Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR PROTEIN ISOLATION
Field of the Invention
The present invention relates to processes for isolating proteins from
biological
sources. More particularly, the invention relates to processes for isolating
proteins from
blood.
Background of the Invention
Plasma is one of nature's most valuable raw materials and proteins purified
from
plasma are essential to.the life and well-being of millions of individuals
around the world.
io The majority of these proteins are produced by Cohn cold-ethanol
fractionation. This
process was developed by Dr Edwin Cohn at Harvard University in the early
1940s.
Cohn found that it was possible to separate proteins in plasma based on their
precipitation
characteristics under different conditions (pH, ionic strength, protein
concentration,
temperature and ethanol. concentration). By varying these parameters,
different proteins
precipitate out in a step by step fashion. Cohn technology has been used in
the plasma
industry for decades but the process presents some limitations in purity and
efficiency.
While this process can produce a quality product, there are limitations to the
flexibility of
the procedure and purity of the products produced. The most common
commercially
produced proteins still using this methodology include albumin,
immunoglobulin, anti-
thrombin III, thrombin and fibrinogen.
One type of technical advance in plasma product manufacture incorporates the
use
of chromatography, which separates the proteins in plasma on the basis of
selective
adsorption of the protein molecules onto a stationary solid surface (solid
phase) as the
liquid (mobile phase) percolates down an enclosed column containing the
stationary
phase. Depending on the efficiency of adsorption or interaction, the movement
of various
proteins is retarded to different extents, enabling their separation and
collection from the
bottom of the column.
Chromatography is essentially a more gentle means of separating proteins than
the
traditional Cohn cold-ethanol process, which involves prolonged exposure of
the proteins
to high concentrations of ethanol. This can denature the proteins and produce
unwanted
aggregates, which in turn have adverse therapeutic consequences to patients
receiving
these products. In contrast, chromatographic fractionation efficiently removes
impurities
. without affecting the native structure of the proteins. Compared with the
repeated large7
scale precipitations involved in Cohn cold-ethanol fractionation,
chromatography is a
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more direct method of separation, enabling increased amounts of protein to be
produced
per litre of plasma.
The chromatographic methods which have been adopted to date still provide
challenges in terms of optimizing yields while retaining or enhancing the
necessary levels
of purity. There remains a need for an efficient and cost effective
chromatographic
technique that selectively elutes plasma components at a superior purity and
yield to
existing methods.
In the early 1960's, it was noticed that when plasma was incubated at low
temperatures, a precipitate was seen to form. This precipitate was established
to contain
Factor VIII, Von Willebrands Factor and a number of other plasma proteins.
Initially this
cryoprecipitate was used to treat Haemophilia A patients but with the. need to
improve
patient tolerance for the product, more purified forms of Factor VIII became
available.
The process of cryoprecipitation remains the first basis of Factor VIII
production used by
industry today. Unfortunately the process is not highly efficient, and while.
various
ts attempts have been made to improve recovery of Factor VIII by directly
using plasma
rather than cryoprecipitate, few of these attempts have been commercially
successful.
Factor IX is used for the treatment of Haemophilia B patients and is isolated
from
supematant I, a side fraction of the well established Cohn plasma
fractionation process.
The Factor IX in supematant I is usually further purified with an affinity
chromatography
step using Heparin Sepharose in essentially all current manufacturing
processes, however
since the overall process is based on- Cohn fractionation, significant
limitations in
manufacturing efficiency still remain.
In this case again, there is a need for more efficient processes for the
isolation of
Factor VIII and/or Factor IX from mixtures containing Factor VIII and/or
Factor IX.
Summary of the Invention
According to a first aspect, the present invention provides a process for
isolating proteins from a solution cornprising said proteins, said solution
being
selected from the group consisting of: crude blood plasma, blood serum,
cryosupernatant derived from plasma, fractionated human' plasma,
cryoprecipitate
derived from plasma and recombinant broths, said process comprising:
(i) providing a solid separation medium having the formula:
M-S-L
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3
wherein M is a matrix backbone, S is an optional spacer arm, and L is a ligand
which
is mercaptonicotinic acid;
(ii) contacting said'solid separation medium with said solution, such that at
least one of said proteins becomes reversibly bound to said solid separation
medium;
s (iii) performing at least one elution step to selectively elute from the
solid
separation medium, at least one protein fraction.
The following features relate to the first aspect of the invention.
In one embodiment the proteins are of mammalian or human origin.
The spacer arm S may be derived from a compound comprising an epoxy group
(e.g. butane dioldiglycidylether or epichlorohydrin) or other suitable
coupling reagent
known in the art for covalent attachment of ligands.
The matrix backbone M may be a resin such that the solid separation medium is
a
resin functionalised with the ligand. Further, the resin may be any resin to
which the
ligand may be attached. Further still, the resin may be a high density resin
suitable for
non-packed bed adsorption, such as for example fluidized bed adsorption and
expanded
bed adsorption, such as a highly cross-linked beaded agarose derivative based
on 6% or
4% agarose, agarose-tungsten carbide conglomerate, an agarose stainless steel
conglomerate, an agarose-quartz conglomerate, porous ceramic beads, porous
zirconia.
beads, beads made of.controlled pore glass and composite beads of poious
inorganic
materials comprising organic polymers within their pores.
In one embodiment the ligand may be 2-mercaptonicotinic acid.
As the binding of the protein(s) to the ligand is reversible, the protein may
be
isolated from the solid separation medium under the appropriate elution
conditions
described below.
The solid separation medium.may comprise a high density resin having a mean
particle size in the range of about 10 micron to about 150 micron and a bead
density in
the range from about 1.5 g/ml to 15 g/ml, or alternatively a mean particle
size in the range
of about 10 micron to 120 micron and a bead density from about 2.0 g/ml to
15g/ml, or
alternatively a mean particle size in the range from about 15 micron to 100
micron and a
bead density in the range from about 2.3 g/ml to 15 g/ml, or alternatively a
mean particle
size in the range from about 15 micron to 80 micron and a bead density in the
range from
about 3 g/ml to 15 g/ml.
Proteins isolated by the process of the first aspect may be selected from the
group
consisting of: immunoglobulin for example, IgG, IgA, IgM, IgD, or IgE,
transferrin (Tf),
fibrinogen or a derivative thereof, plasma protease inhibitor such as an
antithrombin, e.g.,
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antithrombin III, blood pro-coagulation protein, blood anti-coagulation
protein, cytokine,
growth factor, albumin or a derivative thereof, thrombolytic agent, anti-
angiogenic
protein, insulin.or a derivative thereof, a-l-proteinase inhibitor or a
derivative thereof,
such as a-l-antitrypsin, a-2-antiplasmin or a derivative thereof, C-1 esterase
inhibitor,
apolipoprotein, HDL, Fibronectin or a derivative thereof, beta-2-glycoprotein
I,
plasminogen, plasmin, plasminogen activator, plasminogen inhibitor, urokinase
or
derivative thereof, streptokinase or a derivative thereof, inter- a-trypsin
inhibitor, a-2-
macroglobulin, amyloid protein, orosomucoid, ferritin, pre-albumin, GC-
globulin,
haemopexin and C3-complement.
The protein fractions may contain a single protein. Alternatively, the protein
fractions may contain multiple proteins.
The process may comprise from 1 to 50, from 1 to 30, from I to 20, from 1 to
10,
from 1 to 5 elution steps, from I to 3, or a single elution step.
In one embodiment respective eluants may have a pH in the range from about 4.0
to
about 9Ø Alternatively, respective eluants may have a pH in the range from
about 4.0 to
8.5. Alternatively, respective eluants may have a pH in the range from about
4.0 to about
8Ø Further, respective eluants may have a pH in the range from about 5.0 to
about 8Ø
Further, respective eluants may have a pH in the range from about 4.5 to 8Ø
Further
still, respective eluants 'may have a pH in the range from about 5.5 to about
8Ø Yet
further still respective eluants may have a pH in the range from about 6.0 to
8Ø
Respective eluants may have an ionic strength in the range from about 0.00005
Siemens/centimetre (S/cm) to about 10.0 S/cm. Alternatively, respective
eluants may
have an ionic strength in the range from about 0.0005 S/cm to 10.0 S/cm.
Alternatively,
respective eluants may have an ionic strength in the range from about 0.0001
S/cm to
about 6.0 S/cm. -Further, respective eluants may have an ionic strength in the
range from
about 0.001 S/cm to about 5.5 S/cm. Further still, respective eluants may have
an ionic
strength in the range from about 0.001 S/cm to about 5.0 S/cm. Further still,
respective
eluants may have an ionic strerigth in the range from about 0.005 S/cm to
about 5.0 S/cm.
Yet further still the eluants may have an ionic strength in the range from
about 0.01 S/cm
to about 4.0 S/cm.
Respective eluants may have any combinations of pH and ionic strength values
within the above specified ranges.
The first, second or third eluants may have a pH of between about 4.0 and
about
9.0, and an ionic strength of between about 0.00005 S/cm and about 10.0 S/cm.
The first
eluant may have a pH of between about 4.0 and about 8.0 and an ionic strength
of
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between about 0.00005 S/cm and about 0.1 S/cm. Further, the first eluant may
have a pH
of between about 4.5 and about 6.5 and an ionic strength of between about
0.00005 S/cm
and about 0.075 S/cm. Further the first eluant may have a pH of between about
5.0 and
about 6.0 and an ionic strength of between about 0.001 S/cm and about 0.05
S/cm.
5 The second eluant may have a pH of between about 5.0 and about 7.0 and an
ionic
strength of between about 0.0001 S/cm and about 0.1 S/cm. Alternatively the
second
eluant may have a pH of between about 5.5 and about 6.5 and an ionic strength
of
between about 0.0001 S/cm and 'about 0.075 S/cm. Further the second eluant may
have a
pH of between about 5.5 and about 6.5 and an ionic strength of between about
0.001 S/cm
io and about 0.05 S/cm.
The third eluant may have a pH of between about 5.0 and about 9.0 and an ionic
strength of between about 0.0001 S/cm and about 4.0 S/cm. Alternatively the
third eluant
may have a pH of between about 6.0 and about 8.0 and an ionic strength of
between 0.01
S/cm and about 3.0 S/cm. Further the third eluant may have a pH of between
about 6.0
and about 8.0 and an ionic strength of between about 0.05 S/cm and about 2.0
S/cm.
The first eluant may be an eluant that results in no loss of the biological
function of
the proteins, such as demineralised water or an aqueous solution of one or
more inorganic
salts of strong mineral acids, for example salts of hydrochloric acid,
sulfuric acid and
nitric acid, such as sodium chloride, potassium chloride, ammonium chloride,
sodium
sulfate, potassium sulfate and ammonium sulfate. Alternatively, the first
eluant may be
an aqueous buffer solution comprising a salt of an inorganic and/or an organic
acid which
does not result in loss of the biological function of the proteins, for
example a buffer
comprising a citrate, an acetate, a succinate, a lactate, a tartrate, a
formate,, a propionate, a
phosphate or a borate.
The second eluant may be an aqueous buffer solution comprising a salt of an
inorganic and/or an organic acid that results in no loss of the biological
function of the
proteins, for example a buffer comprising a citrate, an acetate, a succinate,
a lactate, a
tartrate, a formate, a propionate, a phosphate or a borate. In one embodiment,
the second
eluant comprises a negatively charged molecule having a non-aromatic, an
aromatic or a
heteroaromatic hydrophobic moiety, such as salts of inedium, to long chain
alkyl-
carboxylic and alkyl-sulfonic acids, and negatively charged detergents, such
as sodium
dodecyl sulphate and sodium deoxycholate. For example, the second eluant may
comprise a salt of one or more acids selected from the group consisting of:
caproic acid,
heptanoic acid, caprylic acid, perlagonic acid and capric acid, undecanoic
acid, lauric
acid, tridecanoic acid, myristic acid and pentadecanoic acid, and also
unsaturated and
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alkyl substituted derivatives thereof. Alternatively, the second eluant may
comprise a salt-
of one or more acids selected from the group consisting of: hexane sulfonic
acid, octane
sulfonic acid, decane sulfonic acid, dodecane sulfonic acid, hexane sulfate,
octane sulfate,
decane sulfate and dodecane sulfate.
The third eluant may be an aqueous buffer solution comprising a salt of an
inorganic and/or an organic acid that results in no loss of the biological
function of the
proteins, for example a buffer comprising a citrate, an acetate, a succinate,
a lactate, a
tartrate, a formate, a propionate, a phosphate, or a borate. Alternatively the
third eluant
may comprise an inorganic or organic salt of a relatively high lyotrophobicity
such as
ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate,
sodium
phosphate, potassium phosphate, ammonium citrate, sodium citrate, potassium
citrate.
The process of'the first aspect may comprise eluting.the solid separation
medium
with a fourth eluant to selectively elute a fourth protein fraction. The
fourth eluant may
have a pH of between about 5.0 and about 9.0 and an ionic strength of between
about 0.01.
S/cm and about 2 S/cm. Alternatively the fourth eluant may have a pH between
about 6.0
and about 8.0 and an ionic strength between about 0.01 S/cm and about 1.0
S/cm.
Further, the fourth eluant may have a pH of between about 6.0 and about 8.0
and an ionic
strength between about 0.05 S/cm and about 1.0 S/cm. The fourth eluant may be
an
aqueous buffer solution comprising a salt of an inorganic and/or an organic
acid
compatible with the proteins to be isolated, for example a buffer comprising a
citrate, an
acetate, a succinate, a lactate, a tartrate, a formate, a propionate, a
phosphate, or a borate.
The fourth eluant may also comprise an aqueous solution of one or more
inorganic
salts of mineral acids,'in particular strong mineral acids, that result in no
loss of the
biological function of the proteins, for example salts of hydrochloric acid,
sulphuric acid
and nitric acid, such as sodium chloride, potassium chloride, ammonium
chloride, sodium
sulphate, potassium sulphate, ammonium sulphate.
The first protein fraction may comprise one or more of the following proteins
selected from the group consisting of: albumin, orosomucoid, pre-albumin, a-l-
proteinase inhibitor (a-1-PI), transferrin and fibrinogen.
The second protein fraction may comprise one or more of the following proteins
selected from the group consisting of: antithrombin, e.g., Antithrombin III,
albumin,
immunoglobulins, transferrin and fibrinogen.
The third protein fraction may comprise one or more of the following proteins
selected from the group consisting of: immunoglobulins, e.g., IgA, IgD, IgE,
IgG and/or
IgM, transferrin and fibrinogen.
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The fourth protein fraction may comprise one or more of the following proteins
selected from the group consisting of: transferrin, a-2-macroglobulin,
immunoglobulins
such as IgM, and fibrinogen.
According to a second aspect, the present invention provides a process for
isolating
s Factor VIII and/or Factor IX from a solution comprising Factor VIII and/or
Factor IX,
said solution being selected from the group consisting of: crude blood plasma,
blood
serum, cryosupernatant derived from plasma, fractionated human plasma,
cryoprecipitate
derived from plasma and recombinant broths, said process comprising:
(i) providing a solid separation medium having the formula:
M-S-L
wherein M is a matrix backbone, S is an optional spacer arm and L is a ligand
which is
xylylenediamine;
(ii) contacting said solid separation medium with said solution such that at
least
Factor VIII and/or Factor IX become reversibly bound to said solid separation
medium;
(iii) performing a first elution step to elute non-bound proteins from the
solid
separation medium;
(iv) performing a second elution step to elute Factor VIII and/or Factor IX
from
the solid separation medium.
M may be a high density resin suitable for non-packed bed adsorption, for
example
fluidized bed adsorption and expanded bed adsorption, for example a highly
cross-linked
beaded agarose derivative based on, 6% or 4% agarose, an agarose-tungsten
carbide
conglomerate, an agarose. stainless steel conglomerate, an agarose-quartz
conglomerate,
porous ceramic beads, porous zirconia beads, beads made of controlled pore
glass and
composite beads of porous inorganic materials comprising organic polymers
within their
pores.
The high density resin may have a mean particle size in the range of about 10
micron to about 300 micron, or from about 15 micron to 150 micron, and a bead
density
in the range from about 1.1 g/ml to 15 g/ml, or from about 1.5 g/ml to 15
g/ml, or
alternatively a mean particle size in the range of about 10 micron to 120
micron and a
bead density from about 2.0 g/ml to 15 g/ml, or alternatively a mean particle
size in the
range from about 15 micron to 100 micron and a bead density in the range from
about 2.3
g/ml to 15 g/ml, or alternatively a mean particle size in the range from about
15 micron to
80 micron and a bead density in the range from about 3 g/ml to 15 g/ml.
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The spacer arm S may be derived from a compound comprising an epoxy group
(e.g. butane dioldiglycidylether or epichlorohydrin) or other suitable
coupling reagent
known in the art for covalent attachment of ligands.
In one embodiment L may be m-xylylenediamine. Alternatively L may be p-
s xylylenediamine. L may also be o-xylylenediamine.
Non-bound proteins may be selected from, but not limited to, the group
consisting
of: IgG, IgA, IgM, IgD, or IgE, transferrin (Tf), fibrinogen or a derivative
thereof, plasma
protease inhibitor such as an antithrombin, e.g., antithrombin III, blood pro-
coagulation
protein, blood anti-coagulation protein, cytokine, growth factor, albumin or a
derivative
io thereof, thrombolytic agent, anti-angiogenic protein, insulin or a
derivative thereof, a-1-
proteinase inhibitor or a derivative thereof, such as a-l-antitrypsin, a-2-
antiplasmin or a
derivative thereof, C-1 esterase inhibitor, . apolipoprotein, HDL, Fibronectin
or a
derivative thereof, beta-2-glycoprotein I, plasminogen, plasmin, plasminogen
activator,
plasminogen inhibitor, urokinase or derivative thereof, streptokinase or a
derivative
15 thereof, inter- a-trypsin inhibitor, a-2-macroglobulin, amyloid protein,
orosomucoid,
ferritin, pre-albumin, GC-globulin, haemopexin and C3-complement.
The eluants used in the elution steps may have a pH in the range of about 5.0
to
about 9Ø The eluants may have a conductivity in the range from about 0.0001
S/cm to
about 10.0 S/cm. The eluants may have a pH in the range of about 6.0 to about
9.0, and a
20 conductivity of about 0.03 S/cm to about 0.2 S/cm.
The eluants may have approximately the same pH but may differ in ionic
strength.
For example, respective eluants may comprise different buffer systems, and/or
optionally
comprise additional salts, for example salts of hydrochloric acid, sulphuric
acid and nitric
acid, such as sodiuni chloride, potassium chloride, ammonium chloride, sodium
sulphate,
25 potassium sulphate, ammonium sulphate.
Respective eluants may have any combinations of pH and ionic strength values
within the above specified ranges. The eluant may comprise an aqueous buffer
solution
comprising a salt of an inorganic or organic acid that results in no loss of
the biological
function of the Factor VIII and/or Factor IX, for example a buffer comprising
a citrate, an
30 acetate, a succinate, a lactate, a tartrate, a formate, a propionate, a
borate or a phosphate.
The eluant may also comprise an aqueous solution of one or more inorganic
salts of
strong mineral acids that result in no loss of the biological function of the
proteins, for
example salts of hydrochloric acid, sulphuric acid and nitric acid, such as
sodium
chloride, potassium chloride, ammonium chloride, sodium sulphate, potassium
sulphate,
35 ammonium sulphate.
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The first and second eluants may have a pH of between about 6.0 and about 8.0,
and
an ionic strength of between about 0.0001 S/cm and about 1.0 S/cm.
Alternatively, the
first eluant may have a pH of between about 5.5 and about 6.0 and an ionic
strength of
between about 0.0001 S/cm and about 0.01 S/cm.
Further, the first eluant may have a pH of,between about 5.5 and about 6.0 and
an
ionic strength of between about 0.0001 S/cm and about 0.05 S/cm. Further the
first eluant
may have a pH of between about 6.0 and about 6.5 and an ionic strength of
between about
0.001 S/cm and about 0.1 S/cm.
The second eluant may have a pH of between about 6.0 and about 8.0 and an
ionic
strength of betweeri about 0.0001 S/cm and about 0.1 S/cm. Alternatively the
second
eluant may have a pH of between about 6.0 and about 8.0 and an ionic strength
of
between about 0.0001 S/cm and about 0.5 S/cm. Further the second eluant may
have a
pH of between about 7.0 and about 9.0 and an ionic strength of between about
0.001 S/cm
and about 1- S/cm.
By controlling the pH and conductivity of the eluants as described in the
above
paragraphs, the process may be used to. isolate Factor VIII and Factor IX as a
mixture,
from a solution comprising Factor VIII and Factor IX as well as other
substances.
Alternatively, where the solution comprises only Factor VIII (for example
where the
solution is cryoprecipitate derived from plasma, or a recombinant broth where
only Factor
VIII has been expressed), by controlling the pH and conductivity of the
eluants as
described in the above paragraph, the process may be used to isolate only
Factor VIII.
Where the solution comprises only Factor IX (for example where the solution is
cryosupernatant derived from plasma, or a recombinant broth where only Factor
IX has
been expressed), by controlling the pH and conductivity of the eluants as
described in the
above paragraph, the process may be used to isolate only Factor IX.
Brief Description of the Drawings
Figure 1 shows a process in accordance with the invention, showing additional
purification of the isolated protein fractions.
Figure 2 shows product recovery using a process in accordance with one
embodiment of the invention.
Figure 3 shows an SDS-PAGE analysis in accordance with one embodiment of the
first aspect of the invention.
Figure 4 shows a Single Radial Immunodiffusion analysis on fractions obtained
from one embodiment of the first aspect of the invention.
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Figure 5 illustrates one embodiment of the invention wherein the first and
second
aspects of the invention are used in series to isolate certain protein
fractions.
Figure 6 shows an SDS-PAGE analysis of fractions from Factor VIII adsorption.
Lane I depicts raw plasma, lane 2 depicts the run-through fraction and lane 3
depicts
s elution of Factor VIII/Factor IX. The Factor VIII/Factor IX eluate was
diluted relative to
the elution volume for direct comparison with the applied plasma, i.e. yields
may be
visually estimated.
Figure 7 shows an SDS-PAGE analysis of protein fractions obtained from the
protein isolation process of the first aspect in which the raw material
(solution comprising
10 said proteins) used is the run-through fraction obtained from the process
of the second
aspect. Lane I depicts human plasma, lane 2 depicts the run-through fraction
((x-1-PI),
lane 3 depicts elution 2 (Albumin), lane 4: depicts elution 3 (IgG) and lane 5
depicts
elution 4 (Fibrinogen).
Figure. 8 shows a single Radial Immunodiffusion analysis of the fractions
obtained
from the protein isolation process of the first aspect in which the raw
material (solution
comprising said proteins) used is the run-through fraction obtained from the
process of the
second aspect. Fractions 1 to 4 represent run-through fraction, elution 2,
elution 3 and
elution 4 respectively. Figure 8A shows quantitation of albumin, and Figure 8B
shows
quantitation of IgG. The upper two rows of 8A and 8B depict a standard curve
of raw
plasma 100-20 % (double determinations). The lower two rows of A and B: depict
the
following: 1: Run-through fraction; 2: Elution 2; 3: Elution 3; 4: Elution 4:
Definitions
The following are some definitions that may be helpful in understanding the
description of the present invention.
In the context of this specification, the term "comprising" means "including
principally, but not necessarily solely". Furthermore, variations of the word
"comprising", such as "comprise" and "comprises", have correspondingly varied
meanings.
In the context of the present invention, the terms "elution step", ."elution"
or
"eluting" may be used interchangeably and are intended to refer to a step of
obtaining a
protein fraction comprising one or more proteins which may, or may not. have
been bound
and subsequently released from the solid separation medium.
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In the context of the present specification, the term "washing step" is
intended to
refer to a step of flushing the solid separation medium with a fluid which
does not
substantially release any proteins from the solid separation medium.
In the context of the present specification, the term "equilibration step" is
intended
to refer to a step wherein sufficient solution is allowed to pass through the
solid
separation medium so that counter-ion concentration, conductivity and pH of
the outgoing
solution is about the same as that of the incoming solution.
In the context of the present specification 'the term "recombinant broth"
refers to
soluble proteins which have been expressed in vitro by genetically manipulated
cells.
The proteins which may be expressed by these manipulated cells within the
recombinant
broth may include: coagulation pathway proteins eg Factor VII, Factor VIII,
Factor IX or
Factor XIII, immunoglobulins for example, IgG, IgA, IgM, IgD, or IgE,
transferrin (Tf),
fibrinogen or a derivative thereof, plasma protease inhibitor such as an
antithrombin, e.g.,
antithrombin III, al-proteinase inhibitor, blood pro-coagulation protein,
blood anti-
coagulation protein,. cytokine, growth factor, albumin or a derivative
thereof,
thrombolytic agent, anti-angiogenic protein, insulin or a derivative thereof,
a-l-proteinase
inhibitor or a derivative thereof, such as a-l-antitrypsin, a-2-antiplasmin or
a derivative
thereof, C-1 esterase inhibitor, apolipoprotein, HDL, Fibronectin or a
derivative thereof,
beta-2-glycoprotein I, plasminogen, plasmin, plasminogen activator,
plasminogen
inhibitor, urokinase or derivative thereof, streptokinase or a derivative
thereof, inter- a-
trypsin inhibitor, a-2-macroglobulin, amyloid protein, orosomucoid, ferritin,
pre-albumin,
GC-globulin, haemopexin and C3-complement.
In the context of the present specification, the term "blood plasma" refers to
the
liquid portion of the blood and is a complex solution comprising more than 90
percent
water. The major solute of plasma is a heterogeneous group of proteins. Other
plasma
constituents include fatty substances (lipids), inorganic electrolytes,
glucose, amino acids,
vitamins, hormones, and waste products of metabolism.
In the context of the specification, the term "blood serum" refers to blood
plasma
from which fibrinogen has been removed in the process of clotting.
In the context of the specification, the terms "cryosupernatant" and
"cryoprecipitate" should be understood according to the following:
Cryosupematant is
that solution produced from plasma following the removal of the
cryoprecipitate.
Cryoprecipitate is composed of those proteins precipitated from plasma by
exposure of
plasma to temperatures of between 1 C and 10 C.
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12
In the context of the specification, the term "fractionated human plasma"
should be
understood as referring to any component or mixture of components of plasma
that is
derived from plasma being subjected to a separation process e.g.,
precipitation, filtration,
chromatography etc.
In the context of the present specification, the term "run-through" should be
understood as referring to a protein fraction obtained from the second aspect
of the
invention comprising the eluate obtained when the plasma solution is loaded
onto the
column, which has been mixed with the eluate obtained from the first elution
step.
Detailed Description of the Invention
Protein Isolation
The f rst aspect of the invention relates to processes for isolating proteins
from
biological solutions. More particularly, the invention relates to processes
for isolating
human proteins from human blood plasma, cryosupernatant derived from human
plasma,
fractionated human plasma, cryoprecipitate derived from human plasma, from
blood
serum, or from i-ecombinant broths.
The solution comprising the proteins may be diluted with another suitable
fluid
prior to contacting said solid separation medium. For example, the plasma may
be diluted
with demineralised water or ari aqueous solution of one or more inorganic
salts of strong
mineral acids compatible with the proteins to be isolated, for example salts
of
hydrochloric acid, sulfuric acid and nitric acid, such as sodium chloride,
potassium
chloride, ammonium chloride, sodium sulfate, potassium sulfate, ammonium
sulfate.
The solution comprising the proteins may be diluted with another suitable
fluid in a
ratio of from about 1000:1 to about 1:1000. For example, the dilution ratio
may be about
1000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 50:1, about
25:1, about
10:1, about 7.5:1, about 5:1, about 3:1, about 2.5:1, about 2:1, about 1:1,
about 1:2, about
1:2.5, about 1:3, about 1:5, about 1:7.5, about 1:10, about 1:25, about 1:50,
about 1:100,
about 1:250, about 1:500, about 1:750, or about 1:1000.
The pH of the solution comprising the proteins may be adjusted prior to
contacting
the fluid with the solid separation medium. Alternatively, the pH may be
adjusted after
the solution comprising the proteins has been contacted with the solid
separation medium. .
The pH may be raised or the pH may be lowered. Alternatively, the pH may
remain
unchanged. The pH may be adjusted to a pH in the range from about 3.0 to about
6.0, or alternatively, the pH may be adjusted to a.pH in the range from about
4.5 to about 6Ø
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13
For example, the pH may be adjusted to a pH of about 3.0, 3.5, 4.0, 4.5, 5.0,
5.5,
6Ø The pH may be adjusted using a suitable acid, e.g, hydrochloric acid,
sulfuric acid;
phosphoric acid, citric acid, succinic acid, acetic acid, etc.
The pH may be adjusted using a suitable base, for example, carbonate,
bicarbonate,
ammonia, hydroxide, etc. The pH may be adjusted with a suitable buffer system.
Buffer
systems are well known to those skilled in the art and include, for example,
citrate,
acetate, phosphate, formate, succinate, MES, ADA, bis-tris propane, PIPES,
ACES,
Imidazole, MOPS, TES, HEPES, HEPPS, TRICINE, Glycine Amide hydrochloride,
TRIS, BICINE, Glycylglycine, Boric Acid, CHES, CAPS. Many buffer systems are
commercially available, and may be obtained, for example from Sigma Chemical
Company. Those skilled in the art would be able to identify suitable buffer
systems in
terms of the desired pH.
The eluants may differ in pH. The elution steps may comprise treating the
solid
separation medium with eluants of increasing pH, or decreasing pH.
The eluants may have approximately the same pH but may differ in ionic
strength.
For example, respective eluants may comprise different buffer systems, and/or
optionally
comprise additional salts, for example salts of hydrochloric acid, sulphuric
acid and nitric
acid, such as sodium chloride,.potassium chloride, ammonium chloride, sodium
sulphate,
potassium sulphate, ammonium sulphate.
The yield of each of the proteins eluted may be at least 50%, or at least 60
%, or at
least 70%, or at least 80%, or at least 90% relative to the amount of protein
present in the
starting material.
The elution steps may be carried out at a temperature in the range of 0 C to
40 C.
For example, the. temperature may be about 5 C, about 10 C, about 15 C,
about 20 C,
about 25 C, about 30 C, about 35 C, or about 40 C.
The process of the first aspect may optionally include one or more washing
steps.
A washing step may be performed at any stage of the process of the first
aspect, for
example, prior to contacting the solid separation medium with the solution
comprising the
proteins, after contacting the solid separation medium with the solution
comprising the
proteins, or between each elution step. The washing step may be carried out
using any
solution that does not result in a loss of the biological function of the
proteins, for
example water, saline, or a buffer solution, for example citrate buffer.
A washing step may be carried out after eluting the solid separation medium
with
the second eluant. The washing buffer may comprise an inorganic- or organic
salt of
relatively high lyotrophobicity such as ammonium sulfate, sodium sulfate,
potassium
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14
sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, ammonium
citrate, sodium citrate, potassium citrate. The ionic strength of the washing
buffer may be
at least 0.001 S/cm, or alternatively at least 0.01 S/cm, or alternatively at
least 0.1 S/cm or
further, at least 1.0 S/cm.
The process of the first aspect may further include one or more equilibration
steps.
An equilibration step may be carried out prior to contacting the solid
separation medium
with said solution comprising the proteins. The equilibration step may
comprise treating
said solid separation medium with water or a suitable buffer solution to
adjust the pH and
ionic strength of the solid separation medium. The solution used for the
equilibration step
may be demineralised water or an aqueous solution of one or more inorganic
salts of
strong mineral acids compatible with the proteins to be isolated, e.g. salts
of hydrochloric
acid, sulfuric acid and nitric acid, such as sodium chloride, potassium
chloride,
ammonium chloride, sodium sulfate, potassium sulfate, ammonium sulfate.
Alternatively, the equilibration buffer may be an aqueous buffer solution
comprising a
salt of an inorganic and/or an organic acid compatible with the proteins to be
isolated, for
example a buffer comprising a citrate, =an acetate, a succinate, a lactate, a
tartrate, a
formate, a propionate, a phosphate, or a borate.
Equilibration steps may be carried out at a temperature in the range of about
10 C
to about 40 C. For example, the temperature may be about 10 C, about 15 C,
about 20
C, about 25 C, about 30 C, or about 40 C.
The pH of the solution comprising the proteins may be the same as the pH of
the
buffer solution used to equilibrate the solid separation medium prior to
cointacting said
solid separation medium with said solution comprising the proteins. The pH of
the
solution comprising the proteins may be different from the pH of the buffer
solution used
to equilibrate the solid separation medium prior to contacting said solid
separation
medium with said solution comprising the proteins.
The process of the first aspect may further include one or more regeneration
steps.
A regeneration step comprises treating the solid separation medium with a
suitable
reagent capable of removing residual material from the solid separation
medium. A
regeneration step may be carried out at any time after the first elution step
is performed.
Suitable regeneration reagents include bases, for example, hydroxide
solutions, such as
sodium hydroxide or potassium hydroxide, solutions of peracids or hydrogen
peroxide,
.solutions comprising active chlorine, such as hypochlorite solutions,
denaturants, such as
guanidinium hydrochloride, organic solvents, e:g, ethanol.
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In accordance with the process of the first aspect, the solid separation media
may be
loaded into a suitable chromatography column apparatus. The process may be
carried out
in a packed bed column in packed bed mode. Alternatively, the process may be
carried
out in an expanded bed absorption (EBA) column in expanded bed mode. In the
context
5 of the first aspect, practically all human plasma proteins can be adsorbed
to the solid
separation medium in a single column. Successive elution steps described
herein can then
be used to selectively elute protein fractions enriched in specific proteins.
EBA columns are, well known in the art and suitable column apparatus and set
up,
including methods of introducing liquids into an expanded bed column, are
available
10 commercially from GE Healthcare, Sweden, or have been described in WO
99/65586,
WO 01/85329and W092/00799, the entire contents of which are incorporated
herein by
cross-reference.
One embodiment of the first aspect comprises selecting an EBA column and
placing
an adequate quantity of solid separation medium into the column. The amount of
solid
15 separation medium used will depend on the amount of solution comprising the
proteins
that is to be applied, and the protein concentration in the protein solution.
When the
protein solution is human plasma or cryoprecipitated plasma, typically 1.0
litre of solid
separation medium is used for every 0.5 to 1.5 litres of plasma. If the
protein solution is a
recombinant fermentation broth, one litre of solid separation medium may be
used for 1
litre of fermentation broth and up to 1000 1'of fermentation broth. A flow
through is
established from the bottom of the column until the solid separation medium is
fluidised.
Suitable column linear flow rates include flow rates in the range 0.5 to 20
cm/min, or
alternatively from about 5 cm/min to 15 cm/min. The solid separation medium
may then
be equilibrated using an appropriate solution (for example, water, an aqueous
electrolyte
solution, or buffer solutiori), after which a solution, such as blood plasma,
blood serum,
cryosupernatant, solubilised cryoprecipitate or recombinant broth may be
introduced to
the bottom of the column. A further washing step inay optionally be performed
once the
solution has been loaded onto the solid separation medium. Elution steps are
then carried
out in order to selectively elute protein fractions enriched in specific
proteins.
Each respective elution step is carried out under conditions suitable to
selectively
elute a protein fraction comprising one or more proteins. For example, by
varying the pH
and/or ionic strength of the eluant used in respective elution steps,
different proteins may
be eluted. The eluant may be adjusted to a suitable pH and ionic strength
using an
appropriate buffer. The ionic strength may also be adjusted using a salt.
Successive
elution steps may comprise treating the solid separation medium with an eluant
of
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.16
increasing pH. Alternatively successive elution steps may comprise treating
the solid
separation medium with an eluant of decreasing pH.
Another embodiment in accordance with the first aspect involves establishing
an
EBA column comprising agarose-tungsten carbide conglomerate 'beads
functionalised
with 2-mercaptonicotinic acid. The agarose-tungsten carbide beads have a size
distribution between 40-120 micron with a mean diameter of 70 micron. The
density of
the beads is 2.9 g/ml. An equilibration step is carried out using a citrate
buffer at a pH of
about 4.5, which is immediately followed by a further equilibration step with
citrate
buffer at a pH of about 5Ø Human plasma that has been diluted with 2 parts
water, and
adjusted to a pH of 5.0 with HCl was then applied to the column in a ratio'
1.5 litres of
diluted plasma per litre of solid separation medium. The column was then
eluted with the
following buffer solutions:
Elution 1. 10mM sodium citrate at pH 5.0
Elution 2. 5g / litre of sodium caprylate and HCl at pH 6.0
1s Elution 3. 1 M sodium citrate at pH 8.0
Elution 4. 20mM sodium c'itrate and 0. I M sodium chloride at pH 8.0
The solid separation medium was then regenerated with 1 M NaOH.
The flow rate for all operations was 7.5 cm/minute, and the amounts of each
solution used
are given below in Table 2.
The volume of eluant used will typically depend on a number of interrelated
factors,
for example:
(i) The flow rate used during sample application, washing, elution,
regeneration
and equilibration;
(ii) The number of product fractions eluted;
(iii) The choice of eluants used in each step, as the choice of eluants
influence the
yield and purity of the individual fractions;
(iv) The optimal gap between individual fractions, which also has an influence
on
the yield and purity of the products obtained;
(v) Bed height of the solid separation medium, as generally the washing and
elution volumes consumed decrease when the bed height of the solid separation
medium
is decreased.
Table 2 shows the optimal solution volume for each step in the above described
embodiment.
Referring to Figure 3, it can be seen from the above embodiment that the
proteins
a-1 proteinase inhibitor, albumin, IgG and fibrinogen can be effectively
isolated.
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17
TABLE 2
Step Volume of Solution*
Equilibration 5.0 CV
Eluant 1 4.2 CV
Eluant 2 2.9 CV
Eluant 3 4.4 CV
Eluant 4 2.1 CV
Re eneration 1.0 CV
TOTAL 19.6 CV
*CV is the column volume
s Table 3 below shows the yields of the proteins relative to the applied raw
plasma as
determined by SRI.
TABLE3
Elution 1 Elution 2 Elution 3 Elution 4
Albumin < 5% 95+%
Immunoglobulin 95+% <5%
a-l-proteinase 95+%
inhibitor
Fibrinogen <5% 95+%
Single radial immunodiffusion (SRI) was performed in order to determine the
relative yield of the individual proteins.in eluant fractions 1 to 4. Figure 4
shows the SRI
analysis for albumin, IgG, a-l-proteinase inhibitor and fibrinogen.
The above described process was found to ruri in a smooth, reproducibl.e and
uncomplicated manner. All final eluates were found to be clear liquids with no
sign of
is significant denaturation/precipitation of any of the proteins. Post column
testing by SDS-
PAGE and SRI as described above showed no sign of break-down, agglomeration or
changes in immunoreactivity of the eluted products.
Those skilled in the art will be aware that the process of the first aspect,
and indeed
the second, aspect, may be monitored by measuring the ultraviolet absorbance
of the
liquid exiting the column. The presence of proteins and other UV-absorbing
material can
be detected and quantified during the processes of the invention and correct
collection of
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18
the different protein fractions can be performed. Also continuous monitoring
of pH,
conductivity and refractive index may be useful in documenting and controlling
the.
processes of the invention.
Isolation of Factor VIII and/or Factor IX
The solution comprising said Factor VIII and/or Factor IX is typically raw
undiluted
plasma which may be directly contacted with the solid separation medium.'
Alternatively,
the raw plasma may be diluted with another suitable fluid prior to contacting
said solid
separation medium. For example, the plasma may be diluted with demineralised
water or
an aqueous solution of one or more inorganic salts of strong mineral acids
compatible
with Factor VIII and/or Factor IX, for example salts of hydrochloric acid,
sulfuric acid
and nitric acid, such as sodium chloride, potassium chloride, ammonium
chloride, sodium
sulfate, potassium sulfate, ammonium sulfate.
The solution comprising the Factor VIII and/or Factor IX may be diluted with
another suitable fluid in a ratio of from about 1000:1 to about 1:1000. For
example, the
dilution ratio may be about 1000:1, about 750:1, about 500:1, about 250:1,
about 100:1,
about 50:1, about 25:1, about 10:1, about 7.5:1, about 5:1, about 3:1, about
2.5:1, about
2:1, about 1:1, about 1:2, about 1:2.5, about 1:3, about 1:5, about 1:7.5,
about 1:10, about
1:25, about 1:50, about 1:100, about 1:250, about 1:500, about 1:750, or about
1:1000.
The pH of the solution comprising Factor VIII and/or Factor IX may be adjusted
prior to contacting with the solid_ separation medium. Alternatively, the pH
may be
adjusted after the solution comprising the Factor VIII and/or Factor IX has
been contacted
with the solid separation medium. The pH may be raised or the pH may be
lowered.
Alternatively, the pH may remain unchanged. The pH may be adjusted to a pH in
the
range from about 5.0 to about 9Ø For example, the pH may be adjusted to a pH
of about
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9Ø The pH may be adjusted using a
suitable acid,
e.g, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, succinic
acid, acetic
acid, etc.
The pH may be adjusted using a suitable base, for example, carbonate,
bicarbonate,
ammonia, hydroxide, etc. The pH may be adjusted with a suitable buffer system.
Buffer
systems are well known to those skilled in the art and include, for example,
citrate,
acetate, phosphate, formate, succinate, MES, ADA, bis-tris propane, PIPES,
ACES,
Imidazole, MOPS, TES, HEPES, HEPPS, TRICINE, Glycine Amide hydrochloride,
TRIS, BICINE, Glycylglycine, Boric Acid, CHES, CAPS. Many buffer systems are
commercially available, and may be obtained, for example from Sigma Chemical
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19
Company. Those skilted in the art would be able to identify suitable buffer
systems in
terms of the desired pH.
The process may optionally include one or more washing steps. A washing
step may be performed prior to contacting the solid separation medium with the
solution
comprising the Factor VIII and/or Factor IX, or alternatively after contacting
the solid
separation medium with the solution comprising the Factor VIII and/or Factor
IX. The
washing step may be carried out using any solution that does not result in a
loss of the
biological function of the Factor VIII and/or Factor IX, for example water,
saline, or a
buffer solution, for example citrate buffer.
The washing buffer may alternatively comprise an inorganic or organic salt of
relatively high lyotrophobicity such as ammonium sulfate, sodium sulfate,
potassium
sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, ammonium
citrate, sodium citrate, potassium citrate. The washing buffer may have a pH
in the range
of about 5.0 to about 9Ø The pH of the washing buffer may be 5.2, 5.4, 5.6,
5.8, 6.0, 6.2,
6.4, 6.6, 6.8, 7.0, 7:2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8 or 9Ø The
washing buffer may
have a conductivity in the range from about 0.1 mS/cm to about 100 mS/cm.
Alternatively, the washing buffer may have a conductivity of about 0.1 mS/cm
to about 40
mS/cm.
The washing buffer may also 'comprise an aqueous solution of one or more
. inorganic salts of strong mineral acids that result in no loss of the
biological function of
the proteins, for example salts of hydrochloric acid, sulphuric acid and
nitric acid, such as
sodium chloride, potassium chloride, ammonium chloride, sodium sulphate,
potassium
sulphate, ammonium sulphate.
The process may further include one or more equilibration steps. An
equilibration
step may be carried out prior to contacting the solid separation medium with
the solution
comprising Factor VIII and/or Factor IX. The equilibration step may comprise
treating
said solid separation medium with water or a suitable buffer solution to
adjust the pH and
ionic strength of the solid separation medium. The solution used for the
equilibration step
may be demineralised water- or an aqueous solution of one or more inorganic
salts of
strong mineral acids . compatible with Factor VIII and/or Factor IX, e.g.
salts of
hydrochloric acid, sulfuric acid and nitric acid, such as sodium chloride,
potassium
chloride, ammonium chloride, sodium sulfate, potassium sulfate, ammonium
sulfate.
Alternatively, the equilibration buffer may be an aqueous buffer solution
comprising a
salt of an inorganic and/or an organic acid compatible with Factor VIII and/or
Factor IX,
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for example a buffer comprising a citrate, an acetate, a succinate, a lactate,
a tartrate, a
formate, a propionate, a phosphate, or a borate.
Equilibration steps may be carried out at a temperature in the range of about
2 C to
about 28 C. For example, the temperature may be about 2, 4, 6, 8, 10, 12, 14,
16, 18, 20,
5 22, 24, 26 or 28 C.
The pH of the solution comprising the Factor VIII and/or Factor IX may be the
same as the pH of the buffer solution used to equilibrate the solid separation
medium
prior to contacting said solid separation medium with said solution comprising
the Factor
VIII and/or Factor IX. The pH.of the solution comprising the Factor VIII
arid/or Factor
10 IX may be different from the pH of the buffer solution used to equilibrate
the solid
separation medium prior to contacting said solid separation medium with said
solution
comprising the Factor VIII and/or Factor IX.
The process may further include one or more regeneration steps. A regeneration
step comprises treating the solid separation medium with- a suitable reagent
capable of
15 removing residual material from the solid separation medium. A regeneration
step may
be carried out after eluting said solid separation medium with an eluant to
elute Factor
VIII and/or Factor IX. Suitable regeneration reagents include bases, for
example,
hydroxide solutions, such as sodium hydroxide or potassium hydroxide,
solutions of
peracids or hydrogen peroxide, solutions comprising active chlorine, such as
hypochlorite
20 solutions, denaturants, such as guanidinium hydrochloride, organic
solvents, e.g, ethanol.
The process may be carried out in a packed bed column in packed bed mode.
Alternatively, the process may be carried out in an EBA column in expanded bed
mode.
In one embodiment, as indicated above, the process may be used to isolate
Factor
VIII and Factor IX as a mixture from a solution comprising Factor VIII and
Factor IX as
well as other substances. In this embodiment an EBA column is selected and
into it is
placed an adequate quantity of solid separation medium. The amount of solid
separation
medium used will depend on the amount of solution comprising the Factor VIII
and
Factor IX that is to be applied, and the concentration of the Factor VIII and
Factor IX in
the solution. When the protein solution. is human plasma or cryoprecipitated
plasma,
30typically 1.01itre of solid separation medium is used for every 5 to
301itres of plasma. If
the protein solution is a recombinant fermentation broth, one litre of solid
separation
medium may be used for 1= litre of fermeritation 'broth and up to 1000 1 of
fermentation
broth. A flow through is established from the bottom of the column until the
solid
separation medium is fluidised: Suitable column linear flow rates include flow
rates in
the range 0.5 cm/min to 40 cm/min, or alternatively from about 3 cm/min to 15
cm/min.
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An equilibration step may then be performed using an appropriate solution (for
example,
water, an aqueous electrolyte solution, or buffer solution), after which a
solution
comprising Factor VIII and Factor IX, for example crude blood plasma, may be
introduced to the bottom of the column, whereby the solution comprising Factor
VIII and
Factor IX is contacted with the solid separation medium. A first elution step
is then
performed in order to elute non-bound protein from the column. A washing step
may
optionally be performed after the first elution step. A second elution step is
then
performed in order to elute the Factor VIII and Factor IX from the solid
separation
medium.
The first and second aspects of the invention can be used in series in a two-
step
process wherein the first step involves use of the second aspect of the
invention for the
isolation of Factor VIII, or its complex with von Willebrand factor, and/or
Factor IX from
a solution.comprising any of the proteins listed above on page 4 of the
specification, with
an enrichment factor of at least 10, and the second step involves use of the
first aspect of
is the invention for the separation of the non-bound proteins obtained from
the first step of
the second aspect, into fractions comprising proteins such as a-l-proteinase
inhibitor,
albumin, IgG, anti-thrombin III and fibrinogen, all in high yield.
The process of the second aspect may be used in series with other protein
isolation
processes. For example, the process of the second aspect may be used to
isolate Factor
VIII and/or Factor IX from a solution of blood plasma, with the remaining
proteins
present in the fraction obtained from the run-through being used as the raw
material from
which further proteins, for example albumin, fibrinogen, immunoglobulins, a-1-
proteinase inhibitor, may be isolated using various other fractionation
processes eg
precipitation; filtration, ion exchange chromatography etc.
Isolated proteins of the first and second aspects of the invention aspects may
be
incorporated into formulations as appropriate, for examples, as powders,
solutions, liquids
etc. Examples of some product formulations are provided in Table 4.
Table 4
Product Formulation
Factor VIII Dried Powder: 250 IU, 500 IU, 1000 IU
a-1-PI Dried Powder, lg
Albumin 5%, 25% solution
Antithrombin III Dried Powder 1000 IU
IVIG 10% liquid, pH 4.25
Fibrino en Dried powder 1.5
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Additional purification of the isolated protein fractions may also be employed
in the
processes of the invention as required, or as appropriate.
One such method may involve the serial inclusion of anion exchange or
hydrophobic interaction chromatography steps. Methods such as these function
to
concentrate and further purify the protein fractions obtained from the
processes of the
invention. By choosing optimal stationary media and elution conditions for the
serial
chromatography step or steps, all of the proteins obtained from the processes
of the
invention could be bound onto a column. Bound protein could then be
selectively eluted
io from the secondary column undet a different set of chemical conditions
resulting in
significant concentration and potentially further purification of the
proteins. Other
chromatographic techniques, such as affinity chromatography, metal chelate
chromatography and gel filtration may also be employed either individually, or
in
combination.
Figure 1 demonstrates some of the additional purification steps that may
feasibly be
employed following the processes of the invention.
The processes of the invention may optionally include viral inaction steps.
For
example, viral inactivation/elimination steps are illustrated in Table 5 for
each product,
wherein each product has been isolated via a process of the invention.
Table 5
Product Step 1 Step 2
Factor VIII Solvent detergent Dry heat, 80 C, 72h
a-1 proteinase Solvent 'detergent Viral filtration
inhibitor
Antithrombin Pasteurization, 60 C, lh Viral filtration
III
Albumin S-200 chromato a h Pasteurisation, 60 C, lh
IVIG Solvent detergent Viral filtration
Fibrinogen Solvent detergent Wet heat, 62 C, l Oh
T'he invention will now be described in more detail by way of illustration
only, with
respect to the following examples. The examples are intended to serve to
illustrate the
invention and should not be construed as limiting the generality of the
disclosure of the
description throuhgout the specification.
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23
Examples
Example 1- Isolation of a-l-proteinase inhibitor, antithrombin Ill,
fibrinogen,
immunoglobulins and albumin
Step 1. Solid separation medium: Agarose - tungsten carbide beads
functionalised
with 2-mercaptonicotinic acid. The agarose-tungsten carbide beads have a size
distribution of between 40-120 micron with a mean diameter of 70 micron. The
density
of the beads were 2.9 g/ml (FastLine UFC NNSDW cat. No.: CS48, UpFront
Chromatography A/S, Copenhagen, Denmark.). The beads are placed in an EBA
column
(FastLine 100, UpFront Chromatography A/S, Copenhagen, Denmark) (10 cm
diameter;
50 cm settled bed height; Settled bed Volume = 3.926 L). Equilibration is
performed at a
temperature of 25 C with 2.5 column volumes (9.8L) of 40 mM sodium citrate
with a pH
4.5, followed by a further 2.5 column volumes (9.8L) of 40 mM sodium citrate
and pH
5.0 at a linear flow rate of 7.5 cm/min.
Step 2. Onto this column was loaded 6L of a diluted plasma solution comprising
2L
of thawed human plasma which had been diluted at a ratio of 1:2 with 4L of
water. The
pH of the diluted plasma solution was adjusted to pH 5.0 with 1M HCl prior to
application to the column and the load ratio was 1.5 litres of diluted plasma
solution per
litre of resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted
with Elution Buffer I comprising 4.2 column volumes (16.49L) of a buffer
solution
comprising 10mM sodium citrate pH. 5Ø This resulted in the removal of non-
bound
protein, lipid and other substances including greater than 95% of the a-l-
proteinase
Inhibitor present in the diluted plasrria solution applied to the column.
Step 4. Following Step 3 the column was eluted with 2.9 column volumes
(11.39L)
of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø Step 4
resulted in
the elution of albumin from the column at a yield of greater than 95% of the
quantity of
the albumin present in the diluted plasma solution applied to the column. This
step also
results in the elution of 60% of the Antithrombin III present in the diluted
plasma solution
applied to the column.
Step 5. Following the elution of the column as described in Step 4 above the
column was eluted with 4.4 column volumes (17.27L) of Elution Buffer 3,
comprising
1 M sodium citrate pH 8Ø This step resulted in the elution of greater than
95% of the
immunoglobulins present in the diluted plasma solution applied to the column.
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24
Step 6. Following the elution of the column as described in Step 5 above, the
column was eluted with Elution Buffer 4, comprising 2.1 column volumes (8.24L)
of
20mM sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This Step
resulted in
the elution of greater than 95% of the fibrinogen present in. the diluted
plasma solution
applied to the.column.
Step 7. Following elution of the column as described in Step 6 the column,
_was
regenerated with I column volume (3.926L) of 1 M sodium hydroxide and re-
equilibrated
with 2.5 column volumes (9.8L) of 40 mM sodium citrate pH 4.5 followed by a
further
2.5 column volumes (9.8L) of 40 mM sodium citrate and pH 5Ø
Example 2 - Isolation of a-1-proteinase inhibitor, albumin, immunoglobulins
and fibrinogen wherein an extra washing step is included.
The solid separation medium used in this Example is the same as that used in
Example 1.
Step 1. An EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a temperature of 21 C with 2.5 column volumes (196.3mL) of 40
mM
sodium citrate pH 4.5 at a linear flow rate of 5.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising 39.3mL of thawed human plasma which had been diluted at a ratio of
1:2 with
78.5mL of water. The pH of the diluted plasma solution was adjusted to pH 5.0
with 1 M
HCI prior to application to the column and the load ratio was 1.5 litres of
plasma solution
per litre of resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted
with 3.3 column volumes (259.2mL) of Elution Buffer 1 comprising 10mM sodium
citrate pH 5Ø This resulted in the removal of non-bound protein, lipid and
other
substances including the a-l-proteinase Inhibitor present in the diluted
plasma solution
applied to the column.
- Step 4. Following Step 3 above, the column was eluted with 2.6 column
volumes
(204.2mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of the albumin present in the diluted plasma solution
applied to the
column.
Step 4a. Following elution of the.column as described in Step 4 above the
column
was washed with 1.0 column volume (78.5mL) of 1 M sodium citrate pH 8Ø
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Step 5. Following the washing of the column as described in Step 4a above the
column was eluted with 4.9 column volumes (384.8mL) of Elution Buffer 3,
comprising
0.3M sodium citrate pH 8Ø This step resulted in the elution of the
immunoglobulins
present in the diluted plasma solution applied to the column.
5 Steu 6. Following the elution of the column as described in Step 5 above,
the
column was eluted with Elution Buffer 4, comprising of 2.6 column volumes
(204.2mL)
of 20mM sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This step
resulted
in the elution of the fibririogen present in the diluted plasma solution
applied to the
column.
10 Step 7. Following elution of the column as described in Step 6 the column
was
regenerated with 1 column volume (78.5mL) of 1M sodium hydroxide and re-
equilibrated
with 2.0 column volumes (157mL) of 40 mM sodium citrate pH 4.5.
Example 3 - Isolation of a-1-proteinase inhibitor, albumin, transferrin,.
15 immunoglobulins and fibrinogen
The solid separation medium used in this Example is the same as that used in
Example 1.
Step 1. An EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
20 Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL),
was
equilibrated at a temperature of 21 C with 2.5 column volumes (196.3mL) of 40
mM
sodium citrate pH 5.0 at a linear flow rate of 15Ø cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising 39.3mL of thawed human plasma which had been diluted at a ratio of
1:2 with
25 78.5mL of water. The pH of the diluted plasma solution was adjusted to pH
5.0 with 1 M
HCl prior to application to the column and the load ratio was 1.5 litres of
plasma solution.
per litre of resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted
with Elution Buffer 1 comprising 9.4 column volumes (738.3mL) of demineralised
water.
This resulted in the removal of non-bound protein, lipid and other substances
including
100% of the a-l-proteinase Inhibitor, 10% of the Albumin, 5% of the
Transferrin and
10% of the Fibrinogen present in the diluted plasma solution applied to the
column.
Step 4. Following Step 3 above the column was eluted with 8.9 column volumes
(699mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of albumin from the column at a yield of 90% of the
quantity of the
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26
albumin present in the diluted plasma solution applied to the column. This
step also
results in the elution of 5% of the immunoglobulins present in the diluted
plasma solution
applied to the column.
Step 5. Following the eluting of the column as described in Step 4 above the
column .was eluted with 9.0 column volumes (706.8 mL) of Elution Buffer 3,
comprising
0.3M sodium citrate pH 8Ø This step resulted in the elution of greater than
85% of the
immunoglobulins present in the diluted plasma solution applied to the column.
This step
also results in the elution of 95% of the transferrin and 30% of the
fibrinogen present in
the diluted plasma solution applied to the column.
Step 6. Following the elution of the column as described in Step 5 above, the
column was eluted with Elution Buffer 4, -comprising of 5.0 column volumes
(392.7mL)
of 20mM sodium citrate comprising 0.1M sodium chloride at pH 8Ø This step
resulted
in the elution of 60% of the fibrinogen and 10% of the immunoglobulins present
in the
diluted plasma solution applied to the column.
Step 7. Following elution of the column as described in Step 6 the column was
regenerated with 1 column volume (78.5mL) of 1 M sodium hydroxide and re-
equilibrated
with 2.0 column volumes (157mL) of 40 mM sodium citrate pH 5Ø
Example 4- Isolation of a-1-proteinase inhibitor, albumin,
immunoglobulins, transferrin and fibrinogen
The solid separation medium used in this Example is the same as that used in
Example 1.
Step 1. An EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a temperature of 21 C with 2.5 column volumes (196.3mL) of 40
mM
sodium citrate pH 5.0 at a linear flow rate of 5.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising
39.3mL of thawed human plasma which had been diluted at a ratio of 1:2 with
78.5mL of
water. The pH of the diluted plasma solution was adjusted to pH 5.0 with 1 M
HCl prior
to application to the column and the load ratio was 1.5 litres of plasma
solution per litre of
resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted with
Elution Buffer 1 comprising 6.8 column volumes (533.8mL) of demineralized
water.
This resulted in the removal of non-bound protein, lipid and other substances
including
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27
100% of the a-1-Proteinase Inhibitor, 10% of the Albumin, 5% of the
Transferrin and
10% of the Fibrinogen present in the diluted plasma solution applied to the
column.
Step 4. Following washing of the column as described in Step 3 above the
column was
eluted with 5.5 column volumes (431.75mL) of Elution Buffer 2, comprising 5
g/L
sodium caprylate/HCI, pH 6Ø Step 4 resulted in the elution of albumin from
the column
at a yield of 90% of the quantity of the albumin present in the diluted plasma
solution
applied to the.column. This step also results in the elution of 5% of the
immunoglobulins
present in the diluted plasma solution applied to the column.
Step 5. Following Step 4 above the column was eluted with 5.0 column volumes
(392.5mL) of. Elution Buffer 3, comprising 0.3M sodium citrate pH 8Ø This
step
resulted in the elution of greater than 85% of the immunoglobulins present in
the diluted
plasma solution applied to the column. This step also results in the elution
of 95% of the
transferrin and 30% of the fibrinogen present in the diluted plasma solution
applied to the
column.
Step 6. Following the elution of the column as described in Step 5 above, the
column was
eluted with Elution Buffer 4, comprising of 3.1 column volumes (243.35mL) of
20mM
sodium citrate comprising 0.1M sodium chloride at pH 8Ø This step resulted
in the.
elution of 60% of the fibrinogen and 10% of the immunoglobulins present in the
diluted
plasma solution applied to the column.
Step 7. Following elution of the column as described in Step 6 the column was
regenerated with I column volume (78.5mL) of 1 M sodium hydroxide and re-
equilibrated
with 2.0 column volumes (157mL) of 40 mM sodium citrate pH 5Ø
Example 5 - Isolation of a-1-proteinase inhibitor, albumin,
immunoglobulins, fibrinoQen and transferrin
The solid separation medium used in this Example is the same as that used in
Example 1.
Step 1. An EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a temperature of 21 C with 2.5 column volumes (196.3mL) of 40
mM
sodium citrate pH 5.0 at a linear flow rate of 10.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising
39.3mL of thawed human plasma which had been diluted at a ratio of 1:2 with
78.5mL of
water. The pH of the diluted plasma solution was adjusted to pH 5.0 with IM
HCl prior
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28
to application to the colunm and the load ratio was 1.5 litres of plasma
solution per litre of
resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted with
Elution Buffer 1 comprising 9.5 column volumes (745.75mL) of demineralized
water.
This resulted in the removal of non-bound protein, lipid and other substances
including
100% of the a=1-Proteinase Inhibitor, 10% of the Albumin, 5% of the
Transferrin and
10% of the Fibrinogen present in the diluted plasma solution applied to the
column.
Step 4. Following Step 3 above the column was eluted with 7.1 column volumes
(557.35mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of albumin from the column at a yield of 90% of the
quantity of the
albumin present in the diluted plasma solution applied to the column. This
step also
results in the elution of 5% of the immunoglobulins present in the diluted
plasma solution
applied to.the column.
Step 5. Following Step 4 above the column was eluted with 5.9 column volumes
(463.15mL) of Elution Buffer 3, comprising 0.3M sodium citrate pH 8Ø This
step
resulted in the elution of greater than 85% of the immunoglobulins present in
the diluted
plasma solution applied to the column. This step also results in the elution
of 95% of the
transferrin and 30% of the fibrinogen present in the diluted plasma solution
applied to the
column.
Step 6. Following the elution of the column as described in Step 5 above, the
column was
eluted with Elution Buffer 4, comprising of 3.1 column volumes (243.35mL) of
20mM
sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This step resulted
in the
elution of 60% of the fibrinogen and 10% of the irimmunoglobulins present in
the diluted.
plasma solution applied to the column.
Step 7.. Following elution of the column as described in Step 6 the column was
regenerated with I column volume (78.5mL) of 1 M sodium hydroxide and re-
equilibrated
with 2.0 column* volumes (157mL) of 40 mM sodium citrate pH 5Ø
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Example 6 - Isolation of a-1-proteinase inhibitor, albumin, immunogloblins,
transferrin and fibrinogen
The solid separation medium used in this Example is the same as that used in
Example 1.
Step 1. An EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a temperature of 21 C with 2.5 column volumes (196.3mL) of 40
mM
sodium citrate pH 5.0 at a linear flow rate of 20.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising
39.3mL of thawed human plasma which had been diluted at a ratio of 1:2 with
78.5mL of
water. The pH of the diluted plasma solution was adjusted to pH 5.0 with 1 M
HCl prior
to application to the column and the load ratio was 1.5 litres of plasma
solution per litre of
is resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted with
Elution Buffer 1 comprising 12.6 column volumes (989.1mL) of demineralized
water.
This resulted in the removal of non-bound protein, lipid and other substances
including
100% of the a-1-Proteinase Inhibitor present in the diluted plasma solution
applied to the
column. It is noted that a significant amount of albumin is being also eluted
in this step at
a flow rate of 20 cm/min.
Step 4. Following Step 3 above the column was eluted with 10.6 column volumes.
(832.1mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of albumin from the column at a yield of 90% of the
quantity of the
albumin present in the diluted plasma solution applied to the column. This
step also
results in the elution of 5% of the immunoglobulins present in the diluted
plasma solution
applied to the column.
Step 5. Following Step 4 above the column was eluted with 6.9 column volumes
(541.65mL) of Elution Buffer 3, comprising 0.3M sodium citrate pH 8Ø This
step
3o resulted in the elution of greater than 85% of the immunoglobulins present
in the diluted
plasma solution applied to the column. This step also results in the elution
of 95% of the
transferrin and 30% of the fibrinogen present in the diluted plasma solution
applied to the
column.
Step 6. Following the elution of the column as described in Step 5 above, the
column was
eluted with Elution Buffer 4, comprising of 6.8 column volumes (533.8mL) of
20mM
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sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This step resulted
in the
elution of 60% of the fibrinogen and -10% of the immunoglobulins present in
the diluted
plasma solution applied to the column.
Step 7. Following elution of the column as described in Step 6 the column was
s regenerated with 1 column volume (78.5mL) of I M sodium hydroxide and re-
equilibrated
with 2.0 column volumes (157mL) of 40 mM sodium citrate pH 5Ø
Example 7 - Isolation of a-1-proteinase inhibitor, albumin and IgG
10 The solid separation medium- used in this Example is the same as that used
in
Example 1.
Step 1. An EBA column. (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a temperature of 21 C with 2.5 column volumes (196.3mL) of 40
mM
15 sodium citrate pH 4.5 at a linear flow rate of 5.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising
39.3mL of thawed human plasma which had been diluted at a ratio of 1:2 with
78.5mL of
water. The pH of the diluted plasma solution was adjusted to pH 5.0 with 1M
HCI prior
to application to the column and the load ratio was 1.51itres of plasma
solution per litre of
20 resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted with 3.3
column volumes (259.2mL) of Elution Buffer I comprising 10mM sodium citrate pH
5Ø
This resulted. in the removal of non-bound protein, lipid and other substances
including
the a-1-Proteinase Inhibitor present in the diluted p.lasma solution applied
to the column.
25 Step 4. Following washing of the column as described in Step 3 above, the
column was
eluted with 2.7 column volumes (211.95mL) of Elution Buffer 2, comprising 5
g/L
sodium caprylate/HCI, pH 6Ø Step 4 resulted in the elution of the albumin
present in the
diluted plasma solution applied to the column.
Step 5. Deleted.
30 Step 6. Following the elution of the column as described in Step 4 above,
the column was
eluted with Elution Buffer 4, comprising of 3.8 column volumes (298.3mL) of
20mM
sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This step resulted
in the
elution of the IgG present in the diluted plasma solution applied to the
column.
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Step 7. Following elution of the column as described in Step 6 the column was
regenerated with I column volume (78.5mL) of 1 M sodium hydroxide and re-
equilibrated
with 2.0 column volumes (157mL) of 40 mM sodium citrate pH 4.5.
Example 8- Isolation of a-l-proteinase inhibitor, albumin, immunoglobulins
and fibrinogen wherein an extra washing step is included
The solid separation medium used in this Example is the same as that used in
Example 1.
Sten 1 EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark)
(2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a
temperature of 21 C with 2.5 column volumes (392.5mL) of 40 mM sodium citrate
pH
4.5 at a linear flow rate of 5.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising
39.3mL of thawed human plasma which had been diluted at a ratio of 1:2 with
78.5mL of
water. The pH of the diluted plasma solution was adjusted to pH 5.0 with 1 M
HCl prior
to application to the column and the load ratio was 1.51itres of plasma
solution per litre of
resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted with 2.9
column volumes (455.3mL) of Elution Buffer I comprising l OmM sodium citrate
pH 5Ø
This resulted in the removal of non-bound protein, lipid and other substances
including
the a-1-Proteinase Inhibitor present in the diluted plasma solution applied to
the column.
Step 4. Following Step 3 above, the column was eluted with 2:8 column volumes
(439.6mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of the albumin present in the diluted plasma solution
applied to the
column.
Step 4a. Following elution of the column as described in Step 4 above the
column was
washed with 1.0 column volume (157mL) of 1M sodium citrate pH 8Ø
Step 5. Following the washing of the column as= described in Step 4a above the
column
was eluted with 4.5 column volumes (706.5mL) of Elution Buffer 3, comprising
0.3M
sodium citrate pH 8Ø This step resulted in the elution of the
immunoglobulins present in
the diluted plasma solution applied to the column.
Step 6. Following the elution of the column as described in Step 5 above, the
column was
eluted with Elution Buffer 4, comprising of -1.9. column volumes (298.3mL) of
20mM
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32
sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This step resulted
in the
elution of the fibrinogen present in the diluted plasma solution applied to
the column.
Step 7. Following elution of the column as described in Step 6 the column was
regenerated with 1 column volume (157mL) of 1M sodium hydroxide and re-
equilibrated
with 1.9 column volumes (298.3mL) of 40 mM sodium citrate pH 4.5.
Example 9 - Isolation of a-1-proteinase inhibitor, albumin, immunoglobulins
and fibrinogen wherein an extra washinct step is included
io The solid separation medium used in this Exarimple is the same as that used
in
Example 1.
Step =1. EBA column (FastLine 20, UpFront Chromatography A/S, Copenhagen
Denmark) (2 cm diameter; 25 cm bed height; Settled bed Volume = 78.5mL), was
equilibrated at a temperature of 21 C with 2.5 column volumes (392.5mL) of 40
mM
1s sodium citrate pH 4.5 at a linear flow rate of 10.0 cm/min.
Step 2. Onto this column was loaded 117.8mL of a diluted plasma solution
comprising
39.3mL of thawed human plasma which had been diluted at a ratio of 1:2 with
78.5mL of
water: The pH of the diluted plasma solution was adjusted to pH 5.0 with 1 M
HCI prior
to application to the column and the load ratio was 1.51itres of plasma
solution per litre of
20 resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted with 3.7
column volumes (580.9mL) of Elution Buffer 1 comprising 1.0mM sodium citrate
pH 5Ø
This resulted in the removal of non-bound protein, lipid and other substances
including
the a-1-Proteinase Inhibitor present in the diluted plasma solution applied to
the column.
25 Step 4. Following Step 3 above, the column was eluted with 3.6 column
volumes
(565.2mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of the albumin present in the diluted plasma solution
applied to the
column.
Step 4a. Following elution of the column as described in Step 4 above the
column was
30 washed with 1.0 column volume (157mL) of 1M sodium citrate pH 8Ø
Step 5. Following the washing of the column as described in Step 4a above the
column
was eluted with 5.4 column volumes (847.8mL) of Elution Buffer 3, comprising
0.3M
sodium citrate pH 8Ø This step resulted in the elution of the
immunoglobulins present in
the diluted plasma solution applied to the column.
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Step 6. Following the elution of the column as described in Step 5 above, the
column was
eluted with Elution Buffer 4, comprising of 1.7 column volumes (266.9mL) of
20mM
sodium citrate comprising 0.1 M sodium chloride at pH 8Ø This step resulted
in the
elution of the fibrinogen present in the diluted plasma solution applied to
the column.
Sten 7. Following elution of the column as described in Step 6 the column was
regenerated with I column volume (157mL) of 1M sodium hydroxide and re-
equilibrated
with 2.6 column volumes (408.2mL) of 40 mM sodium citrate pH 4.5.
Analytical Determinations:
All yields for the above examples were determined by comparative single radial
immunodiffusion (Agnete Ingild in: Handbook of Immunoprecipitation-in-Gel
Techniques, ed. Nils H. Axelsen, Scandinavian Journal of Immunology, Suppl.
No. 10,
Vol 17, pp.41-57, 1983.
Example 10 - Isolation of Factor VIII and Factor IX from raw plasma
Step 1. Solid separation medium: The matrix backbone is an agarose-tungsten
carbide resin, the spacer is derived from an epoxy group and the ligand is p-
xylylene-
diamine or m- xylylene-diamine. The beads are placed in an EBA column
(FastLine 10,
UpFront Chromatography A/S, Copenhagen, Denmark) (1 cm diameter; 25cm settled
bed
height; Settled bed Volume = 20mL). Equilibration is performed at a
temperature of 25 C
20 mM sodium citrate and pH 6.0 at a linear flow rate of 5.0 cm/min.
Step 2 (Loading step). Onto, this column was loaded 300mL of undiluted raw
plasma. The pH of the plasma was adjusted to pH 6Øwith IM HCl.prior to
application
to the column and the load ratio was 15L of plasma per litre of resin.
Step 3 (Elution 1). Following loading of the plasma, the column was eluted
with 9
column volumes (180 mL) of Elution Buffer 1 comprising 20mM sodium citrate, pH
6Ø
This resulied in the removal of non-bound protein.
Step 4. The column was then washed with 6.4 column volumes (128mL) of
washing buffer, comprising of 20 mM sodium citrate + 0.2M sodium chloride, pH
6Ø
Step 5 (Elution 2). Following Step 4 above the column was eluted with 6.0
column
volumes (120mL) of Elution Buffer 2, comprising 20mM sodium citrate + 1.OM
sodium
chloride, pH 8Ø This step resulted in the elution of greater than 45% of the
Factor VIII
activity present in plasma applied to the column. This step also results in
the elution of
85% of the Factor IX activity present in the plasma applied to the column.
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34
Step6. Following elution of the column as described in Step 5 the column was
regenerated with 1 column volume (20mL) of 1 M sodium hydroxide and re-
equilibrated
with 2.0 column volumes (40mL) of 20 mM sodium citrate pH 6Ø
Determination of Factor VIII activity:
Relative Factor VIII activities were determined in the raw plasma and EBA
protein
fractions using a Factor VIII activity kit from Coamatic (cat no 822585-63). A
standard
curve was produced using the raw plasma undiluted (100 % activity) and diluted
to 80 %,
60 %, 40 % 20 % and 0 % (blank) of the initial activity (not shown). Fractions
from run-
through, wash and eluate (Elution 2) from the column were analysed and the
activity yield
in the eluate was determined by reference to the standard curve (see table 7).
From table
7 it can be seen that the solid separation medium binds and elutes 45 % of the
Factor VIII
activity under the conditions applied.
TABLE 7: Factor VIII, fraction volumes and activity yield
Volume Relative Factor VIII Activity
activity yield
Raw plasma 300 ml 100 % 100 %
Run-through 480 ml 13 % 21 %
Wash 128m1 0% 0%
Eluate (Elution 2) 120 ml 113 % 45 %
Determination of Factor IX antigen:
Relative Factor IX antigen concentrations were detennined in the raw plasma
and
EBA protein fractions by a Factor IX sandwich ELISA using commercial
antibodies. A
standard curve was produced using the raw plasma undiluted (100 %) and diluted
to 80
%, 60 %, 40 % 20 % and 0 % (blank) of the initial concentration (not shown).
Fractions
from run-through, wash and eluate (Elution 2) from the column were analysed
and the
antigen yield in the eluate. (Elution 2) was determined by reference to the
standard curve
(see Table 8). From table 8, it can be seen that the solid separation medium
binds and
elutes 85 % of the Factor IX antigen under the conditions applied.
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TABLE 8: Factor IX, fraction volumes and antigen yield
Volume Relative Factor IX antigen concentration Yield
Raw plasma 300 ml 100% 100%
Run-through 480 ml 0% 0%
Wash 128 m1 0% 0%
Eluate (Elution 2) 120 ml 213 % 85 %
5
Selectivity
Figure 8 illustrates by SDS-PAGE that the eluate (Elution 2) comprise a very
small
proportion of the applied protein. When diluted for direct comparison to the
raw plasma
10 only very faint bands of IgG, albumin and other proteins are visible
indicating that the
loss of these proteins to the FVIIUFIX protein fraction will be insignificant.
Also the run-
through fraction is diluted for direct compari son with the raw plasma and no
significant
loss of protein is observed. . RID showed full recovery of a-1-PI and
fibrinogen in the
run-thrdugh fractions as well. (not shown).
Example 11 - Compatibility of the process of the second aspect with the
process of the first aspect
The run-through fraction from Example 10 was used as. a raw material in the
process of the first aspect in order to determine whether the prior removal of
Factor VIII
and Factor IX exerted any negative influence on the isolation process of the
first aspect.
Step 1. Solid separation medium: Agarose - tungsten carbide beads
functionalised
with 2-mercaptonicotinic acid. The agarose-tungsten carbide beads have a size
distribution of between 40-120 micron with a mean diameter'of 70 micron. The
density
of the beads were 2.9 g/ml (FastLine UFC NNSDW cat. No.: CS48, * UpFront
Chromatography A/S, Copenhagen, Denmark.). The beads are placed in an EBA
column
(FastLine 20, UpFront Chromatography A/S, Copenhagen, Denmark) (2cm diameter;
50
cm settled bed height; Settled bed Volume = 157mL). Equilibration is performed
at a
temperature of 25 C with 2.5 column volumes (392.5mL) of 40 mM sodium citrate
with a
3o pH 4.5, followed by a further 2.5 column volumes (392.5mL) of 40 mM sodium
citrate
and pH 5.0 at a linear flow rate of 7.5 cm/min.
CA 02569821 2006-12-07
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36
Step 2. Onto this column was loaded the run-through from the column described
in
Example 10 adjusted to 1 part of raw plasma + 2 parts of water. The pH of the
diluted
plasma solution was adjusted to pH 5.0 with 1 M HCI prior to application to
the column
and the load ratio was 1.51itres of plasma solution per litre of resin.
Step 3. Following loading of the diluted plasma solution, the column was
eluted
with 3.3 column volumes (518mL) of Elution Buffer I comprising 10mM sodium
citrate
pH 5Ø This resulted in the removal of non-bound protein, lipid and other
substances
including the a-1-proteinase Inhibitor present in the diluted plasma solution
applied to the
column.
Step 4. Following Step 3 above, the column was eluted with 2.6 column volumes
(408mL) of Elution Buffer 2, comprising 5 g/L sodium caprylate/HCI, pH 6Ø
Step 4
resulted in the elution of the albumin present in the diluted plasma solution
applied to the
column.
Step 4a. Following elution of the column as described in Step 4 above the
column
was washed with 1.0 column volume (1 57mL) of 1 M sodium citrate pH 8Ø
Step 5. Following the washing of the column as described in Step 4a above the
column was eluted with 4.9 column volumes (769mL) of Elution Buffer 3,
comprising
0.3M sodium citrate pH 7.4. This step resulted in the elution of the
immunoglobulins
present in the diluted plasma solution applied to the column.
Step 6. Following the elution of the column as described in Step 5 above, the
column was eluted with Elution Buffer 4, comprising of 2.6 colunm volumes
(408mL) of
20mM sodium citrate comprising 0.1 M sodium chloride at pH 7.4. This step
resulted in
the elution of the fibrinogen present in the diluted plasma solution applied
to the column.
Step 7: Following elution of the column as described in Step 6 the column was
regenerated with I column volume (157mL) of 1M sodium hydroxide and re-
equilibrated
with 2.0 coliumn volumes (314mL) of 40 mM sodium citrate pH 4.5.
As can be seen from Figure 7, the qualitative pattern obtained with the Factor
VIII/Factor IX depleted plasma is identical to the pattern obtained in Example
4. The
process volumes obtained for each fraction were also equal to those described
in Example
4.
Figure 8A-B illustrates the quantitative analysis of albumin and IgG in the
different
protein fractions, and as can be seen practically all of the albumin is
recovered in elution
2, and practically all of the IgG is recovered in elution 3.. Albumin and IgG
are barely
detectable in the other fractions.
CA 02569821 2006-12-07
WO 2005/121165 PCT/AU2005/000796
a-l-proteinase inhibitor and fibrinogen were also found to behave in the same
way
as described in the process of the first aspect.
The results demonstrate that p-xylylenediamine and m-xylylenediamine are
capable
of selectively extracting Factor VIII and Factor IX from undiluted plasma in
an efficient
manner without significantly reducing the levels of other plasma proteins,
which may be
isolated in a subsequent process(es), such as the process of the first aspect.
The Factor
VIII/Factor IX isolation process is compatible with the
albumin/IgG/Fibrinogen/a-1-
proteinase inhibitor isolation process of the first aspect.
