Fractionation of proteins

The invention is concerned with fractionation of proteins, in particular with fractionation of immunoglobulin containing solutions, such as blood plasma, by continuous flow electrophoresis. Hitherto, such solutions have been fractionated by ethanol precipitation methods (e.g. the Cohn Method). Such methods separate the IgG component of immunoglobulins but cannot separate the useful IgM component. In the invention, the solutions are fractionated by continuous flow electrophoresis at a pH of between 7 and 8.4 and an electrical conductivity of between 1 and 2 mScm.sup.-1, thereby giving rise to fractions containing particular combinations of immunoglobulin components (IgG, IgM, IgA, IgD and IgE) with potentially valuable properties.

This invention relates to the fractionation of immunoglobulin containing 
solutions, such as blood plasma, by continuous flow electrophoresis. 
The immunoglobulins constitute a family of complex proteins contained, for 
example, in blood plasma. They may be made up of several constituents each 
of which has different properties from the others in some respect or 
other. There are five main components found in immunoglobulins and these 
are usually designated as IgM, IgA, IgG, IgD and IgE respectively. Certain 
of the components may be separated by techniques known in the art such as 
an ethanol precipitation method known as the Cohn Method which separates 
the IgG component, but which cannot separate the IgM component which has 
antibacterial antibodies lacking in the IgG component. 
We have now established conditions under which immunoglobulins may be 
fractionated by means of continuous flow electrophoresis and surprisingly 
found that certain of the fractions so obtained may be constituted by 
mixtures of certain components having particularly advantageous 
properties. 
The present invention provides a method of fractionating an immunoglobulin 
containing aqueous solution which comprises the steps of 
(i) adjusting the pH of the aqueous solution to between 7 and 8.4 and the 
electrical conductivity thereof to between 1 and 2 mScm.sup.-1 as measured 
at 20.degree. C.; 
(ii) subjecting the product of step (i) to continuous flow electrophoresis 
by injecting it as a migrant solution into a second aqueous solution, 
laminarly flowing in an annular separation chamber as a carrier solution 
for the migrant solution and stabilised by means of an angular velocity 
gradient, said carrier solution having a pH of between 7 and 8.4, and by 
applying a constant electric field across the resulting mixture to produce 
differential movement of the components of the immunoglobulin relative to 
themselves and to any other major components of the solution perpendicular 
to the direction of flow of the carrier solution; and 
(iii) collecting resulting particular fractions containing one or more 
components of the immunoglobulin. 
The fractions may then, if desired, be concentrated e.g., by hollow fibre 
membrane concentrators, and then freeze dried. The fractions may be 
reconstituted in a small volume of liquid. Analysis of such reconstituted 
fractions for IgG, IgM, IgA, IgD and IgE components has shown that 
particular fractions may contain particular relative proportions of such 
components, which particular fractions may possess specific value for 
immunisation against specific infections. Further details are contained in 
the example of this specification. 
Step (ii) is most conveniently carried out as generally described in U.K. 
Patent Specification No. 1,186,184 (corresponding to U.S. Pat. No. 
3,616,453), which describes a process and apparatus where stabilisation of 
flowing streams in continuous flow electrophoresis is effected by an 
angular velocity gradient. Thus, in our invention, the fractionation may 
be effected in an annular separation chamber defined between a central 
stationary cylinder (a stator) and an outer rotating cylinder (a rotor), 
which results in a gradient of angular velocity across the annular chamber 
giving laminar flow at high throughputs. The constant electric field is 
then applied across the annular chamber to produce the differential 
movement of the immunoglobulin components of the migrant solution. 
Improvements and/or modifications of the apparatus described in U.K. Pat. 
No. 1,186,184 are described in U.K. patent specification Nos. 1,431,887 
and 1,431,888 (corresponding to U.S. Pat. No. 3,844,926). 
The pH of the migrant and carrier solutions in step (ii) is, as stated, 
between 7 and 8.4; very effective fractionation is found to occur in this 
range, which also enables factor VIII, and for albumin, if present, to be 
fractionated at the same time, e.g., where the aqueous solution comprises 
blood plasma. Separation of factor VIII by continuous flow electrophoresis 
is described in the specification of our International Patent Application 
No. PCT/GB78/00038, filed Nov. 10, 1978 (Agents' Reference 11925 M1H) 
which describes inter alia a method of purifying a factor VIII containing 
aqueous solution characterised by the steps of 
(i) reducing the ionic strength of the solution to a level such that it is 
capable of being electrophoresed; 
(ii) adjusting the pH of the solution to within a range where the stability 
of Factor VIII is not adversely affected; 
(iii) subjecting the product of step (ii) to continuous flow 
electrophoresis by injecting the solution as a migrant solution into a 
second aqueous solution, laminarly flowing in an annular separation 
chamber as a carrier solution for the migrant solution and stabilised by 
means of an angular velocity gradient, and applying a constant electric 
field across the resulting mixture to produce a differential movement of 
the factor VIII component of the migrant solution with respect to the 
other major components of the solution perpendicular to the direction of 
flow of the layer; and 
(iv) collecting the separated Factor VIII component. 
It is surprising that our invention operates so satisfactorily when the 
migrant solution has a pH of 7.5. Thus, the isoelectric point of 
immunoglobulins are high (between about pH 7.5 and 8.5) and under 
conditions of electrophoresis at pH 7.5, it might be expected that the 
immunoglobulins would not exhibit any movement or that they might move 
into the membrane in the apparatus described in U.K Patent Nos. 1,431,887 
and 1,431,888. This, however, does not happen in our experience. 
A suitable buffer for the migrant solution is triscitrate and we prefer 
that its electrical conductivity is in the range of 0.75 to 1 mScm.sup.-1 
as measured at 20.degree. C. 
Step (iii) may be carried out as described in U.K. Patent Nos. 1,431,887 
and 1,431,888. Thus, if our method is carried out as described in these 
specifications, the direction of migration of the migrant solution is 
centrifugal and the injection thereof accordingly effected at the inner 
side of the flow of the carrier solution. The direction of flow is 
generally upward and is helical in pattern because of the effect of the 
rotation of the rotor. Particular fractions may then be collected by means 
of an off-take system located in the stator and consisting of a series of 
parallel mazeplates with spacers. A particular fraction may then pass 
through one or more particular mazeplates and hence into collecting 
tube(s).

The invention will now be particularly described, by way of example only, 
as follows. 
Example 
Outdated frozen human blood plasma (250 ml) was thawed rapidly and dialysed 
overnight against an aqueous tris-citrate solution (10 L; pH 7.5; 
conductivity 1 mScm.sup.-1) at 4.degree. C. in order to reduce the salt 
concentration of the plasma. The dialysed plasma was then diluted 
approximately 1.5 times with an aqueous tris-citrate solution to give a 
product of pH 7.5 and an electrical conductivity of 1.0 mScm.sup.-1 at 
20.degree. C. 
The above product, as a migrant solution, was then warmed to 20.degree. C. 
and electrophoresed using a continuous electrophoretic separation 
apparatus of the type generally described in U.K Patent Specification Nos. 
1,431,887 and 1,431,888. The apparatus had 29 outlet ports, a stator 
radius of 40 mm, a rotor radius of 45 mm to give an annular gap of 5 mm, 
and electrodes 304 mm in length. A carrier solution at 2.degree. C. 
comprising an aqueous tris-citrate solution (pH 7.5; electrical 
conductivity 0.75 mScm.sup.-1 at 20.degree. C.) was passed upwardly 
through the annular gap at a rate of 500 ml/minute and the flow stabilized 
by rotation of the rotor. The migrant solution was injected into the 
annular gap at a rate of 10 ml/minute. The electrophoresis was carried out 
at 35 amps and 27 volts giving a temperature rise of carrier solution of 
20.degree. C., i.e., from 2.degree. C. to 22.degree. C. The electrolytes 
were ammonium acetate (1 M; pH 7.5) for the cathode and an equal volume 
mixture (pH 7.5) of ammonium citrate (0.2 M) and ammonium phosphate (0.15 
M) for the anode. 
The particular fractions were each collected and concentrated by hollow 
fibre membrane concentrators and then freeze dried. Each fraction was 
re-constituted in a small volume of distilled water and analysed for the 
immunoglobulin components IgG, IgM, IgA, IgD and IgE by quantitative 
immunoelectrophoresis. The results are shown in Table I below as 
percentage of activity per minute per off-take. 
TABLE 1 
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Fraction 
No IgG IgM IgA IgD IgE 
______________________________________ 
1 0.9 0.1 
2 3.1 0.3 
3 7.4 0.6 0.2 
4 8.6 1.9 0.4 
5 6.5 4.0 0.4 0.6 
6 6.8 5.7 0.1 1.2 0.5 
7 6.8 5.9 0.2 1.7 0.8 
8 10.1 9.0 0.7 5.2 1.4 
9 14.1 17.5 3.4 15.6 5.2 
10 10.0 16.1 6.6 20.3 7.8 
11 9.6 16.4 15.0 23.4 20.2 
12 6.7 9.0 17.9 12.7 24.0 
13 4.5 6.4 20.0 10.8 15.6 
14 3.2 4.5 21.1 5.8 15.0 
15 1.9 2.7 15.0 2.8 8.5 
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It will be seen that, apart from IgG which shows a broad spread across 
about 15 fraction numbers, the bulk of the remaining immunoglobulin 
components are present in about six fraction numbers, specifically 8 to 
13. Also, it will be noted that at the pH of this example (7.5) the 
mobilities of the immmunoglobulin components are sufficiently different 
for fractions to be collected in order to maximise the proportion of any 
one particular component, The order of decreasing mobility is IgA, IgE, 
IgD, IgM and IgG. Table II below shows how fractions may be combined in 
order to obtain mixtures of immunoglobulin components of various 
composition. 
TABLE II 
______________________________________ 
Fractions 
Numbers Total 
Combined Immunoglobulin % 
______________________________________ 
IgG 40 
1-7 IgM 20 
IgG 45 
IgM 60 
8-11 IgA 25 
IgD 65 
IgE 35 
IgG 15 
IgM 20 
12-15 IgA 75 
IgD 35 
IgE 65 
______________________________________ 
Certain of the fractions obtained were combined and analysed for specific 
antibodies, namely anti-tetanus, anti-measles, anti-rubella and anti-polio 
virus type III. The techniques used for analysis were radial 
immuno-diffusion for anti-tetanus antibodies, haemaglutination inhibition 
tests for anti-measles and anti-rubella antibodies, and tissue culture 
neutralization tests for anti-polio virus type III antibodies. The results 
are shown in Table III below. 
TABLE III 
______________________________________ 
Fractions 
Specific Antibodies (%/fraction) 
Numbers Polio virus 
(combined) 
Tetanus Measles Rubella Type III 
______________________________________ 
1, 2 17.7 16.6 17.5 22.6 
3, 4 31.5 44.2 51.1 30.2 
5, 6 23.5 17.4 20.2 16.9 
7, 8 13.8 12.5 7.2 12.3 
9, 10 9.4 5.0 2.9 9.7 
11, 12 4.1 4.4 1.2 8.4 
______________________________________ 
It will be seen that the anti-bodies are principally contained in the early 
fractions up to fraction 6. These early fractions contain mainly the IgG 
and IgM components (see Table I). Thus, by combining specific fractions in 
a pool, high specific activity anti-body preparations can be prepared 
simply, leaving the other fractions for other uses. Also, IgM, which is a 
large molecular weight immunoglobulin, tends to possess good antibacterial 
properties and fractions containing it might be used in treatment where 
the prime aim is to obtain immunity against certain bacterial infections. 
It should be noted that the fractions obtained were not necessarily pure. 
Thus, from fraction numbers 8 to 10 and higher, other plasma proteins were 
present including fibrinogen which may be removed by other means. This 
though is probably not of great practical importance. However, albumin, 
the major plasma protein, was well separated and all of the fractions were 
albumin free.