Fractionation of blood plasma

There is provided in accordance with practice of this invention a process for separating protein fractions from a solution containing proteins and an apparatus for practicing the process. The process and apparatus involve withdrawing from a suspension tank a portion of the suspension contained therein to form a recycle stream. The withdrawn suspension forming the recycle stream contains particles of a selected protein fraction precipitated from a solution containing proteins. Fresh protein solution comprising proteins of the selected protein fraction is mixed into the recycle stream to form a mixed recycle stream. At least a portion of the selected protein fraction proteins in the fresh protein solution precipitate onto previously precipitated selected protein fraction particles suspended in the mixed recycle stream. The mixed recycle stream is introduced back into the suspension tank. A portion of the suspension is withdrawn from the suspension tank in a product stream and precipitated, selected protein fraction particles are recovered from the product stream.

FIELD OF THE INVENTION 
The process and apparatus provided in accordance with practice of this 
invention are useful for continuously separating protein fractions from 
solutions containing proteins. 
BACKGROUND OF THE INVENTION 
Human blood is made up of approximately 35 percent cellular components, 
including red cells, white cells, and platelets with the remaining 65 
percent being a fluid called plasma. The plasma suspends the cells and 
platelets and comprises a solution of approximately 90 percent water, 7 
percent protein, and 3 percent various other organic and inorganic 
solutes. 
The protein portion of plasma consists of various different protein 
fractions including, for example, albumin, fibrinogen, gamma (.gamma.) 
globulin, alpha (.alpha.) and beta (.beta.) globulins, and others. 
For various human therapies, it can be desired to separate and concentrate 
blood protein fractions so that only a selected single fraction is 
administered to a patient. For example, it can be particularly desirable 
to separate albumin from the plasma and provide the separated albumin in a 
concentrated solution for patient therapy. 
Methods for separating protein fractions from plasma are disclosed in U.S. 
Pat. No. 2,390,074 to E. J. Cohn and in E. J. Cohn, L. E. Strong, W. L. 
Hughes, D. J. Mulford, J. N. Ashworth, M. Melin and H. L. Taylor, 
Separation Into Fractions of Protein and Lipoprotein Components, J. Am. 
Chem. Soc. 68 (1946), p 459-475. Both U.S. Pat. No. 2,390,074 and the 
American Chemical Society article are incorporated herein by this 
reference. The protein fractions are separately precipitated by adding a 
precipitating agent or precipitant such as ethanol to the plasma and 
maintaining the plasma solution at a desired pH, temperature, ethanol 
concentration, and ionic strength to precipitate the desired fraction. 
The Cohn process is essentially a batch process where the various proteins 
are precipitated from the solution sequentially. The proteins are grouped 
into fractions with as much as possible of the fibrinogen in the plasma 
precipitated first as Fraction I. Next, Fractions II and III which are 
designated the .gamma. globulin fractions are precipitated together. 
Fraction IV-1, rich in lipid and .alpha.-globulins, is then precipitated, 
followed by precipitation of remaining .alpha.-globulins in combination 
with .beta.-globulins as Fraction IV-4. Albumin remaining in the 
supernatant (centrifugate) is next precipitated as Fraction V to obtain a 
concentrated albumin fraction. 
In each case, after the desired fraction is precipitated, the conditions of 
the supernatant plasma are changed to precipitate the next fraction. 
During the time interval required to adjust the supernatant to its 
equilibrium condition for the next precipitation, protein fractions that 
desirably remain in solution can precipitate. For example, when the 
supernatant is being adjusted to conditions for precipitating an earlier 
fraction, albumin can precipitate, thus contaminating the earlier fraction 
and also reducing the amount of albumin that can finally be recovered. 
Additionally, batch processes can be slower than desired. 
Thus, there is a need in the art for a simple and efficient process for 
precipitating protein fractions continuously that maximizes the amount of 
protein of a desired purity recovered. 
SUMMARY OF THE INVENTION 
There is, therefore, provided in practice of this invention a process for 
continuous separation of protein fractions from a solution containing 
proteins and an apparatus for practicing the process. A suspension that 
contains a precipitation agent and precipitated particles of a selected 
protein fraction is in a suspension tank. The suspension is maintained at 
a selected pH, temperature, and precipitation agent concentration for 
selectively precipitating the selected protein fraction. A portion of the 
suspension is withdrawn from the suspension tank to form a recycle stream. 
A protein solution comprising proteins of the selected protein fraction is 
continuously mixed into the recycle stream to thereby form a mixed recycle 
stream. At least a portion of the selected protein fraction proteins in 
the protein solution precipitate onto previously precipitated selected 
protein fraction particles suspended in the mixed recycle stream. The 
mixed recycle stream is introduced back into the suspension tank. A 
portion of the suspension is also continuously withdrawn from the 
suspension tank in a product stream. Precipitated selected protein 
fraction particles are recovered from the product stream.

DETAILED DESCRIPTION 
Referring to FIG. 1, a generalized schematic diagram is provided showing 
principles of the process of this invention for recovering a selected 
protein fraction from a solution containing proteins. 
A suspension 10 containing particles of a selected protein fraction 
precipitated from a solution comprising proteins is mixed at a selected 
temperature in a tank 12 with a precipitation agent and a buffer solution. 
The amount of buffer and precipitation agent in the suspension provide the 
suspension with a pH and precipitation agent concentration so that, at the 
selected temperature, only the selected protein fraction precipitates. 
Thus, other proteins that are present remain in solution. 
A portion of the suspension is withdrawn from the tank in a recycle stream 
14. Fresh protein solution that includes proteins of the selected protein 
fraction is introduced in stream 16 into the recycle stream 14 forming a 
mixed recycle stream 18. At least a portion of the selected protein 
fraction in the fresh protein solution precipitates onto previously 
precipitated selected protein fraction particles in the mixed recycle 
stream. The mixed recycle stream is introduced back into the suspension in 
the tank 12. 
A portion of the suspension is withdrawn from the tank 12 in a product 
stream 20. The product stream is sent to a separation means 22 where the 
precipitated selected protein fraction particles are separated from a 
supernatant and recovered. 
If desired, the above described process can be provided in a plurality of 
stages in series with the supernatant from a preceding stage being sent to 
the next stage for recovery of a selected protein fraction remaining in 
the supernatant. In such a system, the process can be repeated from stage 
to stage at different combined conditions of pH, temperature, and 
precipitation agent concentration as desired until all of the desired 
protein fractions are separated. 
The basic separation chemistry used in practice of this invention is that 
developed by Cohn and described in E. J. Cohn et al, Separation Into 
Fractions of Protein and Lipoprotein Components, J. Am. Chem. Soc. 68 
(1946), p 459-475. This article is incorporated hereinabove by reference. 
Referring to FIG. 2, there is shown a schematic view of a preferred 
embodiment of an apparatus 24 provided for separating protein fractions 
from a solution containing proteins according to practice of the process 
described above. 
A tank 26 is provided for holding a suspension 28 containing particles of a 
selected protein fraction precipitated from a protein containing solution. 
The precipitated particles are maintained in suspension by an agitator 30 
in the tank. 
Separate supply containers 32, 34, and 36, respectively, are provided for a 
buffer solution, a liquid precipitation agent or precipitant, and fresh 
protein containing solution. 
Precipitation agents useful in practice of principles of this invention are 
alcohols, acetone, and other water miscible organic solvents. Ethanol is 
the preferred precipitation agent and, for purposes of exposition herein, 
the process is described below using ethanol. Other precipitation agents 
can be used if desired. 
A pipeline 38 connects the ethanol or precipitant tank 34 to the top of the 
suspension tank 26. A metering pump 40 in the line 38 pumps the ethanol 
from the tank 34 into the top of the suspension tank 26. Preferably, a 
spray nozzle 42 is on the end of the line 38 for disbursing the ethanol in 
a fine mist as it enters the suspension tank. 
As is described below in greater detail, the level of the suspension in the 
tank 26 is maintained below the top of the tank. Preferably, the spray 
nozzle 42 is located above the top surface 28a of the suspension in the 
tank so that the ethanol is sprayed onto the surface of the suspension. 
Spraying the precipitant onto the surface of the suspension tends to 
eliminate foaming, which has been found to denature the proteins being 
recovered. 
If desired, more than one spray nozzle can be provided for spraying the 
ethanol uniformly across the suspension surface. 
The buffer solution tank 32 is connected to the top of the suspension tank 
26 by a pipeline 44. A metering pump 46 in the line 44 pumps the buffer 
solution from the tank 32 into the top of the tank. As was the case with 
ethanol, preferably a spray nozzle 48 is on the end of the line 44 for 
disbursing the buffer solution in a fine mist as it enters the tank. The 
spray nozzle 48 preferably is located above the top surface 28a of the 
suspension in the tank so that the buffer is sprayed onto the surface of 
the suspension. As was the case with spraying the ethanol, the buffer 
spray tends to eliminate foaming. Additionally, more than one nozzle can 
be provided if desired for spraying the buffer into the tank. 
It is preferred that the particle size of both the ethanol droplets and the 
buffer solution droplets sprayed into the tank are as small as possible so 
that the time it takes to mix the ethanol and buffer into the suspension 
is minimized. It has been found that when mixing time is reduced, 
precipitation of proteins from fractions other than the selected fraction 
is inhibited. Thus, by providing the buffer and ethanol in small droplets 
to reduce mixing time, unwanted precipitation is reduced which, in turn, 
enhances the purity of the product and increases yields. 
In one exemplary embodiment of the apparatus 24, the ethanol spray nozzle 
42 and the buffer spray nozzle 48 provide an average ethanol and buffer 
particle size of between about 50 and 100 microns (.mu.m). Having an 
average particle size between 50 and 100 .mu.believed to result in mixing 
that is rapid enough to substantially eliminate precipitation of unwanted 
fractions. Thus, although spray nozzles providing any size droplets can be 
used, those that provide an average droplet size of less than about 100 
.mu.m are preferred. 
Additionally, it has been found that adding ethanol to a protein suspension 
can create localized overheating which can tend to denature the proteins. 
Spraying the alcohol into the tank in a fine mist reduces such overheating 
and thus reduces the occurrence of protein denaturation. 
As is mentioned above, the selected protein fraction is precipitated by 
maintaining the suspension 28 at a pH, ethanol concentration, and 
temperature so that proteins of the selected fraction are precipitated, 
while other protein fractions remain in solution. 
The pH of the suspension is controlled by an automatic pH control and 
monitoring system generally shown at 50. In the illustrated embodiment, 
the pH system comprises a pH probe 52 in the tank operatively connected 
via a pH controller 54 to the buffer solution metering pump 46. In 
operation, the desired pH for precipitation of the selected protein 
fraction is set on the controller. The metering pump 46 is automatically 
controlled to pump more or less buffer solution from the buffer solution 
tank 32 into the suspension tank 26 to maintain the desired pH. 
Depending on the pH value desired for precipitating a particular protein 
fraction, the buffer solution can be acidic or basic. Buffers useful in 
practice of this invention are described below in greater detail. 
The temperature of the suspension in the tank 12 is monitored and 
controlled by an automatic temperature control and monitoring system (not 
shown). Since the precipitation reaction is exothermic, the suspension 
must be cooled to maintain its temperature within the desired range. 
Preferably, a cooling jacket 56 is provided on the tank 26 through which a 
coolant flows. The automatic temperature monitoring and control system 
maintains proper coolant conditions so that the suspension temperature is 
automatically maintained at its desired value. 
Systems useful for both automatic pH and temperature control are 
commercially available. 
A recycle line 58 is connected at its inlet end 58a to the suspension tank 
for withdrawing the suspension from the tank and is connected at its 
outlet end 58b to the suspension tank for recycling the suspension back 
into the tank. Preferably, the recycled suspension is introduced back into 
the suspension in the tank below its surface 28a to reduce foaming. A 
recycle pump 60 is in the line 58 between the inlet and outlet to recycle 
the suspension. 
Fresh protein solution is added to the system from the protein supply tank 
36. A supply line 62 connects the protein tank 36 to the recycle line 58 
either at or just upstream of a static mixer 64 in the recycle line. A 
protein solution pump 66 pumps the fresh protein solution from the tank 36 
into the recycle stream in the line 58 so that the fresh protein is mixed 
with the recycling suspension in the mixer 64. This forms a mixed recycle 
stream downstream of the mixer in that portion of the recycle line between 
the mixer and the tank 26. The mixed recycle stream is returned to the 
tank. 
Preferably, the fresh protein solution is pumped continuously into the 
system to provide a continuous protein separation process. 
As is mentioned above, the suspension withdrawn from the suspension tank to 
form the recycle stream is at the proper conditions of pH, ethanol 
concentration, and temperature for precipitating the selected protein 
fraction from solution. Thus, proteins of the selected fraction contained 
in the fresh protein solution precipitate on previously precipitated 
selected protein fraction particles in the suspension as it passes through 
the recycle line. 
It is thought that precipitation of the selected protein fraction from the 
fresh protein solution is virtually complete by the time the mixed recycle 
stream re-enters the tank 26. Since the selected protein fraction tends to 
precipitate in the recycle line on previously precipitated particles, the 
average particle size of the precipitate is increased. This enhances the 
ease of separation of the precipitate from the suspension and tends to 
increase yields from the process. 
Additionally, the entering fresh protein solution does not encounter a wide 
range of pH and temperature conditions as is the case in a batch process, 
as the batch is being brought to equilibrium conditions for precipitation. 
Thus, proteins are less likely to be denatured by practice of this 
invention. 
The ethanol concentration in the suspension required to precipitate the 
selected protein fraction is maintained by adjusting the flow rate of 
ethanol into the tank 26, based on the amount of fresh protein solution 
being introduced. 
Precipitated particles of the selected protein fraction are recovered by 
removing the suspension from the tank and sending it to a separator such 
as a centrifuge. 
In an exemplary embodiment, the suspension is removed from the suspension 
tank 26 in a product stream which flows through a pipeline 67 into a 
product receiving tank 68. 
The amount of suspension removed from the tank as product is balanced 
against the amount of material entering the system to maintain the tank at 
a constant level. The desired level is automatically maintained by a level 
control system generally shown at 70. The level control system 70 
comprises a float 72 operatively connected to an outlet valve 74 in the 
line 67 between the suspension tank and the product tank 68. The valve 74 
is throttled open or closed by a signal from the level control system to 
maintain the desired suspension tank level. 
The suspension is pumped from the product receiving tank 68 into a 
centrifuge 76 by a pump 78. The pump is in a line 80 that connects the 
product tank to the centrifuge. If desired, the product receiving tank can 
be eliminated and the suspension can be sent directly to the centrifuge 
from the suspension tank. 
The precipitated selected protein fraction particles are separated in the 
centrifuge from the suspension forming a supernatant or centrifugate and a 
paste comprising the selected protein fraction particles. The selected 
protein fraction particles and the centrifugate are then recovered. 
If desired, after a first protein fraction is recovered from a protein 
solution using the apparatus 24, the same apparatus can be used for 
recovery of another protein fraction from the centrifugate recovered from 
the first separation. This process can be repeated until all desired 
protein fractions are separated. 
Alternatively and preferably, a plurality of apparatus such as the 
apparatus 24 can be provided in series operation. In such a system, each 
apparatus 24 provides one stage or module for separating a particular 
protein fraction from a protein solution For example, the centrifugate 
from a previous stage can be collected in a centrifugate container 77 and 
the centrifugate can be introduced as fresh protein solution into the next 
stage. Any number of stages can be used depending upon the number of 
protein fractions being separated. 
An example of use of the apparatus 24 for sequentially separating a 
plurality of selected protein fractions from the protein containing 
solution blood plasma in accordance with this invention is set forth 
below. 
EXAMPLE 1 
Separating Protein Fraction I From Blood Plasma 
Using an apparatus similar to the apparatus 24 described above, protein 
fraction I was precipitated from blood plasma and recovered. 
A buffer solution comprising 0.1 molar acetic acid and 8 percent by volume 
ethanol was prepared and poured into the buffer solution tank 32. A 
precipitant (precipitation agent) solution comprising 53.3 percent by 
volume ethanol and water was prepared and poured into the ethanol tank 34. 
Fresh blood plasma having a pH of about 7.99 was prepared and poured into 
the fresh protein solution or plasma tank 36. 
To start the process, the buffer pump 46, recycle pump 60, precipitant pump 
40, and protein solution pump 66 were all started simultaneously to 
introduce the respective fluids into the suspension tank 26. The pump 
settings were as follows: the protein pump was set at 1,000 milliliters 
(ml) per minute; the precipitant pump was set at 174 ml per minute; the 
buffer pump was set at 71 ml per minute; and the recycle pump was set at 
10 liters (l) per minute. 
The suspension being formed in the tank 26 was thoroughly mixed by the 
agitator or impeller 30 which was turning at 100 revolutions per minute 
and was cooled to about -2.degree. C. by coolant flowing through in the 
coolant jacket 56 on the tank. The temperature was maintained at 
-2.degree. C. automatically by setting the automatic temperature 
monitoring and control system accordingly. The blood plasma in the tank 
36, the buffer solution in the tank 32, and the ethanol in the tank 34 was 
maintained and pumped into the suspension tank at from about +2.degree. C. 
to about -5.degree. C. 
When the pH of the suspension being formed reached about 7.0, the pH 
controller was set at 7.0 and the pH was thereafter maintained near this 
value (between 6.85 and 7.14 as measured by laboratory sample) by 
automatically operating the buffer pump as needed. 
During the initial formation of the suspension 28 in the tank, fraction I 
proteins precipitated from solution. Although, during startup other 
protein fractions may also have precipitated, they are redissolved in 
solution once equilibrium conditions for the fraction I precipitate are 
met. 
The conditions of the suspension desired for selectively precipitating 
fraction I are a pH of about 7.0, temperature of about -2.degree. C. and 
an ethanol concentration of about 8 percent by volume. 
The level of suspension desired to be maintained in the suspension tank 26 
was set at about 30 liters. At 30 liters, the top of the suspension was 
below the top of the tank so that the buffer solution and ethanol could be 
sprayed into the tank as described above. The level was controlled 
automatically by the liquid level controller 70. Thus, when the level 
reached 30 liters, the discharge valve 74 was throttled open to discharge 
the suspension continuously at the same rate as the fresh plasma, ethanol, 
and buffer were being added to the tank. The estimated retention time of 
the suspension in the tank was 24 minutes. 
The suspension was discharged from the suspension tank into the product 
receiving tank 68 and was then pumped from the receiving tank to the 
centrifuge 76 for separation. The fraction I precipitate was recovered 
from the centrifuge and a centrifugate was recovered and held for further 
processing. 
In each of the following examples, one or more selected protein fraction 
was separated from the centrifugate recovered from a previous separation. 
In Example 2, protein fractions II and III were separated from the 
centrifugate prepared in Example 1. In Example 3, protein fraction IV-1 
was separated from the centrifugate prepared in Example 2. In Example 4, 
fraction IV-4 was separated from the centrifugate prepared in Example 3 
and, in Example 5, protein fraction V was separated from the centrifugate 
prepared in Example 4. 
The operating conditions for Examples 1 through 5 are summarized in Table I 
as follows: 
TABLE I 
__________________________________________________________________________ 
OPERATING CONDITIONS FOR EXAMPLES 1-5 
__________________________________________________________________________ 
Ethanol 
Retention Concentration 
Time in 
Protein 
in pH Temperature 
Buffer 
Example 
Suspension 
Fraction 
Suspension 
Range in 
of Solution 
No. Tank Recovered 
(% by vol.) 
Suspension 
Suspension 
Used 
__________________________________________________________________________ 
1 24 min. 
I 8 6.85-7.14 
-2.degree. C. 
.1 M acetic 
acid in 8% 
ethanol 
2 20 min. 
II & III 
20 6.85-6.95 
-5.degree. C. 
.1 M acetic 
acid in 20% 
ethanol 
3 19 min. 
IV.sub.1 
20 5.24-5.26 
-5.degree. C. 
.3 M acetic 
acid in 20% 
ethanol 
4 14 min. 
IV.sub.4 
40 5.91-5.93 
-5.degree. C. 
.2 M 
sodium 
bicarbonate 
in 40% 
ethanol 
5 13 min. 
V 40 4.81-4.83 
-6.degree. C. 
.8 M acetic 
acid in 40% 
ethanol 
__________________________________________________________________________ 
Ethanol 
Solution Buffer 
Protein 
Concentration 
Suspension Solution 
Solution 
in Ethanol 
Recycle 
Ethanol 
Flow Rate 
Example 
Flow Rate 
Tank Rate Flow Rate 
(ml/min. 
No. (l/min.) 
(% by vol.) 
(l/min.) 
(ml/min.) 
average) 
__________________________________________________________________________ 
1 1.000 53.3 10 174 71 
2 1.207 95.0 10 186 77 
3 1.396 -- 10 No ethanol 
189 
added 
4 1.486 95.0 10 568 178 
5 2.062 -- 10 No ethanol 
182 
added 
__________________________________________________________________________ 
No ethanol was added to the suspension tank in Examples 3 and 5 because the 
centrifugate recovered from Examples 2 and 4 and used in Examples 3 and 5, 
respectively, already had the proper ethanol concentration for 
precipitation of the desired fractions. 
Typical analyses of the selected protein fractions recovered by the 
exemplary process described above are set forth in Table II. 
TABLE II 
______________________________________ 
TYPICAL COMPOSITION OF RECOVERED 
PROTEIN FRACTIONS (% BY WEIGHT) 
Ex- 
am- Serum 
ple Protein Albu- .alpha.-Glob- 
.beta.-Glob- 
.gamma.-Glob- 
Fibrin- 
No. Fraction min ulin ulin ulin ogen 
______________________________________ 
1 I 24.9 9.6 17.4 19.6 28.5 
2 II & III 11.6 15.0 12.6 49.1 11.7 
3 IV.sub.1 23.9 25.2 14.8 36.1 -- 
4 IV.sub.4 34.3 32.8 32.9 -- -- 
5 V 92.4 3.8 3.8 -- -- 
______________________________________ 
It is estimated that at least about 82% of the original albumin that is 
introduced into the system in the fresh plasma can be recovered by 
practice of this invention in Fraction V. 
The above description of a preferred embodiment of a process for separating 
selected protein fractions from a solution containing proteins and 
apparatus useful for practicing the separations is for illustrative 
purposes. Because of variations which will be apparent to those skilled in 
the art, the present invention is not intended to be limited to the 
particular embodiments described above. The scope of the invention is 
defined in the following claims.