Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to hemoglobin preparation,
and more particularly to methods of preparing substantially
pure, stromal free hemoglobin suitable for use in prepara-
tion of blood substitutes.
Hemoglobin in its natural form is a non-cross-
linked protein constituent of red blood cells
(erythrocytes), possessing in solution the fundamental
property of reversible oxygen binding. Thus it functions
in the body as an oxygen transporter, delivering oxygen
from the lungs to other parts of the body where it is
required.
There is a continuing need, and frequent short-
age, of whole blood for administration to patients during
surgical procedures. Because of the difficulties in
obtaining, storing, typing and administering whole blood,
research and development directed towards providing an
acceptable blood substitute has been pursued vigorously
over the past 20 years or so. To date, however, only
limited success has been achieved.
Because an acceptable blood substitute should
have oxygen binding and release characteristics approximat-
ing that of natural blood, hemoglobin presents itself as a
natural candidate on which to base a blood substitute.
Indeed, much effort has been devoted to the development of
blood substitutes based on hemoglobin, as reflected in the
patent and scientific literature. Hemoglobin is, however,
a relatively complicated macromolecule, consisting of a
tetramer of two pairs of sub-units, ~ sub-units and B sub-
units. Each sub-unit has a globin polypeptide chain
capable of adopting various conformations, and a heme
group. The molecular weight of the tetramer is about
64,000, and it is necessary to ensure that, in a blood
substitute, the tetramer does not dissociate into its sub-
units, since these are of too low a molecular weight to
avoid excretion from the kidneys.
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The principal source of hemoglobin is, of course,
natural blood, where it is found in the red blood cells in
association with other compounds such as phospholipids,
cholesterol and other minor proteins. Before using it as
a basis of a clinically acceptable blood substitute,
hemoglobin should be obtained in as pure form as possible,
or at least in a form in which it is substantially free
from biologically harmful impurities present in a condition
which the impurities can harm the patient. Extraction of
hemoglobin from whole blood, and purification thereof, in
an economically satisfactory manner for use on a production
scale, presents difficult problems.
U.S. patent 4.001.200 Bonsen et al, and the
related patents 4,001,401; 4,053,590 and 4,061,736, all of
which have substantially identical disclosures, stress the
importance of obtaining stromal free hemoglobin as a basis
for a blood substitute. Stroma is essentially the cellular
debris from the erythrocytes, obtained after lysing the
cells, mainly comprising proteins, lipids and phospholipids
derived from the cell components and the cell walls. In
the Bonsen et al process, the red blood cells obtained from
centrifugation of whole blood are lysed in cold aqueous
medium. Then the red blood cell suspension is shaken, and
cold toluene is added to it. The mixture is shaken, then
left to stand for 24 - 72 hours to produce a three phase
mixture. The lower, clear red layer is isolated and
centrifuged at 40,000 - 50,000 g for at least 60 minutes at
4 - 6C. Then the upper clear supernatant is separated and
filtered through a diatomaceous earth filter. This filtra-
tion is reported to remove any traces of stroma. Residual
low molecular weight salts and metabolites are removed by
dialysis against an appropriate buffer, e.g. using hollow
cellulosic fihres as the semi-permeable membrane.
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There are serious drawbacks to this process when
it is considered from a production point of view, as
opposed to a laboratory procedure. During the process,
there is created a three-phase system, consisting of two
liquid phases and one solid phase, which presents a diffi-
cult processing problem. A discontinuous, small-batch
approach has to be adopted.
Also, the process of Bonsen et al involves the
use of toluene, as a solvent for removing lipids and
phospholipids. Whilst toluene is a convenient and
efficient solvent for laboratory use, since it readily and
efficiently extracts the lipids and phospholipids and forms
clean phase separations from water, its use is undesirable
on a production scale. It poses a fire hazard. Its fumes
create an unacceptable working environment unless strict
and expensive precautions are taken in its handling.
Noreover, any residual contamination of the hemoglobin
product with toluene can present physiological problems to
the patient.
It is an ob~ect of the present invention to
provide a novel process for purification of hemoglobin.
It is a further object to provide such a process
which is capable of operation on a production scale, to
yield hemoglobin and hemoglobin solutions of exceptionally
high purity.
It is a further object of the in~vention to
provide a novel, substantially pure hemoglobin product.
In the process according to this invention,
centrifugation is avoided, and the use of toluene and
similar organic solvents is avoided. The process of the
present invention involves the sequential steps of passage
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of a suspension of red blood cells through a coarse filter,
to remove debris and fibrinogen clots, then passage of the
suspension through a leukocyte filter, to remove white
blood cells (leukocytes) and their residues. Then the red
blood cells in the suspension are washed and lysed, passed
through a delipid filter to remove lipids and stroma, and
then concentrated by ultrafiltration through a membrane
filter.
The use of a leukocyte filter at the stage
described above enables centrifugation to be eliminated.
Accordingly, the process of the present invention is
readily adaptable to scale up to production size, to run on
a continuous basis to treat a large batch of red blood cell
suspension to obtain substantially pure hemoglobin there-
from. The use of the delipid filter allows one to avoid
the use of toluene or any other similarly objectionable
solvent, thereby eliminating a solvent removal step from
the process. Substantially reduced quantities of biologi-
cally contaminated waste liquids are formed, as compared
with processes involving toluene extraction and
centrifugation, thereby reducing disposal problems.
In addition the above practical advantages of the
process of the invention, however, it has been found that
a superior product, in terms of bio-acceptability of the
purified hemoglobin produced is obtained by this invention.
As mentioned above, the product is totally free from any
aromatic organic solvent residues. It is also substan-
tially totally free, or indeed even totally free, of
leukocyte residues. It has not previously been recognized,
in prior art descriptions of blood substitutes based on
hemoglobin, that it is very important to remove leukocyte
residues. If they are present to any significant extent,
they will cause human leukocyte antigen reactions in the
patient to whom the blood substitute is administered. One
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such reaction is the generation of Complement Factor 3A
(C3A) from Complement Factor 3, a protein constituent of
the natural immune system. When C3A is freed from C3, it
acts as an anaphylactic toxin. By the practice of the
present invention, involving the use of a leukocyte filter
at the appropriate stage, a product is produced which is so
low in leukocyte residues, that the risk of causing human
leukocyte antigen reactions is reduced to a minimal level.
The single accompanying Figure of drawings is a
diagrammatic process flow sheet of the presently most
preferred embodiment of the present invention.
The starting material for the process of the
present invention is a suspension of red blood cells
obtained from whole blood, by conventional processes.
Thus, the whole blood is treated with an anti-coagulant~
centrifuged, and the plasma withdrawn. This yields a red
blood cell concentrate containing all the cellular elements
of blood along with some residual plasma. The concentrate
is mixed to form a suspension with an aqueous electrolyte,
of a concentration suitable to be substantially isotonic
and at a pH of about 7 - 7.2, preferably, to reduce the
risk of premature lysis of the cells. The suspension is
formed by gentle agitation, so that cell rupture is reduced
to a minimum. It is preferred in the process of the
present invention to maintain the integrity of the red
blood cells in which the hemoglobin is contained, whilst
white cell residues and other debris are removed. The
entire process is suitably conducted at a temperature of
from about 2 - 10C, to avoid premature cell rupturing, and
under aseptic conditions. Preferably, the red blood cell
suspension is mixed with the aqueous electrolyte at about
a 1:1 volume ratio.
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Next, the suspension of red blood cells and other
materials is passed through a coarse filter, at which point
some cellular debris and fibrinogen clots are removed.
Suitably, the coarse filter is of polymeric material, e.g.
polyester, polysulfone etc., or of stainless steel, and
suitably has a passage cutoff size of about 50 microns.
The material which is collected on the filter is biological
waste material, for careful disposal in an environmentally
acceptable mannerO Passing through the coarse filter is a
cellular suspension of white blood cells, red blood cells
and platelets, along with some residual plasma. This
material is next passed to the leukocyte filter. Without
the preliminary use of the coarse filter, the leukocyte
filter may clog and be unsatisfactory for use on a continu-
ous or semi-continuous basis.
Leukocyte filters for use in the process of the
present invention are available on the market, and have
previously been used to filter whole blood at a patient's
bedside as the blood is being administered to the patient
by a nurse. They normally comprise charged polyester
filter material through which the suspension is passed. On
the basis of electrokinetic absorption and depth filtra-
tion, the leukocyte filter material selectively retains
white blood cells as opposed to red blood cells, according
to the different electrostatic charge characteristics of
the two types of cell. At least a three log reduction in
the quantity of white blood cells is achieved by a single
passage through the leukocyte filter. Inevitably, a small
quantity of red blood cells are filtered out as well, but
not to any practically significant extent. It is possible
to recover any such retained red blood cells from the
filter material by elution, but the amounts of red blood
cells retained by the filters are usually sufficiently
small that such a process step is not necessary. The
filter material, after use, is discarded. Passage of the
suspension through a leukocyte filter can be repeated if
traces of leukocyte residues are detectable therein, for an
additional three log (1000 fold) reduction therein.
One example of a commercially available leukocyte
filter useful in the process of the present invention is
that manufactured by Asahi Medical Co. Ltd. and sold under
the trade-mark "SEPACELL" by the Fenwal Division, Baxter
Health Care Corporation. Another is that marketed by Pall
Biomedical Products Corporation under the trade-marks "PALL
RC 50" and "PALL RC 100" leukocyte removal filters. Both
are sold as recommended for use for blood transfusion at
bedside.
From the outlet of the leukocyte filter, there is
obtained a suspension of red blood cells and residual
plasma proteins in aqueous electrolyte, and this suspension
is now preferably subjected to one or more washing steps,
to remove all extra-cellular contaminants including plasma.
This is preferably done by washing the suspension in a
tangential flow microfiltration device of 0.1 - 0.45 micron
size with isotonic sodium chloride solution. It is pre-
ferred to conduct at least three separate, sequential such
washes, using in each case a 3:1 v/v ratio of sodium
chloride solution to red blood cell suspension. The sodium
chloride stabilizes the proteins and maintains them in
solution. A substantially pure red blood cell suspension
has now been obtained.
Next, the red blood cells are lysed, to free the
hemoglobin and other contents thereon. As is well known in
the art, there are a wide variety of methods for lysing red
blood cells. In the present process, it is preferred to
conduct a gradual decrease of the ionic strength and hence
osmotic pressure of the solution, by slow addition of a
solution of 5-20 mmolar phosphate at pH 7. Too rapid a
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lysis of the cell causes it to break into very small
components, which can create complications in subsequent
steps of separation of cellular components.
During the lysing procedure, the mixture is
subjected to a tangential flow micro filter, suitably of
0.1 - 0.45 micron size, which are readily available,
standard units. This separates the cell wall residues,
"ghosts". Passing through the microfilter are the red cell
intracellular proteins, along with some residual debris and
some lipids. The steps of washing, lysing and microfilter-
ing are suitably all combined in a single unit.
The filtrate from the micro filter is preferably
fed directly to the delipid filter. Preferred forms of
delipid filters are those containing fumed silica embedded
in cellulose. Lipids, phospholipids and stroma particles
are removed by such delipid filters by affinity mechanisms.
Also, the delipid filter will remove some if not all of the
contaminating pyrogens present in the suspension at this
stage.
Delipid filters commonly require a stabilization
period during start up of a process, so that it is pre-
ferred to arrange for a recycle line in the process of the
present invention, whereby initial quantities of filtrate
from the lipid filter are recycled through the filter until
stabilization occurs.
The filtrate from the delipid filter, once it has
stabilized, is dilute stromal free hemoglobin suspension.
It is nex~ subjected to ultrafiltration, whereby soluble
molecules from the suspension below a certain size and
molecular weight are removed in the filtrate, and soluble
and insoluble molecules above the cut off molecular weight
are retained. The ultrafiltration medium is preferably an
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asymmetric membrane with pores of a size sufficient to
provide a cut-off of about 30,000 molecular weight, i.e. a
membrane which will retain 95% of all molecules of molecu-
lar weight of 30,000 or higher. This preferred choice of
30,000 molecular weight cut-off is made on the basis of the
likely presence in the material of carbonic anhydrase, one
of the intracellular proteins present in red blood cells
along with hemoglobin, in small but significant amounts.
By this choice of molecular weight cut-off, a substantial
amount of carbonic anhydrase can be separated from the
hemoglobin, it being understood that even initially carbon
anhydrase is present in only very minor amounts, compared
with the amount of hemoglobin. The ultrafiltration is
performed until the desired final hemoglobin concentration
of 8 - 15% w/w is achieved.
The preferred embodiment of the process of the
present invention next subjects the concentrated suspension
of stroma free hemoglobin which has not passed through the
ultrafilter to a micro pre-filtration to remove any aggre-
gates which may have been formed during the ultrafiltration
process. This suitably uses a 0.6 micron filter membrane.
Then it is subjected to a sterilizing micro filter, with a
0.2 micron size, to obtain the final, purified, hemoglobin
product.
In the alternative, a step of ion exchange
chromatography may be used, further to reduce traces of
other proteins which may be present, for example carbonic
anhydrase. When chromatography is used, it is preferred to
arrange for hemoglobin to pass through the column, whilst
other proteins are retained, using conditions based upon
the isoelectric point of hemoglobin. Such a chromatography
step can be used in addition to the micro pre-filter and
the sterilizing micro-filtration.
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The resulting product is one which contains no
detectable leukocyte residues, and no organic solvent
contaminants. Whilst the possibility of the presence of
microscopic amounts of other protein contaminants cannot be
ruled out, the product produced by the process of the
present invention can justifiably be termed substantially
pure hemoglobin. The process is operable on a continuous
basis for a given batch of red blood cell suspension,
avoiding centrifugation of a three phase system.
With reference to the accompanying Figure, red
blood cell concentrate RBC obtained in the normal way from
whole blood is contained as a batch in vessel 10, and from
there it is fed to a ~essel 12, where it is mixed with an
equal volume of isotonic aqueous sodium chloride solution
from line 14 and gently agitated at 4 C, to form a re-
suspended dilute RBC suspension. This and all other steps
in the process are conducted under aseptic conditions,
using incoming solutions which have all passed through a
sterilizing filter prior to use.
From the vessel 12, the dilute suspension passes
to a coarse filter 14 which is a 50 micron cut-off
polyester membrane, and removes the fibrinogen clots from
the suspension. The clots so removed are waste material,
for careful disposal. The temperature of the suspension is
maintained at about 4C through this step.
Issuing from the coarse filter is a cellular
suspension with residual plasma, and this is passed into
the leukocyte filter 16, which is a SEPACELL filter, and
serves to absorb the white blood cells preferentially over
the red blood cells, resulting in a 1000 fold reduction in
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the amount of white cell residues, in the product issuing
from the leukocyte filter 16.
Next, the red blood cell suspension is washed
three separate times with isotonic aqueous sodium chloride
solution, again maintaining the temperature at 4C, as
diagrammatically illustrated by inlet lines 18, 20 and 22
from saline storage facility 24. This is conducted in a
single tangential flow filtration device, the red blood
cell suspension being passed tangentially across the filter
membrane, under pressure, with the saline on the same side
of the membrane.
Next, 20 mmolar potassium phosphate solution is
added to the suspension via line 26, and the mixture passes
into a microfilter unit 28 in which lysis of the cells
takes place, the ghosts being removed via line 30 and the
resulting proteinaceous material and accompanying cell
contents passing through the 0.1 - 0.45 micron microfilter
membrane 32.
The lysed material next passes into the delipid
filter 34, which contains fumed silica embedded in cellu-
lose as the affinity removal medium, to which stroma
particles, lipids and phospholipids adhere. During initial
start-up, recycle line 36 is utilized to pass filtrate back
through the delipid filter a second or third time, until
the operation of the delipid filter has stabilized.
The output from the delipid filter is fed into a
30 K ultrafiltration unit 3>3, which removes from the
solution, by passage through the filter membrane, molecular
constituents of molecular weight about 30,000 or lower, to
waste line 40, whilst higher molecular weight materials in
the solution pass on to a 0.6 micro pre-filter 42 where
aggregates formed during the ultrafiltration process are
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removed, and then to a 0.2 micro sterilizing filter 44 for
final treatment. The product issuing from sterilizing
microfilter 44 via outlet line 46 is substantially pure
stromal free hemoglobin product, in aqueous solution.
