U.S. patent application number 09/931342 was filed with the patent office on 2002-01-03 for removal of biological contaminants.
This patent application is currently assigned to Gradipore Ltd.. Invention is credited to Conlan, Brendon, Edgell, Tracey Ann, Lazar, May, Nair, Chenicheri Hariharan, Seabrook, Elizabeth Jean, Turton, Thomas Norman.
Application Number | 20020000386 09/931342 |
Document ID | / |
Family ID | 25645959 |
Filed Date | 2002-01-03 |
United States Patent
Application |
20020000386 |
Kind Code |
A1 |
Conlan, Brendon ; et
al. |
January 3, 2002 |
Removal of biological contaminants
Abstract
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; (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 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
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.
Inventors: |
Conlan, Brendon; (North
Ryde, AU) ; Edgell, Tracey Ann; (Dee Why, AU)
; Lazar, May; (Fairfield West, AU) ; Nair,
Chenicheri Hariharan; (Castle Hill, AU) ; Seabrook,
Elizabeth Jean; (Lane Cove, AU) ; Turton, Thomas
Norman; (Neutral Bay, AU) |
Correspondence
Address: |
ARTER & HADDEN, LLP
1100 HUNTINGTON BUILDING
925 EUCLID AVENUE
CLEVELAND
OH
44115-1475
US
|
Assignee: |
Gradipore Ltd.
|
Family ID: |
25645959 |
Appl. No.: |
09/931342 |
Filed: |
August 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09931342 |
Aug 16, 2001 |
|
|
|
09470823 |
Dec 23, 1999 |
|
|
|
Current U.S.
Class: |
205/701 ;
204/252 |
Current CPC
Class: |
A61L 2/0017 20130101;
B01D 61/425 20130101; B01D 57/02 20130101; B82Y 10/00 20130101 |
Class at
Publication: |
205/701 ;
204/252 |
International
Class: |
A61L 002/03 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 1999 |
AU |
PP7906 |
Claims
What we claim is:
1. A method for removing pathogens from biological liquids, said
biological liquids containing at least one pharmaceutically active
molecule, said method comprising the steps of: providing a
biological liquid, wherein pathogens are potentially present, in an
apparatus comprising an anode and a cathode and a separation means
suitable for separating said pathogens from said pharmaceutically
active molecule, said separation means being positioned between
said anode and said cathode; applying current between said anode
and said cathode, thereby causing one of said pathogens or said
pharmaceutically active molecules to pass said separation means,
and recovering said pharmaceutically active molecule in a form
being essentially free of pathogens.
2. The method according to claim 1 wherein said separation means is
a filtration means.
3. The method according to claim 2 wherein said filtration means is
an ultrafiltration membrane.
4. The method according to claim 2 wherein said filtration means is
a nanofiltration membrane.
5. The method according to claim 1 wherein said pharmaceutically
active molecule is a protein.
6. The method according to claim 5 wherein said protein is a blood
protein.
7. The method according to claim 5 wherein said protein is smaller
than said pathogen and said separation means allows passing of said
protein but prevents passing of said pathogen.
8. The method according to claim 1 wherein said separation means is
a series of filters with different separation characteristics.
9. The method according to claim 8 wherein said different
filtration characteristics are caused by different cut-off values
of the filters in said series of filters.
10. The method according to claim 1 wherein said pathogens are
selected from the group consisting of viruses, bacteria, prions,
and combinations thereof.
11. The method according to claim 9 wherein said cut-off values are
selected to allow a separation between said pharmaceutically active
molecule and aggregate of said molecule.
12. An apparatus for removing pathogens from biological fluids,
said biological fluids containing at least one pharmaceutically
active molecule, said apparatus comprising: a container for uptake
of said biological liquid, an anode, a cathode, and a separation
means suitable for separating said pathogens from said
pharmaceutically active molecule, said separation means being
positioned between said anode and said cathode, and a current
supply and means for applying said current between said anode and
said cathode.
Description
TECHNICAL FIELD
[0001] The present invention relates to methods for the removal of
biological contaminants, particularly removal of biological
contaminants from biological preparations.
BACKGROUND ART
[0002] The modern biotechnology industry is faced with a number of
problems especially concerning the processing of complex biological
solutions which ordinarily include proteins, nucleic acid molecules
and 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, which in one step will both
purify macromolecules and separate these biological
contaminants.
[0003] 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 1.5.times.10.sup.-8 m to 5.0.times.10.sup.-5 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.
[0004] 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 0.5 ng endotoxin per
kilogram body weight or 25 ng endotoxin/dose for a 50 kg 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 amphipathic 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
gel filtration are employed.
[0005] 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
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.
[0006] 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.
[0007] The Gradiflow Technology
[0008] 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.
[0009] In essence 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
[0010] In a first aspect, 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:
[0011] (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;
[0012] (b) selecting a buffer for the first solvent stream having a
required pH;
[0013] (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 biological contaminant is
substantially retained in the first sample stream, or if entering
the membrane, being substantially prevented from entering the
second solvent stream;
[0014] (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 any biomolecules that
have entered the second solvent stream to re-enter first solvent
stream: and
[0015] (e) maintaining step (c), and optional step (d) if used,
until the second solvent stream contains the desired purity of
biomolecule.
[0016] In a second aspect. 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:
[0017] (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;
[0018] (b) selecting a buffer for the first solvent stream having a
required pH;
[0019] (c) applying an electric potential between the two solvent
streams causing movement of the biological contaminant 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;
[0020] (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 biological contaminants that
have entered the second solvent stream to reenter first solvent
stream; and
[0021] (e) maintaining step (c), and optional step (d) if used,
until the first solvent stream contains the desired purity of
biomolecule.
[0022] In the first and second aspects of the present invention,
preferably the biomolecule is selected from the group consisting of
blood protein, immunoglobulin, and recombinant protein.
[0023] The biological contaminant can be a virus, bacterium, prion
or an unwanted biomolecule such as lipopolysaccharide, toxin or
endotoxin.
[0024] Preferably, the biological contaminant is collected or
removed from the first stream.
[0025] Preferably, the buffer for the first solvent stream has a pH
lower than the isoelectric point of biomolecule to be
separated.
[0026] 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. 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 1000 kDa. A number of different membranes
may also be used in a desired or useful configuration.
[0027] 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.
[0028] 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.
[0029] In a third aspect, the present invention consists in use of
Gradiflow in the purification or separation of biomolecule from a
biological contaminant.
[0030] In a fourth aspect, the present invention consists in
biomolecule substantially free from biological contaminants
purified by the method according to the first aspect of the present
invention.
[0031] 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.
[0032] In a sixth aspect, the present invention consists in a
substantially isolated biomolecule substantially free from
biological contaminants.
[0033] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or
steps, but not the exclusion of any other element, integer or step,
or group of elements, integers or steps.
[0034] 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
[0035] FIG. 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 mixing with BCG reagent and
reading the absorbance of 630 nm. The concentration of albumin in
each sample was calculated from the standard curve, and multiplied
by the volume of the up-or downstream to obtain the Total HSA in
the up- and downstream (y-axis). All samples were assayed for prion
using a sandwich ELISA, and recording the absorbance values at 450
nm (second y-axis).
[0036] FIG. 2. Samples from the second phase of an IgG separation
were taken from both up- and downstreams (U/S and D/S respectively)
at 30 minute intervals. The samples were assayed for endotoxin
using a LAL Chromogenic assay (Cape Cod Assoc.)
[0037] FIG. 3. 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 (y-axis) in the samples.
[0038] FIG. 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.
MODES FOR CARRYING OUT THE INVENTION
[0039] Virus Removal During Plasma Protein Purification Using
Gradiflow Technology
[0040] 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.
[0041] Aim. To isolate IgG, HSA, and Fibrinogen from human plasma
spiked with virus, using the Gradiflow, with simultaneous removal
of the contaminating virus.
[0042] IgG Purification Procedure
[0043] 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 150 kDa 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 10 mg/ml.
[0044] 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
.beta.-lipoprotein and transferrin. The product of conventional
protein purification protocols is concentrated using
ultrafiltration. Immunoaffinity can also be used to isolate
specific IgGs.
[0045] Method: 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 .about.10.sup.8 pfu/ml (plaque forming units/ml).
A potential of 250V was placed across a separating membrane with a
molecular weight cut off of 200 kDa (3 kDa 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 500 kDa cut off separating
membrane (3 kDa 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.
[0046] Examination of samples taken at 30 minutes intervals was
made on reduced SDS-PAGE 4-25% gels.
[0047] Virus Testing
[0048] One hundred and fifty .mu.l taken at each time point sample
was mixed with 100 .mu.l of appropriate Escherichia coli culture
(Strain HB101 was used for T7 and strain JM101 for Llambda). The
mixtures were incubated for 15 minutes at 37.degree. C. before each
was added to 2.5 ml of freshly prepared molten soft agar, and
vortexed. The mixtures were poured over culture plates of Luria
Agar and incubated at 37.degree. C. overnight. The 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.
[0049] HSA Purification Procedure
[0050] Albumin is the most abundant protein component (50 mg/m.) 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 67 kDa 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.
[0051] Method: Pooled normal plasma was diluted one in three with
Tris-Borate (TB) running buffer, pH 9.0 and spiked with
.about.10.sup.8 pfu/ml of Llambda or T7 phage. 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 isoelectric point (pI) and its
molecular weight. Thus, a cartridge with a 75 kDa cutoff separation
membrane was placed between two 50 kDa 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
50 kDa restriction membrane. Samples were taken at regular
intervals throughout a 90 minutes run.
[0052] The presence of the purified HSA in the downstream was
demonstrated by examination by SDS-PAGE. Virus was detected as
previously described above.
[0053] Fibrinogen Purification Procedure:
[0054] 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 340 kDa 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.
[0055] 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.
[0056] 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.
[0057] Method: Cryo-precipitate 1, produced by thawing frozen
plasma at 4.degree. C. overnight was removed from plasma by
centrifugation at 10000.times. g at 4.degree. 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
.about.10.sup.8 pfu/ml. A potential of 250V was applied across a
300 kDa 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 500 kDa 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 10 mM and 10NIH unit/ml respectively).
[0058] 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.
[0059] Results of IgG. HSA and Fibrinogen Purification:
[0060] The procedures described successfully purified IgG, albumin
and fibrinogen as judged by electrophoresis. Neither T7 nor Llambda
phage were detected in the downstream products, but were present in
the upstream samples.
[0061] Prion Removal During Plasma Protein Purification Using
Gradiflow Technology
[0062] There is an international concern regarding the
contamination of plasma proteins by prion protein. Prion is a
glycoprotein of 27-33 kDa 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 clinically.
[0063] Aim: To isolate NSA from human plasma using the Gradiflow,
with simultaneous removal of prion.
[0064] 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 its molecular weight. Thus, a cartridge with a 75 kDa cutoff
separation membrane was placed between two 50 kDa 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 50 kDa restriction membrane. Samples were taken at 20
minute intervals throughout a 240 minute run. The buffer stream and
cartridge were replaced after the initial two hours, with identical
solutions and cartridge.
[0065] The presence of the purified HSA in the downstream was
demonstrated by examination by SDS-PAGE, and was measured using a
Bromocresol Green Assay (purchase from Trace Scientific. Prion was
tested for in both up- and down-stream samples using a sandwich
ELISA comprised of prion specific antibodies obtained from Prionics
Inc (Switzerland).
[0066] Albumin Quantitation
[0067] Fifty .mu.l of each time point sample was diluted with 50
.mu.l of PBS buffer. A 20 .mu.l aliquot of each diluted sample was
placed in a microplate well. A standard curve of the kit calibrator
from a maximum concentration of 40 mg/ml was prepared using PBS as
the diluent. The standard curve dilutions were also placed in the
microplate (2 .mu.l plasma/well). The bromocresol green reagent was
added to all the wells (200 .mu.l/well) and the absorbance at 630
nm was read using a Versamax microplate reader. The standard curve
was drawn on a linear scale and the concentration of albumin 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 providing a value for the
total HSA present in each stream.
[0068] Prion Detection
[0069] A solution of 5 .mu.g/ml monoclonal antibody denoted 6H4
(Prionics, Inc. Switzerland) in a 10 mM carbonate buffer was added
to the wells of a microplate (100 .mu.l/well), and incubated
overnight at 4.degree. C. The antibody was later decanted and the
wells washed three times with 25 .mu.l/well of a PBS solution
containing 0.1%(v/v) Tween 20. The plate wells were blocked by
incubating at room temperature for 30 minutes with 200 .mu.l/well
of PBS/T20 containing 1% albumin. The plate was again washed three
times with 250 .mu.l/well of PBS/T20 before the up- and down-stream
time point samples were added (100 .mu.l/well). 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 1:1000(v:v) in PBS/T20,
and added to the wells of the plate (100 .mu.l/well). The mixture
was incubated for 1-2 hours at room temperature, before being
decanted. The plate was washed three times and 100 .mu.l/well of
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.
Any bound HRP conjugate was detected using o-tolidine substrate
solution (100 .mu.l/well) and the reaction stopped by addition of
3M HCl (50 .mu.l/well). The developed colour was measured at 450 nm
in a Versamax plate reader.
[0070] Results
[0071] Albumin was transferred to the downstream and was detected
in the BCG assay (FIG. 1), and visualized on a native 8-16%
electrophoresis gel. Decreasing quantities of Prion were detected
in the upstream during the time-course, and no Prion was detected
in the downstream samples.
[0072] Endotoxin Removal During Plasma Protein Purification Using
Gradiflow Technology
[0073] Contamination with bacterial endotoxin is a major concern
when purifying plasma proteins, such as IgG and HSA. 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.
[0074] Aim: To isolate IgG and HSA from human plasma spiked with
endotoxin, using the Gradiflow, with simultaneous removal of
endotoxin.
[0075] IgG Purification Procedure
[0076] 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 55 ng/ml. A potential of 250V was placed
across a separating membrane with a molecular weight cut off of 200
kDa (3 kDa 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 500 kDa cut off separating membrane (3 kDa
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.
[0077] Examination of samples taken at 30 minutes intervals was
made on reduced SDS-PAGE 4-25% gels. Endotoxin was tested for using
a LAL Pyrochrome Chromogenic assay purchased from Cape Cod
Associates. All samples were diluted 1 in 10 and the endotoxin
assay was performed according to the manufacturer instructions.
[0078] HSA Purification Procedure
[0079] Method: Pooled normal plasma was diluted one in three with
Tris-Borate (TB) running buffer, pH 9.0 and spiked with 55 ng/ml of
purified endotoxin. 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. Thus, a cartridge with a 75 kDa
cutoff separation membrane was placed between two 50 kDa 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 50 kDa restriction membrane. Samples were taken at
regular intervals throughout a 90 minute run.
[0080] 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.
[0081] Results of IgG and HSA Purification
[0082] 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 in the upstream at all time points (FIG. 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.
[0083] Analysis of samples taken at 30 minute intervals during the
purification of HSA from plasma spiked with endotoxin found the
majority of endotoxin remained in the upstream. Only 4% of the
total endotoxin was found in the downstream at the end of the run
(FIG. 3). Native PAGE examination confirmed the presence of
purified HSA in the downstream samples (FIG. 4).
[0084] Bacteria removal during plasma protein purification using
gradiflow Technology
[0085] 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.
[0086] Aim: To isolate IgG. and HSA, from human plasma spiked with
bacteria, using the Gradiflow.
[0087] IgG Purification Procedure
[0088] Method: 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. coil to a concentration of
4.times.10.sup.8 cells/ml. A potential of 250V was placed across a
separating membrane with a molecular weight cut off of 200 kDa (100
kDa 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 500 kDa
cut off separating membrane (3 kDa 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.
[0089] Examination of samples taken at 30 minutes intervals was
made on reduced SDS-PAGE 4-25% gels.
[0090] Bacteria Testing
[0091] Twenty .mu.l of upstream or 100 .mu.l of downstream samples
were spread plated onto Luria agar culture plates. The plates were
incubated for 24 hours at 37.degree. C., and the number of colonies
was counted.
[0092] HSA Purification Procedure
[0093] Method: Pooled normal plasma was diluted one in three with
Tris-Borate (TB) running buffer, pH 9.0 and spiked with
.about.4.times.10.sup.8 cells/ml of E. coil. 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. Thus a
cartridge with a 75 kDa cutoff separation membrane was placed
between two 50 kDa 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 50 kDa restriction membrane.
Samples were taken at regular intervals throughout a 90 minutes
run.
[0094] The presence of the purified HSA in the downstream was
demonstrated by examination by SDS-PAGE. Bacteria were detected as
previously described above.
[0095] Results of IgG and HSA Purification
[0096] The procedures described successfully purified IgG, and
albumin as judged by electrophoretic examination. The downstream
samples containing the purified protein products did not contain
detectable E. coli colonies, while the upstream samples produced
greatly in excess of 500 colonies/plate.
CONCLUSION
[0097] It is possible to purify proteins such as IgG, albumin and
fibrinogen from plasma, while simultaneously removing contaminating
virus by the methods according to the present invention.
[0098] Prion present in plasma can be moved across a 75 kDa
separation membrane with albumin, however, unlike albumin, the
prion is not retained by the 50 kDa restriction membrane. Thus,
albumin can be purified from plasma with simultaneous removal of
Prion protein.
[0099] 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 using the Gradiflow technology.
[0100] Furthermore, it has been found that it is also possible to
purify proteins such as IgG, and albumin from plasma, while
simultaneously removing contaminating bacteria.
[0101] It will be appreciated by persons skilled in the art that
numerous variations 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 and not restrictive.
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