Process for the preparation of blood plasma fractions

A precipitation process for the preparation of blood plasma constituents is disclosed, wherein the plasma to be fractionated and the precipitant are circulated in a closed system.

The present invention relates to a process for the preparation of plasma 
fractions by precipitation. 
Albumins and globulins are being used in human and veterinary medicine for 
some application purposes. These proteins are obtained from human or 
animal plasma by fractional precipitation. The precipitation process has 
been carried out hitherto in the following manner: The plasma was placed 
initially batchwise into the reaction vessel and the precipitant was 
metered in slowly thus causing the individual fractions to precipitate. 
The disadvantages of this discontinuous process reside in the fact that it 
is very time-consuming because of the long dwell times required and that 
it yields only difficulty separable precipitates. 
It was therefore a task to provide a fractionation process which should 
yield easily separable precipitates within an acceptable period of time. 
It has now been found that this task can be solved when circulating the 
plasma to be fractionated, the precipitant and the buffer in a closed 
system. 
Subject of the present invention, consequently, is a process for the 
preparation of plasma fractions by mixing plasma with a precipitant, at a 
constant temperature and in the presence of a buffer, followed by 
separation of the precipitated plasma protein, which comprises circulating 
the mixture consisting of plasma, precipitant, buffer and precipitated 
protein, in a loop reactor consisting of a closed tube circuit connected 
with a circulating pump. 
The particular features of this process reside in the fact that the mixture 
consisting of plasma, precipitant, buffer and precipitated protein is 
circulated in a loop reactor consisting of a stirring tank, the outlet of 
which is connected with the inlet of a circulating pump and the inlet of 
which is connected with the outlet of this circulating pump, that the 
plasma, the precipitant and the buffer are conveyed to the system 
separately in a zone of high turbulence and that a quantity of reaction 
mixture corresponding to the total quantity of plasma, precipitant and 
buffer metered in is withdrawn continuously from the system via a branch 
pipe located between the outlet of the stirred tank and the inlet of the 
pump. 
The process according to the invention is carried out in a "loop-type" 
reactor. This loop reactor consists of a stirring tank and pipings forming 
a closed circular system, a circulating pump being connected with this 
system. 
A further feature of this process is that the reaction mixture withdrawn 
from the loop reactor flows through a dwell zone prior to the separation 
of the precipitated protein. 
The stirring tank corresponds to those commonly used. It is provided with 
means for measuring and controlling the temperature and the pH of the 
precipitant, for heating or cooling, respectively, the contents of the 
tank and for stirring up the contents of the tank. Heating or cooling may 
be done by a double jacket or by exterior or interior tube coils and the 
like. The contents of the tank are stirred up by agitator means driven by 
a motor, by introducing an inert gas or by means of the impulse of the 
precipitant added. The inlet and the outlet for the medium kept 
circulating may be arranged at any point, preferably, however, the outlet 
is located at the bottom of the tank and the inlet pipe entering into the 
tank at the head ends below the liquid level. The outlet of the tank 
connected with the inlet of a circulating pump and the outlet of this pump 
is connected with the inlet of the tank. A branch pipe is located between 
the outlet of the tank and the inlet pipe of the pump. This branch pipe 
may be shut off by means of a valve. It leads to separating means, for 
example a filter or a centrifuge. A zone of particular high turbulence, 
for example, at the pump inlet, is moreover created by the admission pipes 
for the medium to be fractionated, the precipitant and the buffer, these 
pipes passing over the metering pumps. As circulating pump there is used a 
pump with high output, preferably a glandless centrifugal pump, whereas 
suitable metering pumps are hose pumps, gear pumps, reciprocating pumps or 
diaphragm pumps. 
Depending on the size of the apparatus, the pipes and pumps, too, are 
maintained at a certain temperature, by a double jacket, or by heating and 
cooling coils and the like or the whole loop reactor, including the 
metering pumps, is placed in a room kept at a certain temperature. 
Suitable materials for this system are glass, enamel, noncorrosive plastic 
or metal, for example stainless steel. The material chosen depends in the 
first place on the size of the system. Small apparatuses may be made 
completely of metal, glass or enamel. 
The size of the stirred tank depends on the quantities of matter to be 
converted and is in the range of from 0.5 to 100 liters, preferably of 
from 0.5 to 10 liters. The dimensions of the other parts of the system are 
adapted in each case to the size of the stirred tank.

The preferred design of an apparatus to be used according to the invention 
can be seen from the drawing. The stirring tank (1) provided with agitator 
means (2) is connected with the circulating pump (4) via conduit (3). 
Conduit (5) extending from pump (4) returns to the stirring tank (1). 
Conduit (6) branching off conduit (3) leads to separating means (not shown 
in the FIGURE) by passing over dwell zone (6a). Conduit (6) may be sut off 
by valve (7). The metering pumps (8), (10) and (12) are moreover connected 
with the inlet of pump (4) by conduits (9), (11) and (13). The stirring 
tank is kept at a certain temperature by means of a double jacket (14). 
The whole apparatus may alternatively be placed in a room maintained at a 
certain temperature, which is indicated by the dotted line (15). 
The process according to the invention is used preferably for the 
fractionation of human or animal plasma or plasma fractions, respectively, 
but it may also be used for the fractionation of other protein matter. 
The precipitant chosen has to be adapted to the material to be 
precipitated, (NH.sub.4).sub.2 SO.sub.4 solution, alcohol such as ethanol 
or a polyether alcohol, for example polyethylene glycol, being used 
preferably for human or animal plasma. The quantity of the precipitant, 
too, depends on the nature and on the quantity of the protein fractions to 
be precipitated and may be determined by a preliminary test. 
An acid or an alkaline liquor or a commercial buffer, respectively, is used 
for maintaining the optimal pH for the precipitation process. 
The mixture consisting of the medium to be fractionated, the precipitant, 
the buffer and the precipitated protein is withdrawn from the stirred tank 
by means of the circulating pump and recycled thereto at another point. 
Part of this mixture is continuously withdrawn from the stirred tank 
through the branch pipe and is passed to the separating means, where the 
precipitated protein is separated from the mixture. The corresponding 
total quantity of the medium to be fractionated, of precipitant and buffer 
is simultaneously conveyed to the system by means of the metering pumps so 
that the material balance in the system is substantially in equilibrium. 
The individual components may be metered in continuously or at intervals, 
it being important that possible fluctuations of the concentration in the 
whole loop reactor be within the acceptable limits for the protein 
fraction to be precipitated. 
The process according to the invention yields protein flocks that can be 
easily separated. When there is used a centrifuge for the separation 
process, the recipient may be emptied continuously, since the centrifugal 
forces required for separating the protein flocks are very little. The 
output in the apparratus according to the invention may be increased to a 
multiple of that reached in the known precipitation processes, owing to 
the fact that the residence time in the loop reactor is short. It has 
moreover been shown that the process of the invention does not involve an 
increased danger of a bacterial invasion which would have to be expected 
in the case of fairly long operation times. 
EXAMPLE 1 
The stirring tank (1) according to the accompanying drawing is made of 
glass, has a volume of 1.5 liters, is doublewalled and is kept at a 
temperature of -2.degree. C. Conduits (3) and (5) consist of insulated 
glass double-pipes. The contents of the stirring tank (1) and of the 
conduits (3) and (5) are made circulate by the glandless centrifugal pump 
(4) at a rate of 200 liters/h. The hose pumps (8), (10) and (12) convey to 
the system 800 ml/h of a human plasma, 93 ml/h of ethanol, and a 
commercial buffer (pH 4.8) in a quantity such that a pH of 7.4 is reached 
in the system. 900 ml/h of reaction liquid are withdrawn continuously from 
the system through valve (7) and subsequently submitted to centrifugation, 
whereby there are separated about 10 g/h of an enriched fibrinogen 
fraction. 
EXAMPLE 2 
The apparatus used is analogous to that of Example 1, except that the 
vessel has a volume of 3.0 liters. 800 ml/h of human plasma, 280 ml/h of 
ethanol and buffer solution are conveyed to this apparatus at a 
temperature of -6.degree. C. at pH 7.6, while 1,000 ml/h of reaction 
liquid are withdrawn. There are obtained about 50 g/h of an enriched 
immunoglobulin fraction.