CA1038292A - Process for isolating albumin from blood - Google Patents
Process for isolating albumin from bloodInfo
- Publication number
- CA1038292A CA1038292A CA223,373A CA223373A CA1038292A CA 1038292 A CA1038292 A CA 1038292A CA 223373 A CA223373 A CA 223373A CA 1038292 A CA1038292 A CA 1038292A
- Authority
- CA
- Canada
- Prior art keywords
- albumin
- process according
- plasma
- alcohol
- blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
The extraction of pure serum albumin from blood plasma, blood products, other body fluids or tissue extracts, is effected by 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 0.001 to 0.01 moles,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 75°C 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 process of the invention 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.
The extraction of pure serum albumin from blood plasma, blood products, other body fluids or tissue extracts, is effected by 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 0.001 to 0.01 moles,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 75°C 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 process of the invention 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.
Description
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"' .:
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
'~I
-., ~ - .
. 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.
' C
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:
~ ~ .
, - 4 -.~;, .
~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 , ~,.
~""
' ~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.
-/
~ - 6 -,.~.
' ~' 'D
D
: .
., 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.
" ' . ~ .
~ ?
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 -.
'`' C
~. 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.
. . .
-~ .
,.~ .
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.
:
~, ~ .
.~
~ .~
''' . .
, ~, ''' ':
. :-. -:
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 .. . .
; -- 11 --.:' ' ` 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 :- .
' ~?
. ~
` 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 ., .
. . .
~ - 13 -"
- .
.. . .
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.
;'' .
~1 : ' ' , --
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"' .:
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
'~I
-., ~ - .
. 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.
' C
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:
~ ~ .
, - 4 -.~;, .
~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 , ~,.
~""
' ~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.
-/
~ - 6 -,.~.
' ~' 'D
D
: .
., 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.
" ' . ~ .
~ ?
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 -.
'`' C
~. 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.
. . .
-~ .
,.~ .
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.
:
~, ~ .
.~
~ .~
''' . .
, ~, ''' ':
. :-. -:
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 .. . .
; -- 11 --.:' ' ` 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 :- .
' ~?
. ~
` 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 ., .
. . .
~ - 13 -"
- .
.. . .
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.
;'' .
~1 : ' ' , --
Claims (20)
1. 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 consituents of the blood, isolating dissolved non-albumin constituents from the plasma by adding an albumin-stabiliser in a quantity of about 0.004 mole, treating the fluid with a lower ali-phatic alcohol of the formula CH3-(CH2)n-OH in which n is 0,1 or 2 at a temperature of from 60 to 75°C 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.
2. A process according to claim 1, wherein the treatment is effected at a temperature of about 68°C.
3. A process according to claim 1, wherein the alcool used is ethyl alcohol.
4. A process according to claims 1, 2, or 3 wherein the albumin-stabiliser is sodium caprylate.
5. A process according to claim 1, including the addi-tional step of enriching the albumin content of the solution.
6. A process according to claim 5, wherein the albumin is concentrated by precipitation with a protein precipitating medium.
7. A process according to claim 6, wherein use is made of from 20 to 30 % of polyethylene glycol.
8. A process according to claim 6, wherein use is made of from 15 to 20 % of maleic acid.
9. A process according to claim 5, wherein the albumin is concentrated by freeze-drying.
10. A process according to claim 5, wherein the albumin is concentrated by ultra-filtration.
11. A process according to claim 6, wherein the precipi-tated or settled albumin paste is dissolved in a buffered liquid and adjusted to a desired concentration.
12. A process according to claims 5 or 11,wherein as a final step, the albumin solution is heat sterilized without the addition of a stabiliser.
CLAIMS SUPPORTED BY THE
SUPPLEMENTARY DISCLOSURE
CLAIMS SUPPORTED BY THE
SUPPLEMENTARY DISCLOSURE
13. 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 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 CH3- (CH2)n-OH in which n is 0,1 or 2 at a temperature of from 60 to 75°C and pH of from 4.5 to 7.5 to cause precipitation of partially or largely denaturated concomitant proteins, the treatment being carried out a volume concentration of the alcohol of 7 to 14 %, and separating the resul-tant solution containing pure serum albumin from the precipitate.
14. A process according to claim 13, wherein the lower aliphatic alcohol is ethanol.
15. A process according to claim 14, wherein the treat-ment is effected at a volume concentration of the alcohol of 8 to 12 %.
16. A process according to claim 13, wherein the albumin-stabiliser is sodium caprylate in a 0.003 to 0.005 mole concentra-tion.
17. A process according to claim 13, wherein the pH-value of the treatment is 6 to 7.
18. A process according to claim 13, wherein the tempera-ture is 67 to 69°C.
19. A process according to claim 13, wherein the separa-tion of the albumin solution from the precipitate is carried out at a temperature of from 1 to 30°C.
20. A process according to claim 19, wherein the separa-tion of the albumin solution from the precipitate is carried out at room temperature.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2415079A DE2415079C3 (en) | 1974-03-28 | 1974-03-28 | Method for isolating albumin from blood plasma |
| US05/755,126 US4156681A (en) | 1974-03-28 | 1976-12-28 | Process for isolating albumin from blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1038292A true CA1038292A (en) | 1978-09-12 |
Family
ID=25766899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA223,373A Expired CA1038292A (en) | 1974-03-28 | 1975-03-26 | Process for isolating albumin from blood |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1038292A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1983002057A1 (en) * | 1981-12-17 | 1983-06-23 | Quinn, Patrick, James | Contraceptive method and composition |
| US5849874A (en) * | 1991-07-12 | 1998-12-15 | Gist-Brocades, N.V. | Process for the purification of serum albumin |
| US6504011B1 (en) * | 1991-07-12 | 2003-01-07 | Dsm N.V. | Process for the purification of serum albumin |
-
1975
- 1975-03-26 CA CA223,373A patent/CA1038292A/en not_active Expired
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1983002057A1 (en) * | 1981-12-17 | 1983-06-23 | Quinn, Patrick, James | Contraceptive method and composition |
| US5849874A (en) * | 1991-07-12 | 1998-12-15 | Gist-Brocades, N.V. | Process for the purification of serum albumin |
| US6150504A (en) * | 1991-07-12 | 2000-11-21 | Dsm Patents & Trademarks | Process for the purification of serum albumin |
| US6504011B1 (en) * | 1991-07-12 | 2003-01-07 | Dsm N.V. | Process for the purification of serum albumin |
| US6831157B2 (en) | 1991-07-12 | 2004-12-14 | Dsm Ip Assets B.V. | Process for the purification of serum albumin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4156681A (en) | Process for isolating albumin from blood | |
| US4188318A (en) | Simplified method for preparation of high yield, high purity Factor VIII concentrate | |
| CA1111023A (en) | Process for recovering purified albumin from blood plasma | |
| US4216205A (en) | Process of preparing a serum protein composition for intravenous application | |
| CA1323139C (en) | Method of producing substantially pure albumin | |
| GB1473548A (en) | Bovine immunoglobulin isolation process | |
| JPH0676337B2 (en) | Method for producing high-purity antihemophilic factor concentrate | |
| FI72653C (en) | FORMULATION OF FACTORING AV ETT FACTOR VIII (AHF) CONCENTRATE. | |
| US4104266A (en) | Method for preparation of antihemophilic factor | |
| JPH0348888B2 (en) | ||
| CA1054052A (en) | Simplified method for preparation of high yield, high purity factor viii concentrate | |
| CA1293941C (en) | Method for preparing antihemophilic factor (ahf) by cold precipitation and for improving solubility of recovered ahf product | |
| US4075197A (en) | Serum albumin production | |
| CA1112565A (en) | Process for concentrating blood coagulation factor xiii derived from human placentae | |
| US2958628A (en) | Heat treatable plasma protein product and method of preparation | |
| US4097473A (en) | Production of serum albumin | |
| CA1038292A (en) | Process for isolating albumin from blood | |
| US4294826A (en) | Process for the preparation of highly purified antihemophilic factor | |
| US4478825A (en) | Warm ethanol method for preparation of low fibrinogen antihemophilic factor | |
| US2797184A (en) | Process for the recovery of heparin | |
| JPS6242887B2 (en) | ||
| SU743564A3 (en) | Albumin producing method | |
| US4169829A (en) | Process for the preparation of purified albumin and albumin obtained by said process | |
| IE42460B1 (en) | Process for isolating albumin from blood | |
| EP0076870B1 (en) | Process for the preparation of highly purified antihemophilic factor |