0 and was placed in the upstream of a Gradiflow apparatus. Albumin was isolated from platelet free plasma using the charge of albumin at a pH above its pI and ias molecular weight. Thus a cartridge e~~ith a 75kDa cutoff separation membrane was placed between two 50kDa cutoff restriction membranes. The albmnin was removed from high ' molecular tweight COIItaIIllIlaIltS b~T its nugration through the separation membrane nvhilst small molecular weight contaminants dissipated through the 50kDa restriction membrane. Samples were taken at 20 minute intervals throughout a 2.~0 minute run. The buffer streai:o and cartridge were replaced after the initial tw°o hours, tvith identical solutions and cartridge.
r The presence of the 'purified HSA in th.e downstream was demonstrated by examination by SDS-PAGE, and was measured using a Bromocresol Green Assay (purchase from Trace Scientific. Prior was tested for in both up- and dovvn-stream samples using a sandwich ELISA comprised s of prior specific antibodies obtained from Pri:oriics Inc (Switzerland).
.-~Ibumin qucuttitation Fifty ul of each time point sample was diluted with 50.1 of PBS buffer.
A 20u1 aliquot of each diluted sample was placed in a microplate well. A
standard curve of the kit calibrator from a maximum concentration of 10 ~Omg/ml vvas prepared using PBS as the diluent. The standard curve dilutions were also placed iwthe microplate (.2~.1 plasmalwell). The bromocresol green reagent was added to alI th.e wells (200~llwell) and the absorbance at 630nm was read using a Versamax microplate reader. The standard curve was drawn on a liner scale and the concentration of albumin 25 in the up and downstream samples were read from the curve: The volume in the appropriate stream at the time of sampling; was multiplied by the concentration of each sample. Thus providinf; a value for the total HSA
present in each stream.
Prior detection 2o A solution of 5~g/ml monoclonal antibody denoted 6H4 (Priories Inc.
Switzerland) in a lOIIlIVI carbonate buffer was added to the wells of a microplate (IOO~I/vvell). and incubated overnight at :I"C. The antibody was later decanted and the wells washed three times with 250~1/well of a PBS
solution containing 0.1%(v/v) Tween 20.~ The plate wells were blocked by 25 lllCUbatIIlg at room temperature for 30 minutes with 200~1/well of PBS/T20 containing 1% albumin. The plate was again washed three times with 250ulhvell of PBSlT20 before the up- and dowr.E-stream time point samples tvere added (100~1/~-yell). The samples were incubated for 1-2 hours at room temperature before being dispensed, and the plate a=ashed three times as 3o previousl~~ described. A solution of prior-specific polyclonal antibody, denoted 8029 (Priories Inc. Stvitzerland) ~-vas cliluted at 2:1000(v:v) izz PBS/T20. and added to the wells of the plate (100~1/well). The mixture was incubated for 1-2 hours at room temperature. before being decanted. The plate was tvashed three tlIIleS aIld 100iz1/well of a horseradish peroxidase 3s conjugated polyclonal anti-rabbit IgG antiserum (purchased from Dakopatts) was added. The conjugate ivas incubated for 3t7-fi0 minutes at room WO 00/38743 PC'T/AU99/01171 .
temperature and then removed. Any bound HRP conjugate was detected using o-tolidine substrate solution (100ullweal). and the reaction stopped by addition of 3lVi HCl (50~Iiwell). The developed colour was measured at -i50nm in a Versamax plate reader.
Results -Albumin vvas transferred to the downstream and was detected in the BCG assat> (Figure 2). and visualized on a nai:ive &-I6% electrophoresis gel.
Decreasing quantities of Priors wtere detected in the upstream during the time-course. and no Priori was detected in the downstream samples.
Endotoxin removal during ~piasma protein purification using Gradiflow technolog'f Contamination with bacterial endotoxi:n is a major concern when purifying plasma proteins, such as IgG and HSA. EIIdOtOxlIls 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.
r'lim: To isolate IgG and HSA from human plasma spiked with endotoxin, using the Gradiflow, with simultaneous removal of endotoxin.
fgG purification procedure ~Ylethod: 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 55I1g/illl. A potential of 250V was placed across a separating membrane with a molecular weight cut off of 200kDa (3kDa restriction membranes). A
I22eII1bT3Ile Of this size restricts IgG migration 'whilst allowing smaller molecular «>eight contaminants to pass throue;h the membrane, leaving IgG
and other large molecular weight proteins in t:he upstream. A second purification phase was carried out using a GA:BA/Acetic acid buffer, pH 4.6 with a 500kDa cut off separating membrane (3kDa restriction membranes). A
potential of 250V reversed polarity vvas placed across the system resulting in IgG migration through the membrane leaving other high molecular weight COIItaI12111aI1t5 upstreaIll.
Examination of salnpies taken at 30 minutes intervals was made.on reduced SDS-PAGE ~-25% gels. Endotoxin teas tested for using a LAI.
Pvrochrome Chromogenic assay purchased fra:m Cape Cod Associates. All ' b ' WO 00/38743 samples tvere diluted 1 in IO and the endoto:~in assay was performed according to the manufacturer instructions.
HSA puri~cqtion procedure :vletlzod: Pooled normal plasma ivas diluted one in three v~~ith Tris-Borate s (TB) running buffer pH 9.0 and spiked with :i5ng/mI of purified endotoxin.
The mixture was placed in the upstream of a Gradiflowapparatus. Albumin was isolated from platelet free plasma in a one-phase process using the charge of albunun at a pH above its pI and its molecular weight. Thus a cartridge with a 75kDa cutoff separation membrane was placed between two s0 50kDa cutoff restriction membranes. The albumin was removed from high molecular weight contaminants by its migration through the separation meiizbrane tvhilst small molecular weight coni:aminants dissipated through the 50kDa restriction membrane. Samples were taken at regular intervals -- throughout a 90 minutes run.
1s The presence of the purified HSA in the downstream was demonstrated by examination by SDS-PAGE. hndotoxin 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 teas performed according to the manufacturer instructions.
20 Results of IgG and HSA purificafion Up and downstream samples taken at 30 minute intervals during the second phase of an IgG purification from endotoxin spiked plasma were tested for endotoxin using a LAL Chromogenic assay. The results showed that the endotoxin was almost entirely found ir,~ the upstream at all time 25 points (Figure 2). The downstream contained only 0.7% of the initial endotoxin. Reduced SDS-PAGE examination showed that IgG had been successfully isolated in the downstream Analysis of samples taken at 30 minute izatervals during the purification of HSA from plasma spiked with en~dotoxin found the majority of 30 eildOt0x1I1 reIIlaIIled in the upstream. Only 4~,0 of the total endotoxin was found in the downstream at the end of the run (Figure 3). Native PAGE
examination confirmed the presence of purified HSA in the downstream samples (Figure ~).
WO 00138743 PCTlAU99/O1I7I
Results -Albumin vvas transferred to the downstream and was detected in the BCG assat> (Figure 2). and visualized on a nai:ive &-I6% electrophoresis gel.
Decreasing quantities of Priors wtere detected in the upstream during the time-course. and no Priori was detected in the downstream samples.
Endotoxin removal during ~piasma protein purification using Gradiflow technolog'f Contamination with bacterial endotoxi:n is a major concern when purifying plasma proteins, such as IgG and HSA. EIIdOtOxlIls 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.
r'lim: To isolate IgG and HSA from human plasma spiked with endotoxin, using the Gradiflow, with simultaneous removal of endotoxin.
fgG purification procedure ~Ylethod: 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 55I1g/illl. A potential of 250V was placed across a separating membrane with a molecular weight cut off of 200kDa (3kDa restriction membranes). A
I22eII1bT3Ile Of this size restricts IgG migration 'whilst allowing smaller molecular «>eight contaminants to pass throue;h the membrane, leaving IgG
and other large molecular weight proteins in t:he upstream. A second purification phase was carried out using a GA:BA/Acetic acid buffer, pH 4.6 with a 500kDa cut off separating membrane (3kDa restriction membranes). A
potential of 250V reversed polarity vvas placed across the system resulting in IgG migration through the membrane leaving other high molecular weight COIItaI12111aI1t5 upstreaIll.
Examination of salnpies taken at 30 minutes intervals was made.on reduced SDS-PAGE ~-25% gels. Endotoxin teas tested for using a LAI.
Pvrochrome Chromogenic assay purchased fra:m Cape Cod Associates. All ' b ' WO 00/38743 samples tvere diluted 1 in IO and the endoto:~in assay was performed according to the manufacturer instructions.
HSA puri~cqtion procedure :vletlzod: Pooled normal plasma ivas diluted one in three v~~ith Tris-Borate s (TB) running buffer pH 9.0 and spiked with :i5ng/mI of purified endotoxin.
The mixture was placed in the upstream of a Gradiflowapparatus. Albumin was isolated from platelet free plasma in a one-phase process using the charge of albunun at a pH above its pI and its molecular weight. Thus a cartridge with a 75kDa cutoff separation membrane was placed between two s0 50kDa cutoff restriction membranes. The albumin was removed from high molecular weight contaminants by its migration through the separation meiizbrane tvhilst small molecular weight coni:aminants dissipated through the 50kDa restriction membrane. Samples were taken at regular intervals -- throughout a 90 minutes run.
1s The presence of the purified HSA in the downstream was demonstrated by examination by SDS-PAGE. hndotoxin 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 teas performed according to the manufacturer instructions.
20 Results of IgG and HSA purificafion Up and downstream samples taken at 30 minute intervals during the second phase of an IgG purification from endotoxin spiked plasma were tested for endotoxin using a LAL Chromogenic assay. The results showed that the endotoxin was almost entirely found ir,~ the upstream at all time 25 points (Figure 2). The downstream contained only 0.7% of the initial endotoxin. Reduced SDS-PAGE examination showed that IgG had been successfully isolated in the downstream Analysis of samples taken at 30 minute izatervals during the purification of HSA from plasma spiked with en~dotoxin found the majority of 30 eildOt0x1I1 reIIlaIIled in the upstream. Only 4~,0 of the total endotoxin was found in the downstream at the end of the run (Figure 3). Native PAGE
examination confirmed the presence of purified HSA in the downstream samples (Figure ~).
WO 00138743 PCTlAU99/O1I7I
Bacteria removal during plasma protein purification using Gradiflow technology 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, zo using the Gradiflow.
zs IgG purification procedure wlethod: Platelet free plasma was diluted o:ne part ill three with Tris-borate, pH 9.0 l~znning buffer and placed in the upstream of Gradiflow and spiked with E. coli to a concentration of 4x10E' cells/ml. A potential of 250V
was placed across a separating membrane with a molecular weight cut off of 200kDa (100kDa 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 upstxeanl. A second purification phase was carried out using 2o a GABA/Acetic acid buffer, pH 4.6 with a 5003sDa cut off separating membrane (3kDa restriction membranes). A potential of 250V reversed polaritv eras placed across the system resulting in IgG migration through the membrane leaving other high molecular weight contaminants upstream.
ExaIlI111at1011 Of samples taken at 30 IIllIauteS lIlterValS W3S made On 25 reduced SDS-PAGE ~-25% gels.
Bacteria testing Twenty ~l of upstream or 1001 of downstream samples mere spread plated onto Luria agar culture plates. The plate ~-vere incubated for 2:;;
hours at 37"C. and the number of colonies was couni:ed.
3o HSA purification procedure al.lethod: Pooled normal plasma was diluted OI12 111 three with Tris-Borate (TB) running buffer. pH 9.0 and spiked mith -.zxlOa cellShlll of E. coli. The mixture Haas placed in the upstream of a Gradiflow apparatus. Albumin was isolated from platelet free plasma in a one-phase process using the charge of 35 albumin at a pH above its pI and its molecular weight. Thus a cartridge with a iSkDa cutoff separation membrane cv-as placE:d between two 50kDa cutoff ~WO 00/38743 PCT/AU99/01171 restriction membranes. The albumin was removed from high molecular weight contaminants by its migration through the separation nlembrane whilst small molecular weight contaminants dissipated through the 50kDa restriction membrane. Samples v~~ere 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.. Bacteria were detected as previously described above.
l3esults of IgG, arid HSA purification The procedures described successfull~~ purified IgG, and albumin as judged by electrophoretic examination. The downstream samples containing the purified protein products did not contain detectableE. coli colonies, while the upstream samples produced greatly in excess of 500 colonies/plate.
w CONCLUSION
It is possible to purify proteins such as. IgG, albumin and fibrinogen from plasma, while simultaneously removin~; contaminating virus by the methods according to the present invention.
Prior present in plasma can be moved across a 75kDa separation membrane with albumin, however. unlike albumin. the prior is not retained_ by the 50kDa restriction membrane. Thus, albumin can be purified from plasma with simultaneous removal of Prior protein.
Evidence has been provided by the present inventors that it is possible to purify proteins such as IgG and albumin from plasma. while simultaneously removing endotoxin contamination in the starting plasma 'S using the Gradiflow technology.
Furthermore, it has been found that it is also possible to purify proieins such as IgG. and albumin'froin plasma. while simultaneously removing contaminating bacteria.
It will be appreciated by persons skilled in the art that numerous 3o vTariations and/or modifications may be .made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are. therefore. to be considered in all respects as illustrative anii not restrictive.
Aim: To isolate IgG. and HSA. from human plasma spiked with bacteria, zo using the Gradiflow.
zs IgG purification procedure wlethod: Platelet free plasma was diluted o:ne part ill three with Tris-borate, pH 9.0 l~znning buffer and placed in the upstream of Gradiflow and spiked with E. coli to a concentration of 4x10E' cells/ml. A potential of 250V
was placed across a separating membrane with a molecular weight cut off of 200kDa (100kDa 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 upstxeanl. A second purification phase was carried out using 2o a GABA/Acetic acid buffer, pH 4.6 with a 5003sDa cut off separating membrane (3kDa restriction membranes). A potential of 250V reversed polaritv eras placed across the system resulting in IgG migration through the membrane leaving other high molecular weight contaminants upstream.
ExaIlI111at1011 Of samples taken at 30 IIllIauteS lIlterValS W3S made On 25 reduced SDS-PAGE ~-25% gels.
Bacteria testing Twenty ~l of upstream or 1001 of downstream samples mere spread plated onto Luria agar culture plates. The plate ~-vere incubated for 2:;;
hours at 37"C. and the number of colonies was couni:ed.
3o HSA purification procedure al.lethod: Pooled normal plasma was diluted OI12 111 three with Tris-Borate (TB) running buffer. pH 9.0 and spiked mith -.zxlOa cellShlll of E. coli. The mixture Haas placed in the upstream of a Gradiflow apparatus. Albumin was isolated from platelet free plasma in a one-phase process using the charge of 35 albumin at a pH above its pI and its molecular weight. Thus a cartridge with a iSkDa cutoff separation membrane cv-as placE:d between two 50kDa cutoff ~WO 00/38743 PCT/AU99/01171 restriction membranes. The albumin was removed from high molecular weight contaminants by its migration through the separation nlembrane whilst small molecular weight contaminants dissipated through the 50kDa restriction membrane. Samples v~~ere 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.. Bacteria were detected as previously described above.
l3esults of IgG, arid HSA purification The procedures described successfull~~ purified IgG, and albumin as judged by electrophoretic examination. The downstream samples containing the purified protein products did not contain detectableE. coli colonies, while the upstream samples produced greatly in excess of 500 colonies/plate.
w CONCLUSION
It is possible to purify proteins such as. IgG, albumin and fibrinogen from plasma, while simultaneously removin~; contaminating virus by the methods according to the present invention.
Prior present in plasma can be moved across a 75kDa separation membrane with albumin, however. unlike albumin. the prior is not retained_ by the 50kDa restriction membrane. Thus, albumin can be purified from plasma with simultaneous removal of Prior protein.
Evidence has been provided by the present inventors that it is possible to purify proteins such as IgG and albumin from plasma. while simultaneously removing endotoxin contamination in the starting plasma 'S using the Gradiflow technology.
Furthermore, it has been found that it is also possible to purify proieins such as IgG. and albumin'froin plasma. while simultaneously removing contaminating bacteria.
It will be appreciated by persons skilled in the art that numerous 3o vTariations and/or modifications may be .made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are. therefore. to be considered in all respects as illustrative anii not restrictive.
Claims (16)
1. A method of removing one or more infectious agents from a mixture containing a biomolecule and the agent, the method comprising:
(a) placing the biomolecule and agent 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;
(c) applying an electric potential between the two solvent streams causing movement of the biomolecule through the membrane into the second solvent stream while the agent is substantially retained in the first sample stream, or if entering the membrane, being substantially prevented from entering the second solvent stream;
(d) optionally, periodically stopping and reversing the electric potential to cause movement of any agents having entered the membrane to move back into the first solvent stream, wherein substantially not causing any biomolecules that have entered the second solvent stream to re-enter first solvent stream; and (e) maintaining step (c), and optional step (d) if used, until the second solvent stream contains the desired purity of biomolecule substantially free of at least one infectious agent.
(a) placing the biomolecule and agent 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;
(c) applying an electric potential between the two solvent streams causing movement of the biomolecule through the membrane into the second solvent stream while the agent is substantially retained in the first sample stream, or if entering the membrane, being substantially prevented from entering the second solvent stream;
(d) optionally, periodically stopping and reversing the electric potential to cause movement of any agents having entered the membrane to move back into the first solvent stream, wherein substantially not causing any biomolecules that have entered the second solvent stream to re-enter first solvent stream; and (e) maintaining step (c), and optional step (d) if used, until the second solvent stream contains the desired purity of biomolecule substantially free of at least one infectious agent.
2. A method of removing one or more infectious agents from a mixture containing a biomolecule and the agent, the method comprising:
(a) placing the biomolecule and agent 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;
(c) applying an electric potential between the two solvent streams causing movement of the agent through the membrane into the second solvent stream while the biomolecule is substantially retained in the first sample stream, or if entering the membrane, being substantially prevented from entering the second solvent stream;
(d) optionally, periodically stopping and reversing the electric potential to cause movement of any biomolecule having entered the membrane to move back into the first solvent stream, wherein substantially not causing any agents that have entered the second solvent stream to re-enter first solvent stream; and (e) maintaining step (c), and optional step (d) if used, until the first solvent stream contains the desired purity of biomolecule substantially free of at least one infectious agent.
(a) placing the biomolecule and agent 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;
(c) applying an electric potential between the two solvent streams causing movement of the agent through the membrane into the second solvent stream while the biomolecule is substantially retained in the first sample stream, or if entering the membrane, being substantially prevented from entering the second solvent stream;
(d) optionally, periodically stopping and reversing the electric potential to cause movement of any biomolecule having entered the membrane to move back into the first solvent stream, wherein substantially not causing any agents that have entered the second solvent stream to re-enter first solvent stream; and (e) maintaining step (c), and optional step (d) if used, until the first solvent stream contains the desired purity of biomolecule substantially free of at least one infectious agent.
3. The method according to claim 1 or 2 wherein the biomolecule is selected from the group consisting of virus, prion, blood protein, immunoglobulin, and recombinant protein.
4. The method according to any one of claims 1 to 3 wherein the infectious agent is selected from the group consisting of virus, bacterium, yeast, and prion.
5. The method according to claim 1 wherein the infectious agent is a virus.
6. The method according to claim 1 wherein the infectious agent is a bacterium.
7. The method according to claim 1 or 2 wherein the infectious agent is a prion.
8. The method according to any one of claims 1 to 7 further including separating a biological contaminant selected from the group consisting of lipopolysaccharide, toxin, and endotoxin from the biomolecule.
9. The method according to any one of claims 1 to 8 wherein the buffer for the first solvent stream has a pH lower than the isoelectric point of biomolecule to be separated.
10. The method according to any one of claims 1 to 9 wherein the electrophoretic membrane has a molecular mass cut-off of between 3 and 1000 kDa.
11. The method according to any one of claims 1 to 10 wherein the electric potential applied is up to 300 volts.
12. The method according to any one of claims 1 to 11 wherein the infectious agent is collected or removed from the first stream or second solvent stream.
13. The method according to any one of claims 1 to 12 wherein the electrophoretic membrane has a molecular mass cut-off close to the apparent molecular mass of biomolecule.
14. A biomolecule substantially free from infectious agents obtained by the method according to any one of claims 1 to 13.
15. Use of biomolecule according to claim 14 in medical and veterinary applications.
16. Use of biomolecule according to claim 25 in medical and veterinary applications.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPP7906 | 1998-12-23 | ||
AUPP7906A AUPP790698A0 (en) | 1998-12-23 | 1998-12-23 | Separation of microorganisms |
PCT/AU1999/001171 WO2000038743A1 (en) | 1998-12-23 | 1999-12-23 | Removal of biological contaminants |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2356894A1 true CA2356894A1 (en) | 2000-07-06 |
Family
ID=3812113
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002356894A Abandoned CA2356894A1 (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) |
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-
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-
1999
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- 1999-12-23 US US09/470,823 patent/US6464851B1/en not_active Expired - Fee Related
- 1999-12-23 CA CA002356894A patent/CA2356894A1/en not_active Abandoned
- 1999-12-23 JP JP2000590694A patent/JP2002540912A/en active Pending
- 1999-12-23 EP EP99968291A patent/EP1140214A4/en not_active Ceased
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2001
- 2001-06-22 US US09/887,371 patent/US7060173B2/en not_active Expired - Fee Related
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WO2000038743A1 (en) | 2000-07-06 |
US7060173B2 (en) | 2006-06-13 |
AUPP790698A0 (en) | 1999-01-28 |
EP1140214A1 (en) | 2001-10-10 |
JP2002540912A (en) | 2002-12-03 |
EP1140214A4 (en) | 2003-05-02 |
US6464851B1 (en) | 2002-10-15 |
US20020084187A1 (en) | 2002-07-04 |
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