Note: Descriptions are shown in the official language in which they were submitted.
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A METHOD OF PURIFYING PROTEINS
TECHNICAL FIELD
[0001] The present invention relates generally to a method of purifying
proteins. More
specifically, the present invention relates to a method of purifying
haptoglobin and
hemopexin from the same starting material, and uses thereof.
BACKGROUND
[0002] Haemolysis is characterized by the destruction of red blood cells
and is a hall-
mark of anaemic disorders associated with red blood cell abnormalities, such
as enzyme
defects, haemoglobinopathies, hereditary spherocytosis, paroxysmal nocturnal
haemoglobinuria and spur cell anaemia, as well as extrinsic factors such as
splenomegaly,
autoimmune disorders (e.g., Hemolytic disease of the newborn), genetic
disorders (e.g.,
Sickle-cell disease or G6PD deficiency), microangiopathic haemolysis, Gram-
positive
bacterial infection (e.g., Streptococcus, Enterococcus and Staphylococcus),
parasite
infection (e.g., Plasmodium), toxins and trauma (e.g., burns). Haemolysis is
also a
common disorder of blood transfusions, particularly massive blood transfusions
and in
patients using an extracorporeal cardio-pulmonary support.
[0003] The adverse effects seen in patients with conditions associated with
haemolysis
are largely attributed to the release of iron and iron-containing compounds,
such as
haemoglobin (Hb) and heme, from red blood cells. Under physiological
conditions,
released haemoglobin is bound by soluble proteins such as haptoglobin and
transported to
macrophages and hepatocytes. However, where the incidence of haemolysis is
accelerated
and becomes pathological in nature, the buffering capacity of haptoglobin is
overwhelmed.
As a result, haemoglobin is quickly oxidised to ferri-haemoglobin, which in
turn releases
free heme (comprising protoporphyrin IX and iron). Whilst heme plays a
critical role in
several biological processes (e.g., as part of essential proteins such as
haemoglobin and
myoglobin), free heme is highly toxic. Free heme is a source of redox-active
iron, which
produces highly toxic reactive oxygen species (ROS) that damages lipid
membranes,
proteins and nucleic acids. Heine toxicity is further exacerbated by its
ability to intercalate
into lipid membranes, where it causes oxidation of membrane components and
promotes
cell lysis and death.
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[0004] The evolutionary pressure of continuous low-level extracellular
Hb/heme
exposure has led to compensatory mechanisms that control the adverse effects
of free
Hb/heme under physiological steady-state conditions and during mild
haemolysis. These
systems include the release of a group of plasma proteins that bind Hb or
heme, including
the Hb scavenger haptoglobin (Hp) and the heme scavenger proteins hemopexin
(Hx) and
al -microglobulin. However, whilst endogenous Hp and Hx control the adverse
effects of
free Hb/heme under physiological steady-state conditions, they have little
effect in
maintaining steady-state Hb/heme levels under pathophysiological conditions,
such as
those associated with haemolysis.
[0005] The present invention provides a method of purifying Hp and Hx from
the same
starting material. The purified proteins can be used in compositions for
treating conditions
associated with haemolysis and aberrant Hb/heme levels.
SUMMARY OF THE INVENTION
[0006] In an aspect of the present invention, there is provided a method of
purifying
haptoglobin and hemopexin from a solution containing both proteins, the method
comprising:
(i) providing a solution containing both haptoglobin and hemopexin;
(ii) precipitating the haptoglobin from the solution by adding ammonium
sulphate to the solution;
(iii) separating the precipitated haptoglobin from the solution containing
hemopexin; and
(iv) separately purifying the haptoglobin and/or hemopexin in one or more
steps.
[0007] In another aspect of the present invention, there is provided a
composition
comprising the haptoglobin recovered by the methods disclosed herein.
[0008] In another aspect of the present invention, there is provided a
composition
comprising the hemopexin recovered by the methods disclosed herein.
[0009] In another aspect of the present invention, there is provided a
composition
comprising the transferrin recovered by the methods disclosed herein.
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[0010] In another aspect of the present invention, there is provided a
composition
comprising the haptoglobin recovered by the methods disclosed herein and the
hemopexin
recovered by the methods disclosed herein.
[0011] In another aspect of the present invention, there is provided a
composition
comprising a haptoglobin content of at least 95% of total protein. In another
aspect of the
present invention, there is provided a composition comprising a hemopexin
content of at
least 80% of total protein. In another aspect of the present invention, there
is provided a
composition comprising a combined hemopexin and haptoglobin content of at
least 80% of
total protein.
[0012] In another aspect of the present invention, there is provided a
formulation
comprising the composition of the present invention, as disclosed herein, and
a
pharmaceutically acceptable carrier.
[0013] In another aspect of the present invention, there is provided a
method of
treating a condition associated with haemolysis, the method comprising
administering to a
subject in need thereof the composition or the formulation of the present
invention, as
disclosed herein.
[0014] In another aspect of the present invention, there is provided use of
the
compositions or formulations of the present invention, as disclosed herein, in
the
manufacture of a medicament for treating a condition associated with
haemolysis.
BRIEF DESCRIPTION OF THE FIGURES
[0015] Figure 1 is a flow diagram of a Cohn Fractionation Process. The
skilled person
will recognise that variations to the process parameters (e.g. pH, ethanol
concentration,
temperature, etc.) described in Figure I can also be employed to generate Cohn
fractions.
[0016] Figure 2 shows the recovery of transferrin (TRF), albumin (Alb),
hemopexin
(HPX) and haptoglobin (HAP) in the remaining filtrate following precipitation
in the
presence of 2.0M, 2.5M, 3.0M, 3.5M and 4.0M ammonium sulfate.
[0017] Figure 3 shows a desirability plot of the design of experiment
(DOE), showing
the desirable conditions that would precipitate the haptoglobin from the
plasma fraction,
while keeping the hemopexin in solution. A DOE is a set of controlled
experimentation
that was used to evaluate the impact of pH and ammonium sulfate concentration
on the
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ability to separate hemopexin from haptoglobin. A factorial mathematical
design was used
to design and analyze the data. The desirability plot depicted in Figure 3
uses this
mathematical design to determine the most desirable conditions that would
result in the
best separation and recovery of both hemopexin and haptoglobin.
[0018] Figure 4 shows SDS-PAGE electrophoresis of hemopexin recovered
following
the various steps in the purification process disclosed herein. SDS-PAGE
analysis was
performed using pre-cast 10% Tris-Glycine gels (Novex, EC6075). All samples
were
diluted to a concentration of 0.1 mg/mL in Tris-Glycine SDS sample buffer
(LC2676) and
20u1 of each sample was loaded into the sample well of the gel. Run time and
voltage
were set to the gel manufacturer's recommendations (125V constant). Each gel
was
stained with an easy to use type of Coomassie Brilliant Blue stain solution
(Novex, Simply
Blue Safestain, LC6065). Lane 1 (LMWS) contains Novex Sharp Unstained Protein
Standards, LC5801.
[0019] Figure 5 shows an SDS-PAGE electrophoresis (using the method
described
above in Figure 4) of haptoglobin intermediates recovered following the
various steps in
the purification process disclosed herein. The haptoglobin standard in lane 2
is from
Benesis (T043HPX).
[0020] Figure 6 shows a Western Blot of the Capto Q Eluate on a 10% Tris-
Glycine
Non-Reduced SDS-PAGE electrophoresis gel (using the method described in Figure
4).
The separated proteins are then transferred to a nitrocellulose membrane and
the
membrane is blocked to prevent any non-specific binding of antibody. The
nitrocellulose
membrane is then incubated with a solution containing antibodies to Human
Haptoglobin
(rabbit anti-human haptoglobin, Sigma H8636). A secondary antibody linked to
horseradish peroxidase (HRP) is then incubated with the nitrocellulose
membrane (goat
anti-rabbit HRP, Sigma A6154). The nitrocellulose is then developed with a
solution
containing peroxide thereby only visualizing the protein bands that
specifically contain
human haptoglobin. The lanes contain Capto Q ImpRes Eluate (T0209001) at
different
concentrations (Lane 2 - 1:10 dilution, 0.1 mg/mL, 204 loaded; lane 3 ¨ 1:20;
lane 4 -
1:40; lane 5¨ 1:80; lane 6¨ 1:160; lane 7 ¨ 1:320; lane 8¨ 1:640; lane 9
1:1280; and lane
- 1:2560). The Western blot indicates that most of the bands present in the
Capto Q
Eluate are haptoglobin.
[0021] Figure 7 shows SDS-PAGE electrophoresis (using the method described
in
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Figure 4) of transferrin recovered from ion-exchange chromatography (Capto
DEAE)
following the various steps in the purification process disclosed herein. Peak
one (lane 4)
is heavily loaded, but appears to be pure transferrin (compare to lane 2,
human transferrin,
Sigma T8158). This indicates that it is possible to purify transferrin from
the Octyl
Sepharose Wash fraction, which also means that it is possible to purify
hemopexin,
haptoglobin, and transferrin from the same starting material.
[0022] Figure 8 shows a chromatogram of the transferrin linear gradient
from the ion-
exchange chromatographic column during the purification process disclosed
herein. Peaks
labelled 1 to 4 were analysed by SDS-PAGE in Figure 7.
[0023] Figure 9 is a flow diagram of a hemopexin purification process in
accordance
with an embodiment disclosed herein.
[0024] Figure 10 is a flow diagram of a haptoglobin purification process in
accordance
with an embodiment disclosed herein.
[0025] Figure 11 is a flow diagram of a combined
haptoglobin/hemopexin/transferrin
purification process in accordance with an embodiment disclosed herein.
DETAILED DESCRIPTION
[0026] 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 or integer or group of elements or integers
but not the
exclusion of any other element or integer or group of elements or integers.
[0027] The reference in this specification to any prior publication (or
information derived
from it), or to any matter which is known, is not, and should not be taken as
an
acknowledgment or admission or any form of suggestion that that prior
publication (or
information derived from it) or known matter forms part of the common general
knowledge in the field of endeavour to which this specification relates.
[0028] It must be noted that, as used in the subject specification, the
singular forms "a",
"an" and "the" include plural aspects unless the context clearly dictates
otherwise. Thus,
for example, reference to "a resin" includes a single resin, as well as two or
more resins;
reference to "the composition" includes a single composition, as well as two
or more
compositions; and so forth.
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[0029] In the absence of any indication to the contrary, reference made to a
"%" content
throughout this specification is to be taken as meaning % w/w (weight/weight).
For
example, a solution comprising a haptoglobin content of at least 95% of total
protein is
taken to mean a composition comprising a haptoglobin content of at least 95%
w/w of total
protein.
[0030] The present invention is predicated, at least in part, on the finding
that haptoglobin
and hemopexin can be purified from the same starting material. Thus, in an
aspect of the
present invention, there is provided a method of purifying haptoglobin and
hemopexin
from a solution containing both proteins, the method comprising:
(i) providing a solution containing both haptoglobin and hemopexin;
(ii) precipitating the haptoglobin from the solution by adding ammonium
sulphate to the solution;
(iii) separating the precipitated haptoglobin from the solution containing
hemopexin; and
(iv) separately purifying the haptoglobin and/or hemopexin in one or more
steps.
[0031] Haptoglobin (Hp) is a tetrachain (a2132) glycoprotein synthesized by
the adult
liver and secreted into the plasma. The propeptide form of Hp is
proteolytically cleaved
into an a-chain and a 13-chain. Two a-subunits and two 13-subunits of Hp
protein are then
joined by inter-chain disulfide bonds to form the mature peptide, which can be
either an
(4)-dimer or an (4)-multimer. Hemopexin (Hx) is a 60-kD plasma 13-IB-
glycoprotein
comprising a single 439 amino acid long peptide chain, which forms two domains
joined
by an inter-domain linker. It has the highest known affinity for heme (Kd<1
pM) of any
characterized heme-binding protein and binds heme in an equimolar ratio
between the two
domains of Hx in a pocket formed by the inter-domain linker.
[0032] The inventors have found that an ammonium sulfate concentration in
the range
of about 2.0M to about 2.5M, preferable in the range of about 2.2M to about
2.5M, more
preferably about 2.4M, is optimal for separating both proteins from the same
starting
material. Thus, in an embodiment, the method comprises precipitating the
haptoglobin
from the solution by adding about 2.0M to about 2.5M ammonium sulphate to the
solution.
In another embodiment, the method comprises precipitating the haptoglobin from
the
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solution by adding about 2.2M to about 2.5M ammonium sulphate to the solution.
In yet
another embodiment, the method comprises precipitating the haptoglobin from
the solution
by adding about 2.4M ammonium sulphate to the solution. In particular
embodiments the
ammonium sulphate concentration is 2.0M or 2.1M or 2.2M or 2.3 M or 2.4M or
2.5M.
[0033] The inventors have also shown that a pH maintained in the range of
less than or
equal to about 8, preferably about 6 to about 8, more preferably at about 7,
is optimal for
separating both proteins from the same starting material. Thus, in an
embodiment, the
method comprises precipitating the haptoglobin from the solution at a pH of
less than or
equal to 8. In another embodiment, the method comprises precipitating the
haptoglobin
from the solution at a pH within the range of about 6 to about 8. In yet
another
embodiment, the method comprises precipitating the haptoglobin from the
solution at a pH
of about 7. In particular embodiments the method comprises precipitating the
haptoglobin
from the solution at a pH in the range of 6.0 to 8.0, or 6.25 to 7.75, or 6.5
to 8.0, or 6.5 to
7.75, or 6.5 to 7.5, or 6.75 to 7.25, or 6.75 to 7.5. The pH is typically
measured in the
haptoglobin and hemopexin solution and then during the addition of the
ammonium
sulphate and the precipitation of the haptoglobin. If required the pH can be
adjusted
(typically the concentration of the acid (e.g. HCI) or base (e.g. NaOH) used
to adjust the
pH is in the range of 0.05M to 0.6M).
[0034] In the methods disclosed herein, the majority of haptoglobin from
the starting
material will be found within the ammonium sulfate precipitate and the
majority of the
hemopexin from the starting material will be found in the remaining solution
(also referred
to as the suspension). However, persons skilled in the art will understand
that the
precipitate may comprise some hemopexin (e.g., trace amounts of Hx) and that
the
remaining solution (or suspension) may comprise some haptoglobin (e.g., trace
amounts of
Hp). Where trace amounts of Hp and Hx are present in the suspension and
precipitate,
respectively, it may be desirable to remove these by separately purifying the
haptoglobin
and/or hemopexin in one or more steps in accordance with the methods disclosed
herein.
However, persons skilled in the art would understand that trace amounts of Hp
and Hx that
may be present in the suspension and precipitate, respectively, may be
acceptable, for
example, where both proteins will end up in the same composition.
[0035] Any solution comprising both haptoglobin and hemopexin can be used
as the
starting material in the method of the present invention, disclosed herein. In
particular
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embodiments the solution containing both haptoglobin and hemopexin further
comprises
transferrin. Suitable starting material would be known to persons skilled in
the art,
examples of which include plasma fractions such as various supernatants and
precipitates
derived from ethanol fractionation processes. Examples of such ethanol
fractionation
processes include Cohn fractionation and Kistler-Nitschmann fractionation.
Examples of
suitable plasma fractions include those derived from a Cohn fraction I, II,
III, II+III,
I+II+III, IV and V (See Figure 1) or a Kistler-Nitschmann fraction such as a
Precipitate A
or B. In an embodiment, the solution is a human plasma fraction. In another
embodiment,
the solution is a Cohn Fraction IV. In yet another embodiment, the solution is
a Cohn
Fraction IV4. In particular embodiments the Cohn Fraction IV4 is derived from
a Cohn
Fraction II+III or a Cohn Fraction IA1+111. In a particularly preferred
embodiment the
solution is derived from a Fraction IV4 Precipitate.
[0036] It will be understood that, where the starting material is provided
as a
precipitate (e.g., Fraction IV4 Precipitate), it will be necessary to
initially resolublise the
precipitate to provide a suitable starting solution for the methods of the
present invention.
The buffering agent used to resolublise the precipitate can be any agent or
combination of
agents that has a buffering capacity at around pH 7 (examples can include ADA,
PIPES,
ACES, MOPSO, MOPS, BES, TRIS). Typically the buffering agent will be at a
concentration from about 5 mM to about 100mM. In an embodiment the
resolublization
buffer is 50mM Tris, pH 7. In some embodiments the resolublisation buffer can
be added
at about 5 to about 30 grams per gram of the starting material. In particular
embodiments
the resolubilisation buffer is added at 5 to 10 grams per gram of starting
material, or added
at 10 to 15 grams per gram of starting material, or added at 15 to 20 grams
per gram of
starting material, or added at 20 to 25 grams per gram of starting material,
or added at 25
to 30 grams per gram of starting material.
[0037] The methods of the present invention are suitable for the
commercial/industrial
scale purification of hemopexin, haptoglobin and, optionally, transferrin. For
example,
when using plasma fractions as a starting material, employing the method of
the present
invention on a commercial/industrial scale may involve the use of a plasma
fraction
derived from at least about 500 kg of plasma. More preferably, the plasma
fraction will be
derived from at least about 5,000 kg, 7,500 kg, 10,000 kg and/or 15,000 kg of
plasma per
batch.
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[0038] The skilled person will understand that plasma for fractionation is
the liquid
part of blood remaining after separation of the cellular elements from blood
collected in a
receptacle containing an anticoagulant, or separated by any other suitable
means known to
persons skilled in the art, such as by continuous filtration or centrifugation
of
anticoagulated blood in an apheresis procedure.
[0039] In an embodiment, the precipitated haptoglobin and the solution
containing
hemopexin are recovered and stored separately before separately purifying the
haptoglobin
and/or hemopexin in one or more steps, in accordance with the present
invention. In
another embodiment, the precipitated haptoglobin and/or the solution
containing
hemopexin are recovered and subjected immediately to further purification
steps in
accordance with the methods of the present invention; that is, separately
purifying the
haptoglobin and/or hemopexin in one or more steps.
[0040] Thus, in an embodiment, the method further comprises:
(i) dissolving the precipitated haptoglobin in a buffer to obtain a
haptoglobin solution;
(ii) passing the haptoglobin solution through a anion exchange
chromatographic resin under conditions such that the haptoglobin binds
to the resin;
(iii) eluting the haptoglobin from the resin; and
(iv) recovering the eluted haptoglobin.
[0041] In particular embodiments the anion exchange chromatographic resin
of step
(ii) is a strong anion exchange chromatographic resin. In other embodiments
the anion
exchange chromatographic resin of step (ii) is a weak anion exchange
chromatographic
resin.
[0042] Purification of proteins by chromatography can be performed using
either axial
flow columns, such as those available from GE Healthcare, Pall Corporation,
Millipore and
Bio-Rad, or using radial flow columns, such as those available from Proxcys or
Sepragen.
Chromatography can also be conducted using expanded bed technologies known to
persons skilled in the art.
[0043] Most chromatographic processes employ a solid support, also referred
to
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interchangeably herein as a resin or matrix. Suitable solid supports would be
familiar to
persons skilled in the art and the choice will depend on the type of product
to be purified.
Examples of suitable solid supports include inorganic carriers, such as glass
and silica gel,
organic, synthetic or naturally occurring carriers, such as agarose,
cellulose, dextran,
polyamide, polyacrylamides, vinyl copolymers of bifunctional acrylates, and
various
hydroxylated monomers, and the like. Commercially available carriers are sold
under the
names of SephadexTM, SepharoseTM, HypercelTM, CaptoTM, FractogelTM,
MacroPrepTM,
UnosphereTM, GigaCapTM, TrisacrylTm, UltrogelTM, DynospheresTM, MacrosorbTM
and
XADTM resins.
[0044] The chromatography steps will generally be carried out under non-
denaturing
conditions and at convenient temperatures in the range of about -10 C to +30
C, more
usually at about ambient temperatures. The chromatographic steps may be
performed
batch-wise or continuously, as convenient. Any convenient method of separation
may be
employed, such as column, centrifugation, filtration, decanting, or the like.
[0045] Buffers that are suitable for dissolving the haptoglobin precipitate
would be
familiar to persons skilled in the art and may depend on the conditions
required for
performing the chromatographic purification step. Typically the buffer will
have a
concentration of the buffering agent (i.e. Tris) from about 5 mM to about 100
mM. In
particular embodiments the buffering agent is from about 10 mM to about 60 mM.
The
skilled person will also recognise that the buffer may comprise more than one
buffering
agent. Examples of suitable buffers are sodium acetate and Tris with a pH
range of 5.5 to
9Ø Particular embodiments utilize a pH of 7.5 to 9Ø In some embodiments
the pH of
the buffer used to dissolve the haptoglobin precipitate is from pH 7.5 to 9.0,
or pH 7.75 to
9.0, or pH 8.0 to 9.0, or pH 8.25 to 9.0 or pH 8.4 to 8.6. In a preferred
embodiment the
buffer is about 50mM Tris at a pH of about pH 8.4 to about pH 8.6.
[0046] In embodiments the lipid content of the extracted precipitate
comprising
haptoglobin (i.e. the haptoglobin solution) is reduced by exposure to a lipid
removal agent
under conditions that allow the lipid to bind to the lipid removal agent.
Examples of such
lipid removal agents include fumed silica such as Aerosil. In an embodiment
the lipid
removal agent is a fumed silica. In an embodiment the fumed silica is an
Aerosil (e.g.
Aerosil 380). In embodiments the lipid removal agent such as Aerosil can be
added to the
extracted precipitate comprising haptoglobin at about 0.5 g to about 4 g per
liter of plasma
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equivalent. In particular embodiments the lipid removal agent such as Aerosil
is added at
1 to 2 g per liter plasma equivalent to the extracted precipitate comprising
haptoglobin. In
another embodiment the lipid removal agent such as fumed silica (e.g. Aerosil)
is added at
1.6 to 1.8 g per liter plasma equivalent to the extracted precipitate
comprising haptoglobin.
In another embodiment the lipid removal agent such as Aerosil is added at 1.8
to 2.0 g per
liter of plasma equivalent to the extracted precipitate comprising
haptoglobin. In another
embodiment the lipid removal agent such as Aerosil is added at 1.6 g per liter
of plasma
equivalent to the extracted precipitate comprising haptoglobin. It was
determined that lipid
removal is most effective within a specific pH range. A pH range of 5.5 to 9.0
was found
to be effective in conjunction with Aerosil. In some embodiments the pH range
is 6.5 to
8.6. In other embodiments the pH during lipid removal step is from pH 5.5 to
9.0, or pH
5.75 to 9.0, or pH 6.0 to 9.0, or pH 6.25 to 9.0 or pH 6.5 to 9.0, or pH 6.75
to 9.0, or pH
7.0 to 9.0, or pH 7.25 to 9.0, or pH 7.5 to 9.0, or pH 7.75 to 9.0, or pH 8.0
to 9.0, or pH
8.25 to 9.0, or pH 8.4 to 9.0 or pH 8.6 to 9Ø The preferred embodiment
utilizes a pH
range of 8.4 to 8.6.
[0047] The lipid
removal agent can be removed using methods such as filtration and or
centrifugation. In particular embodiments the lipid removal agent is removed
by depth
filtration. An example of a depth filter for use in this application is a Cuno
70CA filter or
one of similar or smaller particle size retention capabilities.
[0048] Persons
skilled in the art will understand that any anion exchange
chromatographic resin can be used to separately purify haptoglobin from the
haptoglobin
solution, as long as the haptoglobin is capable of binding to the
chromatographic resin
while allowing some impurities in the solution to pass though the resin. In
particular
embodiments the anion exchange chromatographic resin is a strong anion
exchange resin.
In other embodiments the anion exchange chromatographic resin is a weak anion
exchange
resin. Persons skilled in the art would also determine that due to the ionic
strength of the
extraction buffer and subsequent pH adjustment of the load solution; dilution,
diafiltration,
chromatographic desalting, or other methods of buffer exchange / ionic
strength reduction
would be required to allow haptoglobin to bind to the resin. Suitable resins
would be
known to persons skilled in the art. Examples of suitable anion exchange
resins are ones
comprising a functional quaternary amine group (Q) and/or a diethylaminopropyl
group
(ANX). In an embodiment, the strong anion exchange chromatographic resin
comprises a
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functional quaternary amine group (e.g., Capto Q ImpResTm).
[0049] Solutions
that are suitable for the equilibration (often referred to as an
equilibration buffer) of the chromatography media usually have a concentration
of a
buffering agent of about 5mM to about 100 mM. The pH of the equilibration
buffer will
normally be in the range of about 5 to about 9 and the conductivity is
typically less than
about 9 mS/cm. These conditions generally allow for the binding of haptoglobin
to the
anion exchange media. In particular embodiments the buffering agent will be in
the
concentration range from about 10 mM to about 60 mM. The pH of the
equilibration
buffer in particular embodiments is in a pH range from about 5.0 to about 9Ø
In some
embodiments the pH is in the range from about 5.0 to about 7.0, or from about
5.0 to about
6.0, or from about 5.3 to about 5.7. In particular embodiments the pH of the
equilibration
buffer is pH 5.3 0.1, or pH 5.4 0.1, or pH 5.5 0.1, or pH 5.6 0.1, or
pH 5.7 0.1, or
pH 5.8 0.1. The conductivity of the equilibration buffer in some embodiments
is less
than about 7.0 mS/cm. In particular embodiments the conductivity of the
equilibration
buffer is less than 6.0 mS/cm or less than 5.0 mS/cm. An example of a
buffering agent for
an equilibration buffer is sodium acetate. Particular embodiments utilize 50mM
sodium
acetate at a pH in the range 5.0 to 6.0, with a conductivity of less than 6.0
mS/cm. Other
embodiments utilize about 10 to about 40 mM sodium acetate at a pH of about 5
to about
6. In a particular embodiment the equilibration buffer comprises 50mM sodium
acetate
with a pH in the range of pH 5.3 to pH 5.7, and a conductivity of less than
5.0 mS/cm.
[0050] The haptoglobin solution to be passed across the anion exchange
chromatographic resin may initially require buffer exchange, desalting or
dilution to
reduce the ionic strength in order to enable binding to the resin. Suitable
solutions for
buffer exchange, desalting or dilution include those having a buffering agent
at a
concentration of about 5mM to about 100 mM. The pH range of these solutions is
typically in the range from about pH 5 to about 9. Whilst the conductivity is
usually less
than about 8.0 mS/cm. In particular embodiments the solution for buffer
exchange,
desalting or dilution will include a buffering agent in the range of about 10
mM to about 60
mM, with a pH range of about 5 to about 9 and a conductivity of less than
about 7.0
mS/cm. An example of a solution for buffer exchange, desalting or dilution is
sodium
acetate. Particular embodiments utilize 50mM sodium acetate at a pH range 5.0
to 6.0,
with a conductivity of less than 6.0 mS/cm. Other embodiments utilize about 10
to about
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40 mM sodium acetate at a of about 5 to about 6.
[0051] Other embodiments include dilution of the haptoglobin load solution
by
addition of water (such as water for injection (WFI)) to reduce ionic
strength. For example
a 1:4 to 1:5 dilution with WFI, the haptoglobin solution comprising 50mM
sodium acetate
at pH 5.3-5.7 would have a final sodium acetate concentration of about 10-
13mM, 5.3
to 5.7, and conductivity of less than 5.0 mS/cm. This generally allows for
binding of the
haptoglobin to the anion exchange chromatographic resin.
[0052] If pH adjustment beyond the buffering range of the buffering agent/s
in the
haptoglobin solution is/are required, then an additional buffering agent can
be added to the
haptoglobin solution. The additional buffering agent will typically have a
concentration of
about 5mM to about 100 mM, and a pH range of about 5 to about 9. In particular
embodiments the additional buffering agent will be in the range of about 10 mM
to about
60 mM, with a pH range of about 5 to about 9. An example of an additional
buffering
agent is sodium acetate. Particular embodiments utilize 50mM sodium acetate at
a pH
range 5.0 to 6Ø In a preferred embodiment the buffer is 50mM sodium acetate
at a pH in
the range of pH 5.3 to pH 5.7.
[0053] Buffers that are suitable for eluting the haptoglobin from the resin
will also be
known to persons skilled in the art. In particular embodiments the suitable
buffer agent
will have a concentration in the range of about 5 mM to about 100 mM. In
particular
embodiments the buffering agent will be in the range of about 10mM to about
60mM. An
example includes sodium acetate. Particular embodiments utilize 50mM sodium
acetate at
a pH of 5.0 to 6Ø Other embodiments utilize about 10 to about 40 mM sodium
acetate
buffers at a pH of about 5.0 to about 6Ø In a preferred embodiment the
buffer is about
50mM sodium acetate at a pH of about pH 5.3 to about pH 5.7.
[0054] In further embodiments, the haptoglobin is eluted from the anion
exchange
resin with an elution buffer comprising from about 100mM to about 200mM NaCl.
This
equates to an elution buffer having a conductivity range of about 10mS/cm
(100mM NaC1)
to about 18mS/cm (200mM NaCl). In particular embodiments the haptoglobin is
eluted in
the presence of about 150 to 170 mM NaCl. In a preferred embodiment the
haptoglobin is
eluted in the presence of about 160 mM NaCl. However, persons skilled in the
art would
know that the NaC1 concentration of the elution buffer will depend upon the
protein load
applied to the column and that adjustments beyond the described limits may be
required to
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achieve the necessary recovery and purity of the haptoglobin eluted from the
resin.
[0055] In an embodiment, the eluted haptoglobin is recovered and stored
separately for
future use. In another embodiment, the eluted haptoglobin is further purified,
for example,
by concentrating and diafiltering the eluted haptoglobin through an
ultrafiltration
membrane and/or sterile filtering the concentrated and/or diafiltering
haptoglobin, as
required.
[0056] In an embodiment, the method further comprises:
(i) passing the solution containing hemopexin through a hydrophobic
interaction chromatographic resin under conditions that allow the
hemopexin to bind to the resin;
(ii) collecting the flow-through fraction from step (i);
(iii) optionally washing the resin following step (ii) and collecting the
flow-
through wash fraction; and
(iv) eluting the hemopexin from the resin following step (ii) and/or
following step (iii); and
(v) recovering the eluted hemopexin.
[0057] Hydrophobic Interaction Chromatography (HIC) is a chromatographic
technique frequently used for the separation of proteins on the basis of a
hydrophobic
interaction between the stationary phase and the protein to be separated. The
level of
hydrophobicity of the target protein will often dictate the type of HIC resin
to be used.
During HIC, a high amount of salt is typically added to the solution to reduce
the solubility
of the target protein and thus increase the interaction of the target protein
with the HIC
resin functionalized with a suitable hydrophobic group (e.g., phenyl, butyl
and octadecyl
groups). Suitable salts typically include sodium sulfate, potassium sulfate,
ammonium
sulfate, ammonium chloride, sodium chloride, sodium bromide, and sodium
thiocyanate.
Suitable hydrophobic interaction chromatographic resins would be familiar to
persons
skilled in the art. Examples include octyl sepharose and capto octyl
chromatographic
resins. The conditions that allow the hemopexin to bind to the resin will be
known to
persons skilled in the art and will be dictated, for example, by the type of
resin used and
the hydrophobicity of the target protein (i.e., hemopexin).
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[0058] The hemopexin load solution contains ammonium sulfate, therefore
suitable
buffers for column equilibration include buffering agents, at a concentration
of about 5mM
to about 100mM, which would maintain a pH of about 6 to about 8 and contain
approximately 2 to 2.5 M ammonium sulfate. These conditions would allow for
the
binding of hemopexin to the hydrophobic interaction chromatography media.
Another
embodiment utilizes an ammonium sulfate concentration of 2.2 to 2.5M, pH 7.0
to 8Ø In
a particular embodiment, the buffer solution is 50mM Tris, containing 2.5M
ammonium
sulfate, at a pH of 7.4.
[0059] The hemopexin load solution typically contains about 2.0 to about
2.5M
ammonium sulfate buffered at pH of about 6 to about 8. These conditions allow
for the
binding of hemopexin to the hydrophobic interaction media. In another
embodiment, the
hemopexin load solution contained 2.2M to 2.5M ammonium sulfate, buffered at a
pH of
7.0 to 8Ø In a particular embodiment, the hemopexin load solution contains
50mM Tris,
as a buffering agent, and 2.5M ammonium sulfate at a pH of 7.4.
[0060] Once the solution containing hemopexin is passed through the
hydrophobic
interaction chromatographic (HIC) resin, the flow through fraction can be
collected and
stored for future use, as disclosed herein.
[0061] The bound hemopexin can be eluted from the resin by means known to
persons
skilled in the art. Prior to eluting the hemopexin from the resin, the resin
can optionally be
washed with a suitable wash solution or buffer under conditions that retain
the hemopexin
bound to the resin. Suitable wash solutions and conditions will be known to
persons skilled
in the art. Wash solution concentrations depend to a certain degree on column
load,
however typical wash solutions will possess a buffering effect at a pH of
about 6 to about 8
and additionally contain approximately 0.8M to 1.5M ammonium sulfate. In
another
embodiment, the wash solution contains 0.9M to 1.3M ammonium sulfate at a pH
of 7.0 to
8Ø In a particular embodiment, the wash solution contains 50mM Tris, as the
buffering
agent, 1.13M ammonium sulfate, at a pH of 7.4. The flow through wash fraction
can also
be collected and stored for future use, as necessary.
[0062] The eluted hemopexin that is recovered from the resin can be stored
for future
use. The eluted hemopexin may also be subjected to further purification to
remove any
impurities in the eluate. Thus, in an embodiment, the method further
comprises:
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(i) passing the eluted hemopexin through a metal ion affinity
chromatographic resin under conditions that allow the hemopexin to
bind to the resin; and
(ii) eluting the hemopexin from the resin; and
(iii) recovering the eluted hemopexin.
[0063] Immobilized
metal ion affinity chromatography (IMAC) is based on the
covalent attachment of amino acids (e.g., histidine) to metals, allowing
proteins with an
affinity for metal ions to be retained in a column containing immobilized
metal ions, such
as zinc, cobalt, nickel or copper. Suitable metal ion affinity chromatographic
resins would
be known to persons skilled in the art. In an embodiment, the metal ion
affinity
chromatographic resin is Ni-Sepharose.
[0064] Buffers
suitable for equilibration and binding are designed to prevent non-
specific binding to the chromatography media and optimized to promote affinity
for
hemopexin while minimizing binding of contaminate proteins. Buffers used
for
equilibration and binding would be generally within a pH range of about 6 to
about 9 and
would include the addition of about 0.5M to about 1.0M sodium chloride along
with the
addition of a small amount (i.e. 1 to 50mM) histidine or imidazole. In
certain
embodiments, the buffer would consist of 0.5mM to 100mM sodium phosphate, at
about
pH 7 to about 8, with about 0.5M to about 1.0M sodium chloride, and about 1mM
to about
50mM imidazole. In a particular embodiment, the equilibration and binding
buffer
consists of 20mM sodium phosphate, 0.5M NaCl, and 30mM imidazole, at pH 7.4.
The
load solution (Octyl Eluate) can be adjusted to these concentrations through
addition of the
desired solid excipients or by addition of a concentrated solution.
[0065] Once the
hemopexin is bound to the metal ion affinity chromatographic
(IMAC) resin, the resin may be washed to remove any residual unbound or weakly
bound
impurities under conditions that retain the hemopexin bound to the resin.
Buffers suitable
for the wash step would be within a pH range of 6.0 to 9.0 and would include
the addition
of 0.5M to 1.0M sodium chloride along with the addition of a small amount
(i.e. 1 to
50mM) histidine or imidazole. In some embodiments, the buffer would consist of
about
0.5mM to about 100mM sodium phosphate, at about pH 7 to about pH 8, with about
0.5M
to about 1.0M sodium chloride, and about 1mM to about 80mM imidazole. In a
particular
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embodiment, the wash buffer consists of 20mM sodium phosphate, 0.5M NaC1, and
30mM
imidazole, at pH 7.4.
[0066] The bound hemopexin can be eluted from the resin by means known to
persons
skilled in the art. Buffers suitable for elution would be within a pH range of
about 6 to
about 9 and would include the addition of about 0.5M to about 1.0M sodium
chloride
along with histidine or imidazole at a concentration high enough to facilitate
elution. In
some embodiments, the elution buffer would consist of about 0.5mM to about
100mM
sodium phosphate, at about pH 7.0 to about pH 8.0, with about 0.5M to 1.0M
sodium
chloride, and less than about 80mM imidazole. In a particular embodiment, the
elution
buffer consists of 20mM sodium phosphate, 0.5M NaCl, and 100mM imidazole, at
pH 7.4.
Alternatively, the bound hemopexin can be eluted through the application of a
low pH
buffer between about pH 4.0 and about pH 6Ø Some embodiments would utilize
about
5triM to about 100mM sodium acetate buffer, with about 0.5M to about 1.0M
NaCl, at a
pH between about 4.0 and about pH 6.0 as the eluting buffer. The eluted
hemopexin can
be further purified, for example, by concentrating and diafiltering the
hemopexin through
an ultrafiltration membrane and/or sterile filtering the concentrated and/or
diafiltering
hemopexin, as required.
[0067] The inventors have also found that any transferrin that may be
present in the
starting material remains in solution (i.e., in the solution comprising
hemopexin) following
the precipitation of haptoglobin in the presence of ammonium sulfate. Thus,
the methods
of the present invention, disclosed herein, can also be used to purify
transferrin from the
same starting material. Conditions are therefore provided that are optimal for
purifying all
three proteins (Hp, Hx and transferrin) from the same starting material. Thus,
in an
embodiment, the solution containing both haptoglobin and hemopexin (e.g., the
starting
material) will further comprise transferrin.
[0068] In an embodiment, the method further comprises:
(a) passing the solution containing hemopexin through a hydrophobic
interaction chromatographic resin under conditions that allow the
hemopexin to bind to the resin;
(b) collecting the flow-through fraction from step (a);
(c) optionally washing the resin following step (b) and collecting the flow-
through wash fraction;
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(d) passing the flow-through fraction from step (b) and/or the flow-through
wash fraction from step (c) through a anion exchange chromatographic
resin under conditions such that transferrin binds to the resin; and
(e) recovering the transferrin from the resin.
[0069] In particular embodiments the anion exchange chromatographic resin
of step
(ii) is a strong anion exchange chromatographic resin. In other particular
embodiments the
anion exchange chromatographic resin of step (ii) is a weak anion exchange
chromatographic resin.
[0070] Suitable strong anion exchange chromatographic resins will be known
to
persons skilled in the art. Examples of suitable anion exchange resins are
ones comprising
a functional quaternary amine group (Q) and/or a diethylaminopropyl group
(ANX). In an
embodiment, the strong anion exchange chromatographic resin comprises a
functional
quaternary amine group (e.g., Capto Q ImpResTm).
[0071] Suitable weak anion exchange chromatographic resins will be known to
persons
skilled in the art. Examples include resins comprising a tertiary or secondary
amine
functional group, such as DEAE (diethylaminoethyl).
[0072] Persons skilled in the art would also determine that due to the
ionic strength of
the HIC Wash fraction; dilution, diafiltration, chromatographic desalting, or
other methods
of buffer exchange / ionic strength reduction would be required to allow
transferrin to bind
to the anion exchange resin. One such method of chromatographic desalting
includes
binding the HIC Wash fraction (transferrin), which contains about 0.8M to
about 1 .5M
ammonium sulfate, directly onto a stronger hydrophobic ligand, such as a
phenyl or butyl
column. Once bound, the transferrin can be eluted in WFI or a low ionic
strength buffer in
preparation for anion exchange chromatography.
[0073] Buffers suitable for anion exchange chromatography equilibration and
binding
allow for the binding of transferrin to the anion exchange media to promote
chromatographic separation of transfen-in from other impurities. Buffers that
are suitable
for the equilibration of the chromatography media have a concentration of
about 5mM to
about 100 mM, a pH range of about 6 to about 9, with a conductivity of less
than about 9.0
mS/cm. These conditions allow for the binding of transferrin to the anion
exchange media.
In particular embodiments the buffering agent will be in the range of about 10
mM to
about 60 mM, with a pH range of about 6 to about 9, with a conductivity of
less than about
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7.0 mS/cm. Examples of suitable buffering agents include Tris and sodium
phosphate.
Particular embodiments utilize 50mM Tris at a pH range in the range from about
7 to about
9, with a conductivity of less than about 6.0 mS/cm. Other embodiments utilize
about 10
to about 40 mM Tris buffers at a pH of about 7.0 to about 9.0, with a
conductivity of less
than about 5.0 mS/cm.
[0074] Buffers that are suitable for eluting the transferrin from the resin
will also be
known to persons skilled in the art. In particular embodiments the suitable
buffer agent
will have a concentration in the range of about 5 mM to about 100 mM. In
particular
embodiments the buffering agent will be in the range of about 10mM to about
60mM.
Examples of suitable buffering agents include Tris or sodium phosphate.
Particular
embodiments utilize 50mM Tris at a pH in the range of about 7 to about 9.
Other
embodiments utilize about 10 to about 40 mM sodium acetate buffers at a pH of
about 7 to
about 9. Persons skilled in the art would know elution buffer NaC1
concentrations depend
to an extent upon the protein load applied to the chromatographic resin.
Methods of
elution would consist of a step gradient, in which the NaC1 concentration of
the buffer is
increased in a stepwise faction to elute off the transferrin, or by use of a
linear gradient
where the NaCI of the buffer is slowly increased in a linear fashion and the
column
effluent is monitored to ensure the collection of transferrin.
[0075] Once the transferrin is recovered from the anion exchange
chromatographic
resin, it can be further purified, for example, by concentrating and
diafiltering the
transferrin through an ultrafiltration membrane and/or sterile filtering the
concentrated
and/or diafiltering transferrin, as required.
[0076] Where a solution comprising haptoglobin and/or hemopexin and/or
transferrin
is to be used for clinical or veterinary applications (e.g., for
administration to a subject
with a condition associated with haemolysis), persons skilled in the art will
understand that
it may be desirable to reduce the level of active virus content (virus titre)
and other
potential infectious agents (for example prions) in the solution. This may be
particularly
desirable where the feedstock comprising haptoglobin and/or hemopexin and/or
transferrin
(i.e., the starting material) is derived from blood plasma. Methods of
reducing the virus
titre in a solution will be known to persons skilled in the art. Examples
include
pasteurization (for example, incubating the solution at 60 C for 10 hours in
the presence of
high concentrations of stabilisers such as glycine (e.g. 2.75M) and sucrose
(e.g. 50%)
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and/or other selected excipients or salts), dry heat treatment, virus
filtration (passing the
solution through a nano-filter; e.g., 20nm cut-off) and/or subjecting the
solution to
treatment with a suitable organic solvent and detergent for a period of time
and under
conditions to inactivate virus in the solution. Solvent detergent has been
used for over 20
years to inactivate enveloped viruses particularly in plasma-derived products.
Thus it may
be carried out using various reagents and methods known in the art (see, for
example, US
4540573 and US4764369). Suitable
solvents
include tri-n-butyl phosphate (TnBP) and ether, preferably TnBP (typically at
about 0.3%).
Suitable detergents include polysorbate (Tween) 80, polysorbate (Tween) 20 and
Triton X-
100 (typically at about 0.3%). The selection of treatment conditions including
solvent and
detergent concentrations depend in part on the characteristics of the
feedstock with less
pure feedstocks generally requiring higher concentrations of reagents and more
extreme
reaction conditions. A preferred detergent is polysorbate 80 and a
particularly preferred
combination is polysorbate 80 and TnBP. The feedstock may be stirred with
solvent and
detergent reagents at a temperature and for a time sufficient to inactivate
any enveloped
vii uses that may be present. For example, the solvent detergent treatment may
be carried
out for about 4 hours at 25 C. The solvent detergent chemicals are
subsequently removed
by for example adsorption on chromatographic media such as C-18 hydrophobic
resins or
eluting them in the drop-through fraction of ion exchange resins under
conditions which
adsorb the protein of interest.
[0077] The
virus inactivation step can be performed at any suitable stage of the
methods disclosed herein. In an embodiment, the feedstock comprising
haptoglobin and/or
hemopexin and/or transferrin is subject to a viral inactivation step prior to
step (ii) from the
first described aspect. In another embodiment, the solution comprising
haptoglobin and/or
hemopexin and/or transferrin that is recovered from the ammonium sulphate
precipitation
step (i.e., from steps (ii) and/or (iii)) is subject to a viral inactivation
step. In other
embodiments a viral inactivation step is conducted after step iii).
[0078] In an
embodiment disclosed herein, the viral inactivation step comprises
pasteurisation and/or treatment with an organic solvent and detergent. In an
embodiment,
the method of the invention further comprises heating a solution comprising
either
haptoglobin and/or hemopexin and/or transferrin at 55 C to 61 C for about 30
minutes to
about 12 hours. In a particular embodiment the solution is heated for about 10
to about
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10.5 hours. In another embodiment disclosed herein, the virus inactivation
step comprises
virus filtration. In a particular embodiment, the method of the invention
further comprises
filtering a solution comprising either hemopexin and/or transferrin through a
virus filter
having a pore size ranging from 15 rim to 35 nm. Where virus filtration is
used, the
inventors have found that the addition of a free amino acid (e.g., arginine)
prior to the
filtration step can significantly improve the flux rate and recovery of
haptoglobin and/or
hemopexin and/or transferrin through the filter. An example of such method is
described
in US7919592.
[0079] In an embodiment disclosed herein, the feedstock or solution
comprising
haptoglobin and/or hemopexin and/or transferrin is subject to a viral
inactivation step
before it is passed through a chromatographic resin. The advantage of
employing a virus
inactivation step such as solvent detergent treatment prior to passing the
treated solution or
feedstock through a chromatographic resin such as an anion exchange resin is
that it allows
for the removal of the organic solvent and detergent from the treated solution
by utilizing
conditions that promote binding of the haptoglobin and/or hemopexin and/or
transferrin to
the resin and removal of the organic solvent and detergent with the flow-
through (drop-
through) fraction.
[0080] Pasteurization can generate protein aggregates and polymers.
Therefore, it may
be desirable in some instances to reduce the level of aggregates/polymers in a
pasteurized
solution. This can be achieved by any means known to persons skilled in the
art, although
conveniently can be achieved by further chromatographic purification. In an
embodiment
disclosed herein, the pasteurized solution or feedstock is passed through an
anion exchange
chromatographic resin in positive mode with respect to the haptoglobin and/or
hemopexin
and/or transferrin such that any aggregates or polymers are removed with the
flow-through
(drop-through) fraction.
[0081] In particular embodiments, unless explicitly stated otherwise, the
methods of
purifying haptoglobin, and/or hemopexin and/or transferrin are conducted
generally in the
temperature range of about 18 C to about 26 C.
[0082] In another aspect of the present invention, there is provided a
composition
comprising the haptoglobin recovered by the methods disclosed herein. In an
embodiment,
the composition comprises a haptoglobin content of at least 80% of total
protein. In
another embodiment, the composition comprises a haptoglobin content of at
least 90% of
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total protein. In another embodiment, the composition comprises a haptoglobin
content of
at least 95%. In yet another embodiment, the composition comprises a
haptoglobin content
of at least 98%. In a particular embodiment the composition comprising
haptoglobin
comprises less than 0.03 mg of IgA per mg of haptoglobin as determined by
immunonephelometry. In particular embodiments the composition comprising
haptoglobin
when at a haptoglobin concentration of 26 mg/mL contains less than 0.067 mg/mL
IgG,
less than 0.042 mg/mL IgM, less than 0.050 mg/mL alpha- 1 -acid glycoprotein,
less than
0.018 mg/mL pre-albumin, less than 0.021 mg/mL ceruloplasmin, less than 0.051
mg/mL
hemopexin, less than 0.053 mg/mL apolipoprotein A-I, less than 0.227 mg/mL
apolipoprotein B, less than 0.031 mg/mL antithrombin III, less than 0.1 mg/mL
transferrin,
less than 0.07 mg/mL alpha- 1 -antitrypsin, less than 0.1 mg/mL alpha-2-
macroglobulin,
less than 0.7 mg/mL IgA, and less than 0.15 mg/mL albumin (as determined by
immunonephelometry).
[0083] In another aspect of the present invention, there is provided a
composition
comprising the hemopexin recovered by the methods disclosed herein. In an
embodiment,
the composition comprises a hemopexin content of at least 80% of total
protein. In another
embodiment, the composition comprises a hemopexin content of at least 90% of
total
protein. In another embodiment, the composition comprises a hemopexin content
of at
least 95%. In another embodiment, the composition comprises a hemopexin
content of at
least 97%. In yet another embodiment, the composition comprises a hemopexin
content of
at least 98%.
[0084] In particular embodiments the composition comprising hemopexin is at
least
98% pure and comprises less than 0.067 mg/mL IgG, less than 0.066 mg/mL IgA,
less than
0.042 mg/mL IgM, less than 0.048 mg/mL alpha- 1-antitrypsin, less than 0.090
mg/mL
transferrin, less than 0.050 mg/mL alpha-1-acid glycoprotein, less than 0.018
mg/mL pre-
albumin, less than 0.021 mg/mL ceruloplasmin, less than 0.053 mg/mL
apolipoprotein A-I,
less than 0.227 mg/mL apolipoprotein B, less than 0.031 mg/mL antithrombin
III, less than
0.17 mg/mL haptoglobulin and less than 0.045 mg/mL albumin (as determined by
immunonephelometry).
[0085] In particular embodiments the composition comprising hemopexin is at
least
97% pure and contains less than 1.7% haptoglobin, less than 0.4% transferrin,
less than
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0.2% albumin, and less than 0.1% alpha-2 macroglobulin as determined by
immunonephelometry.
[0086] In another aspect of the present invention, there is provided a
composition
comprising the transferrin recovered by the methods disclosed herein. In an
embodiment,
the composition comprises a transferrin content of at least 80% of total
protein. In another
embodiment, the composition comprises a transferrin content of at least 90% of
total
protein. In another embodiment, the composition comprises a transferrin
content of at least
95%. In yet another embodiment, the composition comprises a transferrin
content of at
least 98%.
[0087] In another aspect of the present invention, there is provided a
composition
comprising the haptoglobin recovered by the methods disclosed herein and the
hemopexin
recovered by the methods disclosed herein. In an embodiment, the composition
comprises
a combined haptoglobin and hemopexin content of at least 80% of total protein.
In another
embodiment, the composition comprises a combined haptoglobin and hemopexin
content
of at least 90% of total protein. In another embodiment, the composition
comprises a
combined haptoglobin and hemopexin content of at least 95% of total protein.
In yet
another embodiment, the composition comprises a combined haptoglobin and
hemopexin
content of at least 98% of total protein.
[0088] In an embodiment, the composition further comprises the transferrin
recovered
by the methods disclosed herein. In an embodiment, the composition comprises a
combined haptoglobin, hemopexin and transferrin content of at least 80% of
total protein.
In another embodiment, the composition comprises a combined haptoglobin,
hemopexin
and transferrin content of at least 90% of total protein. In another
embodiment, the
composition comprises a combined haptoglobin, hemopexin and transferring
content of at
least 95% of total protein. In yet another embodiment, the composition
comprises a
combined haptoglobin, hemopexin and transferrin content of at least 98% of
total protein.
[0089] In another aspect of the present invention, there is provided a
composition
comprising a haptoglobin content of at least 80%, 90%, 95%, or 98% of total
protein. In
another aspect of the present invention, there is provided a composition
comprising a
hemopexin content of at least 80%, 90%, 95%, or 98% of total protein. In
another aspect of
the present invention, there is provided a composition comprising a combined
hemopexin
and haptoglobin content of at least 80%, 90%, 95%, or 98% of total protein. In
yet another
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aspect of the present invention there is provided a composition comprising a
combined
hemopexin, haptoglobin and transferrin content of at least 80%, 90%, 95%, or
98% of total
protein.
[0090] The compositions comprising haptoglobin, hemopexin and/or
transferrin
recovered by the methods of the present invention disclosed herein will be
substantially
free of other components with which they are normally associated (e.g., other
plasma-
derived proteins). Thus, in an embodiment, the composition comprising
haptoglobin,
hemopexin and/or transferrin will comprise less than 20% of total protein,
preferably less
than 10% of total protein, and more preferably less than 5% of total protein
of other
components with which they are normally associated (i.e., impurities). The
skilled person
will understand that the level of impurities present in the compositions of
the present
invention may depend on the intended use of the compositions. For example,
where the
compositions are to be administered to a human subject in need thereof (i.e.,
for clinical
use), it would be desirable that the composition comprises less than 5%
impurities (of total
protein). Conversely, where the proteins are to be used in vitro, it may be
acceptable if the
composition comprises more than 5% of impurities (of total protein).
[0091] In another aspect of the present invention, there is provided a
formulation
comprising the composition of the present invention, as disclosed herein, and
a
pharmaceutically acceptable carrier.
[0092] Suitable pharmaceutically acceptable carriers, diluents and/or
excipients are
known to those skilled in the art. Examples include solvents, dispersion
media, antifungal
and antibacterial agents, surfactants, isotonic and absorption agents and the
like.
[0093] The pharmaceutical formulation may also be formulated by the
addition of (or a
combination of) suitable stabilisers, for example, an amino acid, a
carbohydrate, a salt, and
a detergent. In particular embodiments, the stabiliser comprises a mixture of
a sugar
alcohol and an amino acid. The stabilizer may comprise a mixture of a sugar
(e.g. sucrose
or trehalose), a sugar alcohol (e.g. mannitol or sorbitol), and an amino acid
(e.g. proline,
glycine and arginine). In a preferred embodiment, the formulation comprises an
amino
acid such as arginine. In other embodiments, the formulation comprises
divalent metal
ions in a concentration up to 100mM and a complexing agent as described in
US7045601.
In embodiments the pH is preferably about 6.5 to 7.5 and the osmolality is at
least 240
mosmol/kg.
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[0094] The pharmaceutical formulation may also be sterilised by filtration
prior to
dispensing and long term storage. Preferably, the formulation will retain
substantially its
original stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36
or more months.
For example, formulations stored at 2-8 C or 25 C can typically retain
substantially the
same molecular size distribution as measured by HPLC-SEC when stored for 6
months or
longer. Particular embodiments of the pharmaceutical formulation can be stable
and
suitable for commercial pharmaceutical use for at least 6 months, 12 months,
18 months,
24 months, 36 months or even longer when stored at 2-8 C and/or room
temperature.
[0095] The compositions described herein may be formulated into any of many
possible dosage forms such as injectable formulations. The formulations and
their
subsequent administration (dosing) are within the skill of those in the art.
Dosing is
dependent on the responsiveness of the subject to treatment, but will
invariably last for as
long as the desirable effect (e.g., a reduction in the level of free Hb/heme)
is desired.
Persons of ordinary skill can easily determine optimum dosages, dosing
methodologies and
repetition rates.
[0096] In an embodiment disclosed herein, the pharmaceutical formulation of
the
present invention is a solution that has a volume of at least 5 mL and
comprises at least 5
mg/mL haptoglobin and/or hemopexin and/or transferrin. In another embodiment,
the
pharmaceutical formulation has a volume of at least 5 mL and comprises at
least 20
mg/mL haptoglobin and/or hemopexin and/or transferrin. In particular
embodiments, the
pharmaceutical formulation has a volume of at least 5 mL and comprises
haptoglobin
and/or hemopexin and/or transferrin at a concentration of about 20 mg/mL, 25
mg/mL, 30
mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL,
70 mg/mL, 75 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL or 200 mg/mL. In
another aspect, there is provided a vessel containing at least 5 mL of a
stable
pharmaceutically acceptable haptoglobin and/or hemopexin and/or transferrin
solution,
wherein the concentration of haptoglobin and/or hemopexin and/or transferrin
is at least 20
mg/mL.
[0097] In another embodiment of the present invention, the pharmaceutical
formulation comprising haptoglobin and/or hemopexin and/or transferrin is
lyophilized.
Due to the presence of lyophilization stabilizer, like sugars (e.g. sucrose),
sugar alcohols
(e.g. mannitol), and an amino acid (e.g. glycine or proline) or combinations
thereof, the
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lyophilisation yields in a stable powder having a long shelf life. This powder
may be
stored, used directly or after storage as a powder or used after rehydration
to form the
pharmaceutical formulation. The lyophilized pharmaceutical formulation of the
present
invention may be formed using any method of lyophilization known in the art,
including,
but not limited to, freeze drying, i.e. the haptoglobin and/or hemopexin
and/or transferrin-
containing formulation is subjected to freezing followed by reduced pressure
evaporation.
The lyophilized formulations that are provided can retain substantially their
original
stability characteristics for at least 2, 4, 6, 8, 10, 12, 18, 24, 36 or more
months. For
example, lyophilized formulations stored at 2-8 C or 25 C can typically
retain
substantially the same molecular size distribution as measured by HPLC-SEC
when stored
for 6 months or longer. Particular embodiments of the haptoglobin and/or
hemopexin
and/or transferrin pharmaceutical formulation can be stable and suitable for
commercial
pharmaceutical use for at least 6 months, 12 months, 18 months, 24 months, 36
months or
even longer when stored at 2-8 C and/or room temperature. In a particular
embodiment
the lyophilised pharmaceutical formulation comprises hemopexin. The invention
may be
used for large scale production of lyophilised pharmaceutical formulations.
The
lyophilized product may be prepared for bulk preparations, or alternatively,
may be
apportioned in smaller containers (for example, single dose units) prior to
lyophilization,
and such smaller units may be used as sterile unit dosage forms. The
lyophilized
formulation can be reconstituted in order to obtain a solution or suspension
of the protein.
The lyophilized powder is rehydrated with an aqueous solution to a suitable
volume.
Preferred aqueous solutions are water for injection (WFI), phosphate-buffer
saline or a
physiological saline solution. The mixture can be agitated to facilitate
rehydration.
Preferably, the reconstitution step is conducted at room temperature.
[0098] In another aspect of the present invention, there is provided a
method of
treating a condition associated with haemolysis, the method comprising
administering to a
subject in need thereof the composition or the formulation of the present
invention, as
disclosed herein.
[0099] The term "subject", as used herein, refers to an animal which
includes a primate
(a lower or higher primate). A higher primate includes human. Whilst the
present
invention has particular application to targeting conditions in humans, it
would be
understood by those skilled in the art that non-human animals may also benefit
from the
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compositions and methods disclosed herein. Thus, it will be appreciated by the
skilled
addressee that the present invention has both human and veterinary
applications. For
convenience, an "animal" includes livestock and companion animals such as
cattle, horses,
sheep, pigs, camelids, goats, donkeys, dogs and cats. With respect to horses,
these include
horses used in the racing industry as well as those used recreationally or in
the livestock
industry.
[00100] The compositions or formulations of the present invention may be
administered
to the subject a number of ways. Examples of suitable routes of administration
include
intravenous, subcutaneous, intra-arterial or by infusion. In an embodiment,
the molecules
are administered intravenously.
[00101] Where necessary, the methods of the present invention may further
comprise
administering a second therapeutic agent. The second therapeutic compound may
be co-
administered to the subject sequentially (before or after administration of
the compositions
or formulations disclosed herein) or concurrently. In an embodiment, the
second
therapeutic agent is an iron chelating agent (e.g., deferrioxamine or
deferiprone).
[00102] In another aspect of the present invention, there is provided use
of the
compositions or formulations of the present invention, as disclosed herein, in
the
manufacture of a medicament for treating a condition associated with
haemolysis. Such
compositions or formulations are preferably suitable for use in human
patients.
[00103] Conditions associated with haemolysis and which are at risk of
haemoglobin/heme-mediated toxicity, are known in the art. In an embodiment,
the
condition is selected from an acute haemolytic condition and/or a chronic
haemolytic
condition. In an embodiment, the condition is selected from the group
consisting of
haemolytic anaemia, transfusion-induced haemolysis, haemolytic uraemic
syndrome, an
autoimmune disease, malaria infection, trauma, blood transfusion, open heart
surgery using
cardiopulmonary bypass and burns, including in the treatment of hemoglobinemia
or
hemoglobinuria accompanied with hemolysis after burn. In an embodiment, the
condition
is selected from the group consisting of sickle cell anaemia, hereditary
spherocytosis,
hereditary elliptocytosis, thalassemia, congenital dyserythropoietic anemia
and Paroxysmal
nocturnal hemoglobinuria, systemic lupus erythematosus and chronic lymphocytic
leukemia.
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[00104] Those
skilled in the art will appreciate that the invention described herein is
susceptible to variations and modifications other than those specifically
described. It is to
be understood that the invention includes all such variations and
modifications which fall
within the spirit and scope. The invention also includes all of the steps,
features,
compositions and compounds referred to or indicated in this specification,
individually or
collectively, and any and all combinations of any two or more of said steps or
features.
[00105] Certain embodiments of the invention will now be described with
reference to
the following examples which are intended for the purpose of illustration only
and are not
intended to limit the scope of the generality hereinbefore described.
EXAMPLES
Example 1
[00106] Starting Material: Cohn Fraction IV4 Precipitate was used as starting
material
for the purification of haptoglobin, hemopexin, and transferrin (see Figure
1). .
[00107] Precipitate Extraction: Extraction of the precipitate was performed by
the
introduction of 20 grams of Extraction Buffer per gram of Fraction IV4
Precipitate (20x
Extraction Ratio). The buffer and the precipitate were mixed for a minimum of
1 hour.
Extraction Buffer consisted of 50mM Tris adjusted to a pH of 7.0 with
Concentrated HC1.
The Extraction Buffer was prepared at a temperature of 20-25 C. The
precipitate
extraction was also performed at a temperature of 20-25 C. The pH during the
extraction
was maintained between 7.0 to 8.0 (preferably 7.0) for the duration of the 1
hour extraction
time.
[00108] Ammonium Sulfate Precipitation: Solid ammonium sulfate was added to
the
Fraction IV4 extract in order to achieve a final concentration of 2.0 to 2.5M
(preferably
2.4M). Under agitation, the ammonium sulfate was slowly added to the extract
and
allowed to continually mix for a minimum of 1 hour. The ammonium sulfate
precipitation
was performed at a temperature of 20-25 C.
[00109] An ammonium sulfate concentration of 2.5M was utilized to precipitate
lipids,
and clarify the Fraction IV4 Extract, while keeping hemopexin and transferrin
soluble.
Coincidently, this ammonium sulfate concentration resulted in the
precipitation of a
significant portion of the haptoglobin present in the Fraction IV4 Extract
(see Figure 2).
The precipitation of haptoglobin at 2.2M to 2.5M ammonium sulfate, while
hemopexin
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remained soluble, allows for the co-purification of hemopexin and haptoglobin
from the
same starting fraction.
[00110] To further narrow the acceptable ammonium sulfate concentration range,
a
design of experiment (DOE) was performed which looked at the impact of pH and
ammonium sulfate concentration. Figure 3 shows the desirability plot of the
DOE. This
plot gives the most desirable conditions that would precipitate the most
haptoglobin while
keeping the most hemopexin soluble. The desirability plot shows that a pH
maintained
between 7-8 (preferably 7.0) and an ammonium sulfate concentration between
2.2M and
2.5M (preferable 2.4M) is optimal for separating both proteins from the same
staring
material. Moreover, transferrin remains soluble at concentrations greater than
2.5M,
hence, these conditions were optimal for the purification of all three
proteins from the
same starting material.
[00111] Filtration:
To prepare the ammonium sulfate treated extract for filtration, 10
grams of C1000 filter aid was added per litre of plasma equivalent utilized
for the batch.
The C1000 filter aid was allowed to mix for a minimum of 15 minutes prior to
the
execution of filtration. A plate and frame filter press was utilized for the
filtration to allow
for collection of the haptoglobin-enriched precipitate. The plate and frame
filter press was
assembled with a sheet of type 175 filter paper in front of a 3M (Cuno) 70CA
depth
filtration filter sheet. For every 3L of plasma equivalent input into the
batch, 0.193 mL of
precipitate collection area was required (3L Plasma/4" Ertel 4S Filter Frame).
The
ammonium sulfate treated extract was then pumped into the filter press through
the use of
a double diagram pressurized air actuated pump.
[00112] After the completion of the C1000 mix time, the treated extract was
pumped
into the filter press and the filtrate was collected after a single pass
through the filter press.
The filter press was then post-washed with 1 to 2 press volumes of 2.4M
ammonium
sulfate, 50mM Tris, adjusted to pH 7Ø The filtration process was performed
at a
temperature of 20-25 C. The resulting filtrate contains hemopexin and can be
stored at 2-
8 C until it is carried forward for further purification. The resulting
precipitate contains
haptoglobin and can be stored at less than or equal to -20 C until it is
carried forward for
further purification.
Purification of Hemopexin (Hx) from the Filtrate Fraction:
[00113] The Hx process scheme is highlighted in Figure 9.
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[00114] (a) Octyl Sepharose Chromatography (HIC): The filtrate obtained from
the
extraction and ammonium sulfate treatment of Fraction 1\74 precipitate was
further clarified
using a 0.22 tim filter. The filtrate was then loaded onto an Octyl Sepharose
column (GE
Lifesciences) that had been equilibrated with three column volumes of 2.5M
ammonium
sulfate, 50mM Tris, at pH 7.4. Alternatively a capto octyl column can be used
for this
step. Eight to 12 column volumes (Target = 10) of filtrate were loaded onto
the Octyl
Sepharose column. Impurities and any unbound protein were washed off of the
column
with 3 column volumes of 0.9M to 1.2M (Target 1.13M) ammonium sulfate, 50mM
Tris,
at pH 7.4 (Wash). The wash fraction contained transferfin, which can be saved
for further
purification. The hemopexin containing fraction (Eluate) was then eluted off
of the
column with three column volumes of water (WFI). The Eluate was then stored at
2-8 C
until used for further transferrin purification. The Octyl Sepharose (HIC)
purification was
performed at a temperature of 20-25 C.
[00115] (b) Ni-Sepharose Chromatography (IMAC): 20mM sodium phosphate, 500mM
sodium chloride, and 30mM imidazole were added to the Octyl Sepharose Eluate.
Once
added, the pH of the Octyl Sepharose Eluate was adjusted to 7.4. Two to 3
column
volumes of the Octyl Sepharose Eluate were then loaded onto a Ni-Sepharose (GE
Lifesciences) column that had been equilibrated with buffer containing 20mM
sodium
phosphate, 500mM sodium chloride and optionally 30mM imidazole, adjusted to pH
7.4.
The addition of imidazole to the Octyl Sepharose Eluate (load) reduces the
affinity of the
Ni-Sepharose to albumin and other impurities, while maintaining its binding
affinity to
hemopexin. Therefore, during the load step, the impurities flowed through the
column,
while the hemopexin bound to the resin. After the completion of the load, the
column was
washed with 2 column volumes of 20mM sodium phosphate, 500mM sodium chloride
and
30mM imidazole, pH 7.4 to remove any unbound impurities. The hemopexin was
then
eluted from the column using 20mM sodium phosphate, 500mM sodium chloride and
100mM imidazole, at pH 7.4. The hemopexin present in the Ni-Sepharose eluate
was
estimated to be greater than 95% by SDS-PAGE (see Figure 4). The Ni-Sepharose
chromatography (IMAC) process was performed at 20-25 C and the resulting
eluate was
stored at less than or equal to -20 C until use.
[00116] (c) Concentration/Diafiltration: The Ni-Sepharose Eluate was
concentrated to a
desired concentration (1-20% w/v), then diafiltered with 10 volumes of
phosphate
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buffered saline per volume of concentrate. The concentration and diafiltration
were
performed using a 301cD ultrafiltration membrane. Once the diafiltration was
completed
and the concentrate was at the desired concentration, the hemopexin was
optionally
formulated with a sugar and or amino acid, sterile filtered and stored at less
than or equal
to -20 C. The purified hemopexin can be in pre-clinical animal and cellular
studies in this
form.
[00117] The final yield of hemopexin recovered from the process was estimated
to be
approximately 0.151 g/L plasma.
[00118] The concentrated hemopexin preparation (approximately 2.3% w/v) was
characterised by immune-nephelometry. Plasma proteins such as IgG (Limit of
detection,
LOD = <0.067 mg/mL), IgA (LOD = <0.066 mg/mL), IgM (LOD = <0.042 mg/mL),
alpha-l-antitrypsin (LOD = 0.048 mg/mL), transferrin (LOD = <0.090 mg/mL),
alpha-1-
acid glycoprotein (LOD = 0.050 mg/mL), pre-albumin (LOD = 0.018 mg/mL),
ceruloplasmin (LOD = 0.021 mg/mL), apolipoprotein A-I (LOD = <0.053 mg/mL),
apolipoprotein B (LOD = <0.227 mg/mL) and antithrombin III (LOD = 0.031 mg/mL)
were below detectable levels. Only trace amounts were detected for other
plasma proteins,
such as haptoglobulin (0.165 mg/mL) and albumin (0.038 mg/mL). These results
indicate
that Hx accounted for at least 99% of the total protein in the preparation.
[00119] A further batch of hemopexin was processed according to the methods
described above. The batch was concentrated to 35 mg/mL protein. Analysis of
the batch
by immunonephelometry indicated a hemopexin purity of about 98% with the
impurities
including 1.6% haptoglobin, 0.3% transferrin, 0.2% albumin, and 0.1% alpha-2
macroglobul in.
Purification of Haptoglobin (Hp) from the 2.5M Ammonium Sulfate Precipitate
[00120] The Hp process scheme is highlighted in Figure 10.
[00121] (a) Precipitate Extraction and Filtration: Haptoglobin was
extracted from the
precipitate by the introduction of 20 grams of extraction buffer/gram of
precipitate (20x
ratio). The extraction buffer consisted of 50mM Sodium Acetate, adjusted to pH
5.5. The
buffer and the precipitate were mixed for a minimum of 1 hour. After 15
minutes, the pH
was adjusted to within the range of 5.4 to 5.6. The low pH extraction buffer
was utilized to
reduce lipid extraction while the low pH was utilized during the subsequent
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chromatography step. To remove remaining filter aid and undissolved protein
and lipids,
the extract was then passed through a Cuno 70CA filter or equivalent depth
filter. The
filtrate was then clarified by use of a 0.2 ttm filter. The resulting filtrate
was ready for the
subsequent chromatography step. The extraction buffer preparation, the
extraction
process, and the filtration process were all performed at 20-25 C.
[00122] In another embodiment the Haptoglobin was extracted from the
precipitate by
the introduction of 20 grams of extraction buffer/gram of precipitate (20x
ratio). The
extraction buffer consisted of 50mM Tris, adjusted to pH 8.5. The buffer and
the
precipitate were mixed for a minimum of 1 hour. After 15 minutes, the pH was
adjusted to
within the range of 8.4 to 8.6. To reduce the lipid content and aid in
clarification of the
extracted precipitate a lipid adsorption agent, Aerosil (fumed silica), was
added at about
1.6 to about 1.8 grams per liter of plasma equivalent. The Aerosil treated
extract was then
allowed to mix for a minimum of 1 hour. To remove remaining filter aid and any
undissolved protein and lipids, the extract was passed through a Cuno 70CA
filter or other
similar depth filter. The filtrate was then clarified by use of a 0.2 p.m
filter. The resulting
filtrate was ready for the subsequent chromatography step. The extraction
buffer
preparation, the extraction process, and the filtration process were all
performed at 20-
25 C.
[00123] (b) Capto Q ImpRes Chromatography Step: Sodium acetate was added to
the
filtrate obtained from the above step, to a final concentration of 50 mM, and
the pH of the
filtrate is adjusted to pH 5.5, using glacial acetic acid. The pH adjusted
filtrate was then
diluted 1:4 to 1:5 with cold water (WFI) and loaded at a temperature of about
2-8 C onto a
GE Healthcare Capto Q ImpRes chromatography column that was equilibrated with
50mM
sodium acetate, pH 5.5. The load requires sodium acetate as a low pH buffer
and dilution
with water is required in order to reduce the conductivity of the load so that
haptoglobin
can bind to the column. After completion of the load, the column was washed
with 2
column volumes of 50mM sodium acetate, pH 5.5 to remove any unbound
contaminate
proteins. The haptoglobin was then eluted with 4 column volumes of 50mM sodium
acetate, 100-200 mM NaC1 (preferably 162mM), pH 5.5. A wide NaCl concentration
range of the elution buffer is required as the elution conditions are
partially dependant on
the load volume utilized. The eluate can be stored at 2-8 C, short term, and
at less than or
equal to -20 C, long term, until concentration/diafiltration. The haptoglobin
content of the
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Capto Q ImpRes eluate was estimated to be greater than 95% by SDS-PAGE (see
Figures
and 6). The chromatography buffers and chromatography steps were all performed
at
20-25 C.
[00124] (c) Concentration/Diafiltration: The Capto Q ImpRes Eluate was
concentrated
to a specified concentration, then diafiltered with 10 volumes of phosphate
buffered saline
per volume of concentrate. The concentration and diafiltration were performed
using a
301(1) ultrafiltration membrane. Once the diafiltration was completed and the
concentrate
was at the desired concentration, the purified haptoglobin solution was
sterile filtered and
stored at less than or equal to -20 C.
[00125] Optionally, a lipid adsorption step can be conducted before or after
the anion
exchange chromatography step or after the concentration/diafiltration step. An
example of
a suitable lipid adsorption agent is a fumed silica like Aerosil (e.g. Aerosil
380).
[00126] The final yield of haptoglobin recovered from the process was
estimated to be
approximately 0.285 g/L plasma input.
[00127] The concentrated haptoglobin preparation (approximately 2.6% w/v) was
characterised by immune-nephelometry. Plasma proteins such as IgG (limit of
detection,
LOD -= <0.067 mg/mL), IgM (LOD = <0.042 mg/mL), alpha-1 -acid glycoprotein
(LOD =
<0.050 mg/mL), pre-albumin (LOD = <0.018 mg/mL), ceruloplasmin (LOD = <0.021
mg/mL), hemopexin (LOD = <0.051 mg/mL), apolipoprotein A-I (LOD = <0.053
mg/mL),
apolipoprotein B (LOD = <0.227 mg/mL) and antithrombin III (LOD = <0.031
mg/mL)
were below detectable levels, whilst only trace amounts were detected for
other plasma
proteins such as transferrin (0.099 mg/mL), alpha- 1-antitrypsin (0.062
mg/mL), alpha-2-
macroglobulin (0.086 mg/mL), IgA (0.65 mg/mL), and albumin (0.123 mg/mL).
These
results indicate that Hp accounted for at least 96% of the total protein in
the preparation.
Purification of Transferrin from the Octyl 1.13M Ammonium Sulfate Wash
Fraction:
[00128] Prior to performing ion-exchange chromatography on the Octyl Wash
Fraction,
the ammonium sulfate was diafiltered out and exchanged with a lower ionic
strength
buffer. To this end, the Octyl Eluate was concentrated and diafiltered against
10 volumes
of 50mM Tris, pH 7.0 per volume of eluate. The concentration and diafiltration
were
performed using a 30kD ultrafiltration membrane.
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[00129] The diafiltered wash fraction was then loaded onto a GE Healthcare
Capto
DEAE column equilibrated with 50mM Tris, pH 7Ø To determine if it was
feasible to
obtain pure transferrin from this fraction, a linear gradient was performed
over 10 column
volumes using 50mM Tris, pH 7.0 as the starting buffer and ending with 50mM
Tris, 0.5M
NaC1, pH 7.0 (see Figure 8). Fractions were collected that correspond to each
peak on the
chromatogram. SDS-PAGE analysis was performed to determine if one of the peaks
contained pure transferrin. As seen in Figure 7, peak one was heavily loaded,
but appeared
to be pure transferrin. This indicates that it is possible to purify
transferrin from the Octyl
Sepharose Wash fraction, which also means that it is possible to purify
hemopexin,
haptoglobin, and transferrin from the same starting material (see also Figure
11).
34
