Note: Descriptions are shown in the official language in which they were submitted.
103~i~9Z
This invention relates to the recovery and isolation of
serum or blood a]b~min, from blood, blood products, other body
fluids or tissue extracts,for use in the therapy applied to men.
To replace the less effective and more dangerous treatment
with stored whole blood, component therapy is coming into its own
in modern medicine. In addition to being waste of the basic
material, whole blood has the highest risk of causing transfusion
reactions, immunizations, and disease transfer, particularly
hepatitis. By administering blood components these risks are either
10 completely avoided (e.g. human serum albumin) or diminished (e.g.
buffycoat free packed red cells).
So in recent years, the separation and concentration of
blood protein fractions has been of interest for a variety of uses.
Blood is a fluid which consists of solid and liquid constituents.
The solid constituents include red and white blood corpuscles and
blood platelets. The plasma or liquid part of the blood contains
about 90% of water and 10% of solids. The substance dissolved
in the plasma include, inter alia, albumin, which is the sole ,
protein constituent of plasma which is stable to temperatures -
20 in excess of about 60C. It is in itself known to separate the
~; blood plasma frcm the red and white blood corpuscles and from the
blood platelets. Furthermore it is known to remove gammaglobulins
as well as coagulation-promoting substances, such as, for example,
~ fibrinogen, from the plasma. For therapeutic or diagnostic purposes
.$~i it is desirable to obtain as pure an albumin solution or albumin
~ paste as possible, which should, as far as possible, not contain
r any further protein constituents of the blood plasma.
: ?
In the past, the separation of albumin from protein and
other components of human plasma has been accomplished by control of
~ 30 the relative solubilities of the components of the plasma. This
;, conccpt is used by a process known as COHN-method. The COHN-
; method was developed in 1946 (J. Am. Chem. Soc. 68 (1946)
~k"'
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1()3~ 9Z
pp. 459-475) and got world-wide acceptance for the technical
fractionation of plasma proteins to be used for human therapy.
Human plasma is separated into five fractions by using different
concentrations of ethanol, employed as a non-toxic protein preci-
pitant, while simultaneously changing the pH with buffers of
different ionic strength. The concentration of alcohol increases
from about 8% to 40%, the pH varies from 7.2 to 4.6. The ethanol
can subsequently be easily removed by freeze-drying, but the -
" disadvantage lies in the fact, that it readily denatures proteins,
especially when used in high concentrations.
This disadvantage may be reduced by lowering the
temperature to between -3 and -7C during fractionation. To
~ accomplish this for large scale fractionation, either the entire
,~ process must take place in a cold room (an uncomfortable and
unhealthy situation for the personnel), or self-cooling machinery
must be used. In additlon to this, all electrical equipement must
be specially protected when working with large volumes of alcohol.
.. .
i The first COHN fractionation step removes mainly
`~ fibrinogen (Fract. l); the second-third, gammaglobulin (Fract.
II-III); the fourth alpha- and beta-globulins (Fract. IV). The
-~ remaining supernatant contains albumin which is precipitated with
40% ethanol as Fract. V. An additional purifying precipitation
is required if high albumin concentrations (20%) are desired
' (Fract. VI).
,. ' .
- Fract. V albumin paste (crude or refined) must be
lyophilized to remove the ethanol. The powder is solved, the
solution is cleared through filtration, and the pH, osmolality, and
protein concentration are adjusted. Also, aliphatic carbonic acids
(e.g. caprylic acid) must be added to protect the protein from heat
. . .
denaturation during pasteurization. Caprylic acid is most often
used for stabilization usually in concentrations between 0.004 M
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. 103829Z
albumin) and 0.04 M (20% albumin).
Generally speaking, the five-steps COHN-method isolates
four useless fractions, thus making production of the fifth, albumin,
unnecessarily expensive.
Consequently, less expensive methods for albumin frac-
tionation have been looked for. In particular achieving a situation
is desirable, where the working time is shortened, the number of
working steps is reduced, the nuisance to personnel is limited and
- the purity is increased without lowering the yield. In addition
it is also desirable that the technical effort required should be
substantially reduced.
The present invention therefore proposes to provide a
commercially feasible process for the recovery of serum albumin
from the other protein constituents of human blood or other
suitable fluids. The process of the invention is applicable not
~, only for the extraction of pure serum albumin from donor blood, - -
but also from various forms of raw materials, such as haemoly~ed
- blood plasma, placental sera and placental extract.
In accordance with the invention, there is thus provided - `
a process for the extraction of pure serum albumin from blood
-~ plasma, blood products, other body fluids or tissue extracts,
which comprises the steps of separating the plasma from the solid
constituents of the blood, isolating dissolved non-albumin-
constituents from the plasma, adding an albumin-stabiliser in a
quantity of about 0.004 mole, treating the fluid with a lower
aliphatic alcohol of the formula CH3-(CH2)n-OH in which n is 0,1
or 2 at a temperature of from 60 to 75C and pH of about 6.5 to cause
precipitation of partially or largely denaturated concomitant
proteins, the treatment being carried out at a volume concentration
of the alcohol of about 9~, and separating the resultant solution
containing pure serum albumin from the precipitate.
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1038Z92
Known in the art are the mentioned protein- and albumin-
stabilisers, which even at heating of a pure albumin-solution
up to 60C will prevent any visual alteration of the albumin.
The work of BALLOU et al. (J. Clinical Invest. 23 (1944); pp 454;
` J. Biol. Chem. 153 (1944); pp. 589) hints,that sodium-salts of
- propionate, butyrate, valerate, caproate, caprylate phenylacetate ~
and phenylbutyrate have the desired stabilising property. ~ --,
As a result, the concomitant proteins are being preci-
pitated at about 68C, while the albumin stays in solution in the
presence of alcohol and stabilisers. It is possible to dispense
with cooling during the separation. In addition, numerous working
1 steps are saved, since fractional precipitation is dispensed with.
-~ Instead, all globulins can be removed in one working step.
;~ A-temperature of 68C - 3C has proved.to be the
.-~! optimum working temperature for precipitating the unwanted proteins
in the presence of sodium caprylate and ethyl alcohol.
For enriching-of the albumin from the residual albumin-
containing fluid, maleic acid or polyethylene glycol are known
in the art to be of particular usefulness as precipitating media;
however, it is also possible to use for this concentration step
still other polymerized, aliphatic multivalent alcohols, organic
., .; -
~ acids, specific salts, monomeric alcohols, or other methods suitable
; . ,
:~ for protein (albumln) concentration, including freeze-
~ drying and ultra-filtration.
~ ",
,~ In order to convert the precipitated albumin paste into
-~ a solution of the desired concentration, the paste is dissolved -
following the above mentioned steps - in a preferably buffered
liquid. The albumin solution can then preferably be heat-sterilised,
-l without adding a stabiliser.
.,
Variants of the process sequence and further features
and advantages of the invention are illustrated with the aid of
the accompanying drawings, wherein:
~ ~ .
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~03829Z
Figure la shows the precipitation process according to
the so-called Cohn method;
Figure lb schematically shows a process according to the
invention; and
Figure 2a-2h show immuno-electrophoresis diagrams (IEP)
of different albumin preparations produced in accordance with known
processes as well as with the new process.
Figure la schematically shows, in stages, the process of
the so-called Cohn method. This starts from a mixed plasma to which
8~ of ethanol is added and which is precipitated at a pH value
of 7.2 and at minus 3 C. This results in fraction I separating
out. The ethanol content of the supernatant liquid is then
increased to 19% at minus 5C and pH value of 5.8. This causes
fraction II/III, which in the main consists of gamma-globulins, to
separate out. The supernatant liquid is again treated at a higher
alcohol content, at a pH value of 5.8 and at a temperature of minus
7 C. This gives fraction IV, consisting in the main of alpha-
and beta-globulins. The supernatant liquid is subjected to a
further treatment, at a pH value of 4~8 and a temperature of minus
7C (-ethanol content 40%). Hereupon, the so-called crude albumin
separates out as a sediment. The supernatant liquid is discarded.
After freeze-drying to remove the alcohol, the crude albumin can be
taken up in, or converted into a 5% strength solution. However, it
is also possible again to take up the crude albumin paste and con-
vert it into a purified albunim paste in two further steps (for
which the conditions can be seen from the diagram).`
In total, about 6 - 8 days are required for the prepa-
ration of the purified paste if a normal 8 hour day is worked.
Furthermore, very careful cooling is required, which demands
correspondingly high expenditure on technological apparatus.
In contrast, the new process can be carried out
substantially more simply (see Figure lb). Here again, a mixed
--5--
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' ~03~Z9Z
pl~sma or other fluids containin~l albumin and globulin are used
as the starting material; it is first subjected to a heat treatment
at a temperature of 68C in the presence of aliphatic, low moleeular
aleohols and of stabilisers. Suitable stabilisers are, as
mentioned, certain aliphatic carboxylic acids and their salts, such
as say, sodium eaprylate, and other eonstituents.
In the first process step, which is carried out at a pH
value of 6.5 and an ethanol eontent of 9~, all globulins are
denatured and separated out as a sediment. After cooling, the
supernatant liquid is acidified to pH 4.8 and a precipitation medium,
such as, say, 22~ of polyethylene glycol (PEG), or 18% maleic acid,
is added. After an appropriate reaction time, a very pure albumin
paste separates out and this is either freeze-dried or taken up
to form a 5% strength solution.
As can be seen from the above description, the new
proeess requires a substantially shorter time and substantially
less teehnological effort. In addition, however, the albumin
obtained is also substantially purer than that obtained according
to known methods. Figures 2a to 2h show immuno-electrophoresis
diagrams (IEP), from which the following can be discerned:
Figures 2a and c show IEP diagrams of a natural plasma,
in whieh the siekle-shaped mark of albumin is formed on the left;
the thinner siekle-shaped marks whieh follow on the right originate
from globulins whieh must be regarded as impurities in a "pure"
albumin solution. Albumin solutions prepared aeeording to a known
proeess are shown in Figures 2g and h. It can be seen elearly
that a part of the impurities has been removed; however, far from
all the undesired protein constituents have been removed.
Figures 2b and d show albumin solutions whieh have been
obtained in aeeordance with the new proeess. Here, almost 100%
purity is aehievable.
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103BZ92
Figure 2 f shows the supernatant liquid which was obtained
after the last process step (precipitation of the albumin). It can
be seen that this liquid virtually no longer contains any albumin.
It follows that the first process step (heating to 68C in the
presence of alcohol and sodium caprylate) already achieves
practically complete separation of albumins and other proteins of
the plasma.
`; The following non restrictive Examples further illustrate
the invention:
Example 1
The starting material is pooled donor plasma, from which
the coagulation factors have been removed. The coagulation factor
VIII and the fibrinogen have been removed by cryoethanol sedimen-
tation. The prothrombin complex is removed by DEAE-Cellulose
adsorption. The original plasma is Hepatitis -Bl antigen negative,
has normal transaminase values and does not contain any visible
haemoglobin.
Sodium caprylate is added to the plasm until the concen-
tration is 0,004 molar. The mixture is heated in a stainless steel
container within 3 hours to 68C, heat being supplied at an even
rate. At the beginning of the heating, the concentration of ethanol
., .
is brought to 9% by volume, and the pH value is set to 6.5,
~ which is obtained by adding 0.5 n HCl. The plasma is continually
; stirred during the heating process.
-~ When a plasma temperature of 68C is reached, the plasma
~ is transferred by compressed air into a container which is connected
Y to a cooling system. The plasma is stirred until the temperature
- is brought down to +10C (about 4 hours), at which time the pH
is lowered to 4.4 with 0.5 n HCl. The acidified plasma is now
30 left to stand overnight at about 10C. During this time the
unwanted proteins coagulate. The pure albumin stays dissolved in
the fluid.
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1038292
The fluid with the sediments is pumped into continuous
flow centrifuges through silicone tubes, where it is separated by
centrifugation. The proteins which have separated out collect
in the rotors and the albumin remains in the supernatant liquid.
The proteins which have separated out in the rotors still contain
albumin, which can be isolated additionally by reeluting and renewed
centrifuging.
The supernatant liquid is filtered through so-called
SSK filters (Seitz, Bad Kreuznach, Germany) to remove remaining
lipids and modified proteins. To the clear fluid a precipitant
is then added, preferably maleic acid or polyethylene glycol
in a concentration of 18% at room temperature. The albumin
precipitates.
: .,
r The albumin suspension is again centrifuged. This leaves
the polyethylene glycol and the salts in the protein-free liquid.
The albumin collects as a paste in the rotors. It is taken up in
distilled water in a container, and converted into a solution
of approx. 8% strength. After a clarifying filtration, the albumin
can be converted directly into a 4 - 5% strength solution for use,
. .. - .
i 20 the osmolality (osmotically measured molar strength) being adjusted
.,
. with glucose or other suitable substances. The product is then
sterile-filtered, filled into bottles and pasteurised for at least
10 hours at 60C.
Also, the supernatant liquid may be filtered through SSK
filters in order to remove remaining lipids and modified proteins.
Then, polyethylene glycol having a molecular weight of from about
~ 4000 to 6000 is added as the precipitating agent in a concentration
`~ of 22% at room temperature. The albumin precipitates within a
period of 30 minutes. The albumin suspension is centrifuged again.
Hereby, the polyethylene glycol and the salts are retained in the
~- - 8 -
.
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1038292
~. ein-fl^co liqui~ The albumil~ is collected as a paste in the
rotors. The albumin is re-dissolved in a container in distilled
water and converted into an about 8~ solution. After a clarifying
filtration, the 8% albumin is freeze-dried. The dried powder is
dissolved in distilled water, whereby the osmolality may be
adjusted, if desired.
Examples 2 and 3
Preliminary remarks:
Two currently discarded Cohn plasma fractions are known
to contain albumin in amounts which might be economical to salvage
if simple technique, like heating, would give an acceptable product.
These fractions are fraction IV-l and fraction IV. Fraction IV-l
isprecipitated from Supernatant II-III at a content of alcohol of
19~, temp. -7C, pH 5.2. The yield is about 20 to 25 grams of moist
paste per liter of starting plasma, containing about 30~ protein.
~? Fraction IV is precipitated from Supernatant II I III
in two steps (first: Fraction IV-l is won as above; two: alcohol
is added to 40~, -6C, pH 6.5, to bring down Fraction IV-4). The
. precipitates are removed together in a single centrifugation step
~ 20 as Fraction IV. Yield is 30 to 35 grams of paste per liter of
`~ plasma. Paste is 30% protein.
Example 2
~`~ 1.O kg of FRACTION IV-l paste is suspended with 2.0 L of
water containing 0.004 M sodium caprylate stabiliser. The pH
~ is adjusted to 7 - 7.6 with sodium hydroxide. The mixture is
i stirred gently at room temperature. pH is periodically checked and
readjusted upwards. Most of the paste will dissolve within one hour.
; The resulting crude solution will contain about 10% protein and
4 - 5~ alcohol and is adjusted to 9~ alcohol.
The mixture is heated to 68C as described, and centrifuged.
; The precipitate is washed to remove entrapped albumin as described.
.,
-' _ 9 _
B.
. . .
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,.~ .
1038292
m~e combined supernatallts are filtered to clarify and precipitated
with a suitable agent to collect the albumin.
Example 3
Likewise, a suspension of 1.0 kg of Fraction IV paste in
; water containing stabilisers is dissolved similarly, using 3.0 L per
` kg for a final solution containing about 7.5~ protein and about
7~ alcohol. To experience, approx. 20% of the protein in Fraction
- IV-I is albumin. If the above procedure is quantitative, 1 to 2
grams additional albumin might be recovered per liter of starting
plasma. The yield of albumin from Fraction IV is in the same range.
Instead of the concentration of the albumin by PEG-precipi-
tation and/or freeze-drying as specified in the examples, it is also
possible to perform an ultrafiltration or a careful evaporation
of the water of the solution by rotational evaporation.
~-~ If the concentration of the supernatant liquid produced
after the globulin separation is effected by other methods than by
albumin precipitation (e.g. by ultra-filtration, rotation
~-~ evaporation), the desired osmolality, the salt composition and the
hydrogen ion concentration must be adjusted by dialysis with a
corresponding liquid.
The yield amounts according to experiments, to more
-~ than 90% of the originally present albumin, whereas in the
conventional methods, in spite of lower degrees of purity, a yield
of at best of from 60 to 70% can be expected.
:
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~ .~
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1038Z92
SUPPI,EMENTA~Y DISCLOSURE
It is known to effect the recovery of serum or blood
albumin from the whole blood, plasma, or serum of cattle, hogs,
and sheep (mammalians), by the steps of selectively denaturing
and coagulating other protein constituents than albumin by heating
the solution to a temperature of about 45C to 75C, while the
solution contains about 0.0075 to 0.0~ mol concentration of capry-
late ions, and then separating the coagulum of the other protein
constituents fromthe supernatant solution of the serum albumin.
In the process, no alcohol is used. Only for precipitating the
albumin after the heatlng process, alcohol (ethanol) can be used
to a concentration of 40%. The precipitated albumin paste is
lyophilized and the dry powder processed anew.
Apparently due to the lack of a sufficient alcohol concen-
tration during the heating process in connection with the relatively
high concentration of caprylate ions, the standard of purity in this
process is too low. The percentage of albumin in the final product
lies between 90 and 96~ of the total protein. The minimum
albumin eontent of Normal Serum Albumin (Human~ for therapeutic
;, .
purposes is 96% of the total protein according to current FDA
- 20 regulations.
~ Beeause some of the preliminary steps may be earried out
Y at low temperatures and an alcohol content of 4~, residues of this
,.
r alcohol may be left in the aqueous solution of the serum albumin.
~,~ The presence of 4% alcohol in the solution does not interfere
.,
in the process. On the other hand, this amount of alcohol is
not enough to gain the desired 100% precipitation of globulins.
~'. Further, in US Patent 3.926.939 (Ivanov et al) a method
~- for extraeting pure serum albumin from biological fluids is known,
- which eomprises treating such fluids with a lower aliphatic
~ 30 alcohol and a salt of a carboxylld acid, whereby the treatment is
- carried out at a volume concentration of alcohol of 15 to 33% in
, .
an presence of 0.1 to 0.6% of the aliphatic carboxylate having an
.. . .
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` 10:~8292
~ ~-toxication and an anion comprising from 6 to 12 carbon
atoms at a temperature of from 1 to 30 C and pH of from 2 to 5,
the concomitant proteins being partly or largely denatured, depending
on the pH value selected from the process; the denatured and native
concomitant proteins are precipitated at a pH value of from 4 to 5
and at a temperatureof from 1 to 30C, with the non-protein
impurities which form complexes with the albumin being split off
therefrom and adsorbed by the precipitate; the resultant solution
containing pure serum albumin is separated from the precipitate
at a temperature of from 1 to 30 C and pH of from 4 to 5.
Experiments show that with the method mentioned the yield
of serum albumin is comparatively low, especially when the purity
to be achieved is high (more than 99%). At optimal conditions,
only about 55% recovery ~ achieved, compared to the theoretical
achievable amount of 100%. In addition, the high alcohol content
in the solution of more than 15% is difficult to remove, resulting
in high costs of purification.
The present invention provides a process which is
applicable to the direct recovery of serum albumin from the
plasma with high percentage of yield, high purity making the
product applicable to the human body, even for intravenous
application. It has been found that the treatment described in
the original disclosure for recovering pure serum albumin can be
carried out at a volume concentration of the alcohol of 7 to 14%
in the presence of 0.001 to 0.01 moles of the stabiliser and at a
pH of from 4.5 to 7.5.
Accordingly, the invention is directed to a process for
the extraction of pure serum albumin from blood plasma, blood
products, other body fluids or tissue extracts, which comprises the
steps of separating the plasma from the solid constituents of the
blood, isolating dissolved non-albumin-constituents from the-~p~sma by
adding an albumin-stabiliser in a quantity of 0.001 to 0.01 moles,
treating the fluid with a lower aliphatic alcohol of the formula
:- .
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` 1038292
C..3-(CH2)n-OH in which n is 0,1 or 2 at a temperature of from 60
to 75C and pH of from 4.5 to 7.5 to cause precipitation of
partially or largely denaturated concomitant proteins, the
treatment being carried out at a volume concentration of the
alcohol of 7 to 14~, and separating the resultant solution
containing pure serum albumin from the precipitate.
The alcohol is preferably ethanol taken to the extent
of 8 to 12% by volume; the preferred carboxylic acid is sodium
caprylate in a 0.003 to 0.005 mole concentration. The preferred pH
10 value of the treatment is 6 to 7 and the preferred temperature is
67 to 69C.
The separation of the albumin solution from the precipi- -
' tate is generally carried out at a temperature of from 1 to 30C,
preferably at room temperature.
The following table shows, that at a temperature of 68C
and a pH-value of 6.5 a maximum recovery of 96% albumin (according to
i 100% content in a human blood plasma) may be achieved from the
plasma. The table also shows, that it may be possible to work
within a certain range of alcohol contents without dQparting from
~ 20 scope of the invention.
-~, Table (recovery of albumin, when treating human blood plasma
at 68C and pH = 6.5; 0.004 mol sodium-caprylate at
different alcohol contents)
recovery of albumin (~) alcohol content (~ by volume)
~ 5.0
'', ~ (*)
6.0
88 7.0
89 8.0
..:
-~ 96
.~ 92 10.0
.-.,
'` 11.0
:,
82 12.0
13.0
., .
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1038292
lcl of albu~ ) alcohol content (~ by volume)
68 14.0
63 15.0
(*) with a concentration of alcohol lower than 8% the separation
of gammaglobulin from albumin is not complete; therefore, the
albumin is not applicable.
Other series of tests show similar results. The optimum
of alcohol content lies at about 9~ for similar conditions.
As may be seen, the maximum of recovery lays at 9%. A
lower or higher content result in worsening of purity and/or yield
of the albumin.
An advantage over the known process proposed by Ivanov et
` al. (US-Pat. 3,926,939) is that a higher yield is achieved by
.~ using a lower alcohol content, which is easier to remove.
As mentioned in the original disclosure, a temperature of
68C + 3C has proved to be the optimum working temperature for
precipitating the unwanted proteins in the presence of sodium
caprylate and ethyl alcohol. Surprisingly, heating up to this
20 temperature does not cause irreversible denaturation changes in
the albumin, as predicted by Ivanov et al. (US-Pat. 3.926.939). In
the contrary, immuno-electrophoresis and other tests show, that
` the biophysical properties of the albumin rendered are the same
of any untreated albumin.
The following additional Examples are illustrative of the
process according to the invention.
Example 4
. ' .
The starting material is a placental serum. After the
~,
~- globulinshave been removed by known methods, the concentration
of ethanol in the solutlon is brought to 8.2~ by volume, and
sodium caprylate is added until its concentration is 0.005 mol.
Then the pH is set to 6.0, which is obtained by adding 0.5 N HCl.
,~
-! 14 -
. . .
103~Z92
rl..en the mixture is heated evenl~ in 150 minutes up to 65C. The
mixture is continually stirred during the heating process.
After reaching this temperature, the mixture is held for
30 minutes. Then the mixture is transferred and cooled down to
20C and the pH is lowered down to 4.5. The acidified plasma is now
- left to stand for three hours to let coagulate the unwanted proteins.
The solution containing pure albumin is separated from the resultant
precipitate at a temperature of 20C and pH 4.5 by centrifugation.
The albumin in the supernatant may be separated as
mentioned in Example 1, e.g. by precipitation with PEG.
Example 5
:
Methanol is added to 1 1 of placental serum until the
concentration of the alcohol has reached 10%; then sodium caprylate
is added until its concentration has reached 0.003 M, the
temperature is increased to 67.5C and maintained for
20 minutes, the pH is adjusted to 6.5 after which the mixture
is held at this pH for 20 min. Then the pH is adjusted to 4.6 and
; the concomitant proteins and non-protein impurities are precipitated.
i, The solution containing pure albumin is separated by centrifugation.
; 20 Example 6
Ethanol and sodium caproate are added to 1 1 of placental
. serum until their respective concentrations have reached 8 and
0.004 M; the pH is adjusted to 6.3; subsequently the process is
carried on in the manner described in Example 1.
~. Example 7
: .;
Ethanol and sodium caproate are added to 1 1 of placental
serum until their respective concentrations have reached 7.5 and
' 0.003 M; the pH of the mixture is adjusted to 6.3; subsequently
~ the process is carried on in the manner described in Example 1.
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