Method for the purification of interferon

Human fibroblast interferon may be purified to a high degree by using a simple two-step purification method comprising (a) subjecting an aqueous interferon solution to chromatography on porous glass beads, and (b) subjecting the resulting aqueous interferon solution to chromatography on immobilized zinc chelate. Overall recoveries of about 45-76% of the initial interferon activity may be achieved and the end product will be free of any skin reactive agents.

BACKGROUND AND SUMMARY OF THE INVENTION 
Interferon is a glycoprotein produced by living cells in defense against 
virus infection. Its chemical structure may vary slightly depending upon 
the cell type that is used for its production. This interferon has various 
biochemical activities, such as antiviral, antiprotozoal, cell growth 
inhibitory and immunosuppressive activities and may, therefore, be applied 
successfully in medicine. For a general review of the present knowledge of 
interferon, reference may be made to the book "Interferons and their 
actions" by W. E. Stewart II, CRC Press, Inc., Cleveland, Ohio. 
For medical application in human patients, interferon should be prepared 
from human cells and the following methods for this purpose are now in 
use: 
1. Production by infection of freshly collected leukocytes from human blood 
donors with Sendai-virus. This results in the so-called leukocyte 
interferon as described by Cantell et al, In vitro, 3, 35-38, (1974). 
2. Production on cultivated diploid human fibroblast cells with the aid of 
a so-called "Poly-I:C-superinduction schedule". The resulting fibroblast 
interferon differs from leukocyte interferon by several biological and 
physiochemical criteria (see A. Billiau et al, J. Gen. Virol., 19, 1-8, 
(1973)). 
3. Production in lymphoblastoid cell lines using a viral interferon 
inducer. The resulting lymphoblast interferon has a strong resemblance to 
leukocyte interferon (see Strander et al, J. Clin. Microbiol., 1, 116-117, 
(1975)). 
The present invention relates to human fibroblast interferon, that is 
interferon produced by the second of the above-mentioned methods, and more 
specifically, it relates to a method for the purification of such 
interferon. 
Purification of interferon is necessary both for studies on the chemical 
character of interferon and for clinical application thereof, since a 
crude interferon solution may contain contaminating proteins that have a 
negative effect on the results of such studies and application. 
For both purposes, rather large amounts of purified interferon are needed. 
Although many techniques for partial purification and a few techniques for 
complete purification of interferon have been described, all of them 
suffer from deficiencies. These deficiencies are in general, either the 
necessity of using complex adsorbents or reagents, or the use of a 
multiple step procedure which is not applicable to large scale production, 
or the fact that only a small fraction of the total initial interferon 
activity is recovered in purified form. 
Therefore, a need exists for a purification method of interferon, and 
especially a purification method of human fibroblast interferon, which 
leads to a high recovery of activity in purified form, which does not 
require complex reagents or a complexity of steps, and which is capable of 
being used on a rather large scale. 
As a result of extensive research, it has now been found that a high 
recovery of interferon activity in the form of completely purified 
interferon may be obtained by treating human fibroblast interferon with a 
simple two-step purification method which is a combination of two earlier 
known methods. This two-step purification method comprises (a) subjecting 
an aqueous interferon solution to chromatography on porous glass beads, 
and (b) subjecting the resulting aqueous interferon solution to 
chromatography on immobilized zinc chelate. 
If, in the first step, an interferon solution containing contaminating 
proteins is contacted with porous glass beads at neutral or slightly 
alkaline pH, the interferon will be selectively adsorbed onto these glass 
beads and the bulk of contaminating proteins will remain in solution and 
may be washed away. The adsorbed interferon may thereafter be eluted from 
the glass beads at an acidic pH. 
If thereupon, in the second step, the eluted interferon solution is 
contacted with an immobilized zinc chelate gel at neutral or slightly 
alkaline pH, the interferon will be selectively adsorbed onto this zinc 
chelate and contaminating proteins that might still be present will remain 
in solution and may be washed away. The adsorbed interferon may thereafter 
be eluted from the zinc chelate at acidic pH and will result in an end 
product of extremely high purity. 
By using this two-step purification method, a high degree of purification 
may be reached since the end product is of such high purity that it may be 
considered to be substantially completely pure. Further, a high recovery 
of initial interferon activity may be achieved. This recovery will be 
about 50-70% in the first step, and 90-95% in the second step, thus 
resulting in an overall recovery of about 45-67%. 
A special advantage is that the end product of the invented purification 
method will be free of any skin reactive agent. The products of earlier 
purification methods always incited skin reactions upon clinical 
application to patients but it has appeared that the product of the 
present invention will not incite such reactions and this is quite 
important for clinical use. 
A further advantage is that the reagents are easily available and may be 
used many times in succession since both the glass beads and the 
immobilized zinc chelate may be reclaimed or regenerated after use. 
Further, the invented method uses only two steps and all these facts 
result in a simple method that can be used on a rather large scale. 
Thus, the invention provides a method for the purification of interferon, 
which comprises (a) subjecting an aqueous solution of human fibroblast 
interferon to chromatography on porous glass beads, and (b) subjecting the 
resulting interferon solution to chromatography on immobilized zinc 
chelate. 
It should be noted here that both steps of the invented method are known 
individually and have been used earlier for purification of human 
fibroblast interferon. Compare A. Billiau et al, Antimicrobial Agents and 
Chemotherapy, 16, 49-55 (1979) for the first step, and V. G. Edy et al, J. 
Biol. Chem. 252, 5934-5935 (1977) for the second step. At the time of 
these publications, however, the said steps were used quite independently 
and there was nothing to suggest that a substantially complete 
purification would be brought about by combining these steps in a simple 
two-step method. 
Moreover, several other methods are known for purification of interferon 
and it would be impossible to predict that a combination of the aforesaid 
two steps without necessity of adding further steps, would lead to the 
desired result. Further, the absence of any skin reactivity in the end 
products of the invented method can be termed surprising and unexpected 
because until now, all purified fibroblast interferon showed such skin 
reactivity upon clinical application to patients (compare A. Billiau et 
al, Antimicrobial Agents and Chemotherapy, 16, 56-63 (1979)). 
Furthermore, it should be noted that the combination of both steps in a 
two-step method has led to a slight modification of the first step; as far 
as the eluate of the glass beads is concerned. In the above-mentioned 
first Billiau paper, this eluate was dialyzed against polyethyleneglycol 
in a sodium acetate buffer in order to prepare it for lyophilization and 
clinical use. In the present invention, however, such eluate will be 
dialyzed against a phosphate buffer in order to prepare it for 
chromatography in the next purification step, as will be described later 
on in this specification. 
DETAILED DESCRIPTION OF THE INVENTION 
The method of the invention will now be described in more detail. 
The starting solution for the invented purification method may be any 
aqueous solution of human fibroblast interferon which contains 
contaminating proteins and which will need purification. Such solution may 
result from a conventional production method of the interferon and from 
any stage thereof. It should be noted that the method is applicable to 
human fibroblast interferon only and that other interferon types will need 
a different purification method. 
Although the starting solution may comprise any aqueous medium for keeping 
the interferon in solution, good results have been obtained with a 
solution of interferon in Eagles's minimal essential medium (compare 
Science, 130, 432 (1959)). A stabilizer may be added to this medium, if 
desired, and the preferred stabilizer in this respect is a human plasma 
protein fraction such as supplied by blood banks. 
The initial protein content and interferon activity of the starting 
solution are not bound to critical limits. However, the solution may be 
diluted or concentrated first, if the protein content is deemed to be so 
high as to render a risk of precipitation or to be so low as to render the 
method uneconomical. It should be noted here that the amount of 
contaminating proteins may exceed that of the interferon and that it may 
well be over 85% of the total amount of dissolved substances. 
The first step of the invented method comprises chromatography on porous 
glass beads. Such porous glass beads are available on the market under 
several Trade marks and are frequently indicated as having a "controlled 
pore size", i.e. a rather uniform pore size concentrated around a specific 
average value. Such average value may be e.g. 350 or 900 A although in 
general any value from 170-1700 A or more may be used (compare W. Haller, 
Nature, 206, 693-696 (1965) and H. G. Bock et al, Science, 191, 380-383 
(1976)). The bead diameter may be less uniform and may range in general 
between about 50 .mu.m and about 500 .mu.m. These beads may be packed in 
columns but it will be more easy to use them in free form for batch-type 
operation. 
In the first step of the method, the starting solution is contacted with 
the porous glass beads for selective adsorption of interferon onto the 
beads. Such contact may be most economically effected by shaking the 
starting solution with an amount of glass beads in a container, but 
passing the starting solution over a column packed with glass beads is 
also possible. The contact leads to an adsorption of interferon from the 
solution onto the glass beads while the bulk of contaminating proteins 
which are present in the starting solution will not be bound by the beads 
and will remain in solution. 
The pH during contact will be the pH of the starting solutin. This pH will 
normally be about 7.4 if Eagle's minimal essential medium has been used 
although in general the system will work well for starting pH's between 
7.0 and 8.2. Above pH 8.2, part of the interferon is inactivated and below 
pH 7.0 much of the applied interferon is not adsorbed onto the glass 
beads. 
The duration of the contact is not bound to critical limits although it 
should of course be sufficient to have nearly all interferon absorbed onto 
the beads. This duration may normally vary between 0.5 and 26 hours. 
After contact, the remaining solution may be removed, e.g. by decantation 
or by discharging. Then, the glass beads may be washed with a conventional 
washing fluid such as a phosphate-buffered saline solution of 
substantially neutral pH to remove any unbound contaminating proteins. The 
molar strength of this fluid should be rather low in order to prevent 
elution of adsorbed interferon. Good washing results have been obtained 
with a phosphate-buffered saline solution containing Ca and Mg salts 
(compare R. Dulbecco et al, J. Exp. Med., 99, 167-182 (1954)) but other 
fluids may be used with the same effect. Further washings may be effected 
with a special buffer solution of lower pH, such as a 0.01 M glycine-HCl 
buffer of pH 3.5 for removing any contaminating proteins that 
inadvertently have been bound to the column. The pH of this washing fluid 
should not be lower than about 3.0 and its ionic strength should not be 
higher than about 0.05 M since lower pH values and higher ionic strengths 
would provoke the elution of large amounts of interferon. 
After the washing step, the adsorbed interferon may be eluted from the 
glass beads with the aid of an acidic aqueous buffer solution. This 
solution should in general have a pH between 1.5 and 2.7 and preferably a 
pH of 2.0. No good elution will be obtained at pH values above 2.7 and 
instability of the eluted interferon will be caused at pH values below 
1.5. The ionic strength (molarity) of the eluting solution is somewhat 
less critical and may vary in practice between 0.05 M and 0.5 M. Values 
below 0.05 M will tend to give insufficient buffering, thus resulting in a 
loss of pH control, and higher values than 0.5 M will tend to result in 
crystallization of constituents. 
Any suitable water-soluble buffering agent, or combination of agents, may 
be used for elution, provided that the aforesaid requirements of pH and 
ionic strength are satisfied. Thus, the eluting solution may contain a 
combination of an aminoacid and an inorganic acid, or a combination of an 
inorganic salt and an inorganic acid, or any other combination or single 
substance. Good results have been obtained with a 0.1 M KCl-HCl buffer 
solution of pH 2.0 and still better results with a 0.3 M glycine-HCl 
buffer solution of pH 2.0. Further, the elution solution may contain a 
suitable stabilizer, e.g. in the form of a human plasma protein fraction, 
as mentioned above. 
With the aid of such an acidic eluant, substantially all of the adsorbed 
interferon may be eluted from the glass beads. 
After elution, the glass beads may be washed and reclaimed, e.g. by rinsing 
them with strong acids for several days or heating them on a steam bath 
with 10% nitric acid, in both cases followed by repeated washing with 
water to neutrality. Thereafter, they may be used again for chromatography 
of interferon in the above-described way. 
The result of the eluting treatment is an aqueous interferon solution which 
comprises the major part of the interferon activity of the starting 
solution and which further comprises only a small proportion of 
contaminating proteins. In practice, the recovery of interferon activity 
in this first step of the invented method will range from 50 to 70% and 
the specific activity of the product per milligrams of protein will be 
substantially increased in view of the starting material. 
The eluate as obtained in the first step has an acidic pH and comprises 
glycine or another substance which makes it less suitable for immediate 
use in the second step. Therefore, it should be neutralized and freed of 
glycine before further processing. This can be achieved by dialyzing the 
eluate against a phosphate-buffered saline solution of substantially 
neutral pH. Good results have been obtained here by using a buffer 
solution containing 0.02 M sodium phosphates (disodium and monosodium) and 
1 M sodium chloride of pH 7.4 but other buffer solutions may be equally 
suitable. The high content of sodium chloride in this buffer solution will 
be transmitted to the interferon solution and will have a favorable effect 
during the next step. 
The second step of the invented method comprises chromatography on 
immobilized zinc chelate. 
The starting solution for this second step is an aqueous solution of human 
fibroblast interferon such as resulting from the above-mentioned first 
step after neutralization and removal of glycine. This solution may be 
diluted or concentrated if desired, but normally such treatments will not 
be necessary. 
Any type of zinc chelate immobilized on a suitable carrier may be used for 
the second step of the invented method but the preferred material is a 
zinc chelate of iminodiacetate immobilized on Sepharose (compare J. Porath 
et al, Nature, 258, 598-599 (1975) and V. G. Edy et al, J. Biol. Chem. 
252, 5934-5935 (1977) which are here incorporated by way of reference). 
Such adsorbant may be produced by first coupling the disodium salt of 
iminodiacetic acid to epoxy-activated Sepharose (i.e. agarose having side 
chains with terminal epoxy groups and then introducing zinc ions to be 
chelated by the iminodiacetate groups. If the coupled material is already 
packed in a column, the zinc can be introduced easily by passing a zinc 
chloride solution through the column and the product is immediately ready 
for use. In this product, the zinc ions are bound by strong bonds and are 
capable of reversibly binding interferon molecules, the latter process 
being pH-dependent. 
More specifically, the chelated and immobilized zinc ions are capable of 
binding human fibroblast interferon quite selectively at neutral or 
slightly alkaline pH and at low ionic strength of mineral additives. Upon 
reduction of the pH and increase of ionic strength, however, the 
interferon is eluted. A good elution can be obtained by using a pH 
gradient at constantly high ionic strength (compare the aforesaid Edy 
paper). 
In the second step of the invented method, the starting solution is 
contacted with the immobilized zinc chelate for selective adsorption of 
interferon thereto. Such contact may be effected by passing the starting 
solution over a column packed with the adsorbant or by shaking it with an 
amount of adsorbant in a container. The use, of a column is preferred here 
since the adsorbant may easily be prepared in this form as shown above. 
The contact leads to an adsorption of interferon from solution onto the 
zinc chelate while contaminating proteins as far as still present will not 
be bound and will remain in solution. 
The pH during contact will normally be 7.4 if the starting solution has 
been dialyzed against the aforesaid phosphate-buffered saline solution, 
although in general the system will work at starting pH's from 7.0 to 8.2. 
Above pH 8.2, some activity is lost and below pH 7.0 much of the 
interferon is not adsorbed and passes straight through the column. 
The duration of contact is not bound to critical limits although it should 
of course be sufficient to have substantially all interferon adsorbed. 
This duration may vary between 0.5 and 26 hours. 
After contact, the remaining solution is removed and the column may be 
washed with a conventional washing agent such as a phosphate-buffered 
solution of pH 7.4 to remove residual unadsorbed proteins from the column. 
Further washings may be effected with a buffer solution of lower pH such 
as a 0.1 M sodium acetate/acetic acid solution of pH 5.9 to remove any 
proteins that have inadvertently been bound to the column. The pH of this 
buffer solution should not be lower than about 5.9 to prevent elution of 
interferon. All these washing fluids may contain 1 M sodium chloride, just 
as the above-mentioned dialyzing fluid in order to prevent non-specific 
binding of proteins to the adsorbant. 
After washing, the interferon may be eluted from the zinc chelate adsorbant 
with the aid of an acidic aqueous solution. This solution may in general 
have a pH between 4.0 and 6.0 and for best results, a pH gradient from 6.0 
down to 4.0 may be used. Good elution will not be obtained at values above 
pH 6.0 and instability of the eluted interferon will occur at values below 
pH 4.0. 
The ion strength (molarity) of the eluant is not very critical and may vary 
in practice between 0.05 M and 0.5 M. Lower values will tend to give 
insufficient buffering, thus resulting in a loss of pH-control, and higher 
values will tend to raise difficulties by crystallization of constituents. 
Good results have been obtained with a 0.1 M sodium acetate solution 
adjusted with glacial acetic acid to pH 4.2, although other solutions like 
those mentioned in the Edy paper are not excluded. If this eluant of pH 
4.2 is used immediately after the washing fluid of pH 5.9, a pH gradient 
will be automatically achieved. As an alternative, 0.1 M sodium acetate 
solutions of gradually decreasing pH could be used in succession. All 
these eluting solutions should contain 1 M sodium chloride in order to 
maintain the NaCl equilibrium in the column. 
With the aid of such an eluant, substantially all of the adsorbed 
interferon may be eluted from the zinc chelate adsorbant. 
After elution, the adsorbant may be washed and regenerated, e.g. by rinsing 
the column with a buffered solution of ethylenediamine tetracetate (EDTA), 
washing out the EDTA with a phosphate-buffered saline solution of pH 7.4, 
treating the column with an acidic zinc chloride solution until the 
chelate is saturated again with zinc, and washing out the excess of zinc 
chloride with a sodium acetate buffer solution and equilibrating the 
column to substantially neutral pH values. 
The result of the elution treatment is an aqueous interferon solution which 
still comprises all or the majority of the interferon activity of the 
starting solution and wherein the content of contaminating proteins has 
been reduced to a very small value. The recovery of interferon activity in 
the second step may amount to 90 to 95%, thus making the overall recovery 
of the whole two-step method about 45 to 67%. 
The specific activity of the product may amount to 10.sup.9 units/mg of 
protein which is higher than any other value obtained until now. The 
purity of the final interferon solution may be established by several 
methods. One of these methods is a normal protein assay, e.g. with 
fluorescamine. Another method comprises labelling the purified interferon 
with a radioactive tracer such as .sup.125 I and subjecting the interferon 
to acrylamide gel electrophoresis followed by X-ray autoradiography. This 
indicates whether the purified interferon is a single or multiple product. 
Further, it can be tested whether the final product incites a skin 
reaction upon application to the human body. 
All these methods indicate a very high purity when applied to the end 
products of the invented purification method. Thus, it is shown e.g. in 
the Examples of this specification that remaining impurities could not be 
measured by protein assays, and that the end products appeared to be a 
homogeneous product by means of electrophoresis. Further, the products did 
not produce any fever or skin reaction upon application to humans. 
In conclusion, the invented two-step purification method leads to a high 
recovery of initial interferon activity in a very pure form. The product 
is suitable for clinical therapy owing to the absence of skin reactivity 
therein. Large scale production has become possible now since the reagents 
are readily available and easy to regenerate. This means that large 
amounts of human fibroblast interferon may be supplied in the future, as 
required for chemical characterization and clinical use. 
The invented method will be further illustrated by the following examples 
which should not limit the scope of the invention.

EXAMPLE I 
The starting material was an aqueous interferon solution derived from human 
embryo fibroblast type cells stimulated with polyinosinic-polycytidylic 
acid. The interferon was in Eagle's minimal essential medium of pH 7.4, 
containing 1% by volume of human plasma protein fraction, supplied by the 
Belgian Red Cross. The interferon activity and protein content of this 
solution in a small scale experiment are stated in Table A. 
This starting solution was mixed with porous glass beads 
(Electro-Nucleonics Inc) having a pore size of about 350 A and a bead size 
of 75 to 120 .mu.m, at a ratio of about 30 ml solution to about 1 ml of 
glass beads. The mixture was gently agitated for 2 hours in order to keep 
the beads in suspension and to cause a selective adsorption of interferon 
from solution onto the beads. Thereupon, the beads were allowed to settle 
and the supernatant was removed by decantation. The beads were washed 
twice with a phosphate-buffered saline solution containing 8 g NaCl, 1.15 
g Na.sub.2 HPO.sub.4, 0.2 g KH.sub.2 PO.sub.4, 0.2 g KCl, 0.12 g 
MgSO.sub.4 and 0.10 g CaCl.sub.2 per liter (compare Dulbecco et al, J. 
Exp. Med., 99, 167-182 (1954)) at a ratio of 20 ml solution per ml of 
beads. Then, they were washed one time with a 0.01 M glycine-HCl buffer 
solution of pH 3.5, at the same ratio. The beads were eluted by 2.times.5 
minutes stirring with a 0.3 M glycine-HCl buffer solution of pH 2.0 and 
2.times.30 minutes stirring with the same buffer solution, each time at a 
ratio of 2 ml buffer solution per ml of beads. This buffer solution 
contained 0.09 mg/ml of human plasma protein fraction as a stabilized. The 
protein content and interferon activity of the combined eluates are shown 
in Table A and it appears that the recovery of interferon activity in this 
experiment was 68%. 
After elution, the glass beads were regenerated by rinsing with strong 
acids for several days and subsequent washing with distilled water to 
neutrality. Before reuse, they were sterilized by autoclaving. 
A portion of the combined eluates was dialyzed against 4.times.500 ml of a 
phosphate-buffered saline solution containing 0.02 M sodium phosphate and 
1.0 M sodium chloride and having a pH of 7.4. This buffer solution was 
changed every 6-8 hours. As a result, the pH became about 7.4 and the 
glycine concentration was decreased from 0.3 M to about 0.0001 M, while a 
sodium chloride content of 1 M was introduced. 
The resulting interferon solution was passed at a flow rate of 20 ml/h 
through an immobilized zinc chelate column of 0.9.times.8 cm. The 
immobilized zinc chelate had been prepared by coupling iminodiacetate to 
epoxy-activated Sepharose 6 B, and adding a zinc chloride solution, as 
disclosed by Edy et al, in J. Biol. Chem. 252, 5934-5935 (1977). 
Following application of the interferon, the column was washed with one bed 
(10 ml) volume phosphate-buffered saline solution (the same as used for 
dialysis) to remove unadsorbed proteins. Then, it was washed with 3 bed 
volumes of a 0.1 M sodium acetate buffer of pH 5.9 containing 1 M sodium 
chloride for removing any adsorbed undesired proteins. 
Then, the column was eluted with 2 bed volumes of 0.1 M sodium acetate 
buffer solution containing 1 M sodium chloride and showing a pH gradient 
of 6 to 4. The fractions within a pH range of 5.2 to 4.5 were collected as 
they contained the bulk of interferon activity. 
The protein content and interferon activity of the resulting eluate are 
shown in Table A. It appears that the recovery of interferon activity in 
this second step was 94%, thus resulting in an overall recovery of 64%. 
The eluate showed a specific interferon activity of about 
1.1.times.10.sup.9 units/mg which means that the product was of high 
purity. 
The zinc chelate column was regenerated by washing it with 5 bed volumes of 
0.05 M EDTA in a phosphate-buffered saline solution of pH 7.4. The 
remaining EDTA was removed by washing with 5 bed volumes of 
phosphate-buffered saline solution (pH 7.4). Then, zinc was reintroduced 
by washing with 0.1 M sodium acetate (pH 4.0) containing 1 M NaCl and 1 mM 
ZnCl.sub.2 until saturation. The saturation point was tested by mixing a 
drop of the eluate from the column with a small quantity of sodium 
carbonate solution and observing the formation of a precipitate. 
Thereafter, the excess of zinc was removed by washing with sodium acetate 
buffer (pH 4.0) and the column was equilibrated to pH 7.4 for application 
of a new sample by washing with 5 bed volumes of phosphate-buffered saline 
solution. 
The purity of the end product was tested by protein assay, acrylamide gel 
electrophoresis, and application to human skin. In the protein assay 
(fluorescamine method), the amount of protein was nearly unmeasurable (the 
limit value in this method is about 2 .mu.g per milliliter). 
Electrophoresis in acrylamide gel after labeling with a radioactive tracer 
and followed by X-ray autoradiography revealed the existence of only one 
radioactive band which had a molecular weight of about 22,000. No skin 
reactivity at all was shown upon application to humans. This leads to the 
conclusion that the product of this Example was of high purity and could 
be considered as being completely pure. 
TABLE A 
______________________________________ 
Total Total 
Volume protein activity 
Spec. activity 
Material ml mg units units/mg 
______________________________________ 
Starting solution 
125 67.92 4 .times. 10.sup.6 
5 .times. 10.sup.4 
Glass beads: 
unadsorbed 300 60.81 0 0 
+ washings 
Eluates 34 2.04 2.7 .times. 10.sup.6 
1.5 .times. 10.sup.6 
Dialyzed solution 
34 2.04 2.7 .times. 10.sup.6 
1.5 .times. 10.sup.6 
Zinc chelate: 
unadsorbed 44 2.03 0 0 
+ 1st washings 
2nd washings 
30 0 0 0 
Eluates 3 .about.0.002 
2.55 .times. 10.sup.6 
.about.1.1 .times. 10.sup.9 
______________________________________ 
EXAMPLE II 
The same starting material as in Example I was used for two large scale 
experiments. The procedure was similar to that of Example I with the 
exception that the eluates from the glass beads were dialyzed against 
4.times.4 liters of phosphate-buffered saline solution, and that a zinc 
chelate column of 1.5.times.16 cm was used. This zinc chelate column (bed 
volume 30 ml) was washed first with one bed volume of phosphate-buffered 
saline and then with 5 bed volumes of sodium acetate buffer. Elution was 
effected with 2 bed volumes of 0.1 M sodium acetate buffer solution of pH 
4.2 containing 1 M sodium chloride, which resulted in pH gradient in the 
eluate. The fractions within the pH range of 5.2 to 4.5 were collected and 
contained the bulk of interferon activity. 
The protein contents and interferon activities of the starting solution and 
its products are shown in Table B. It appears from this Table that the 
recovery of interferon activity in the first step was 57.5% and 58% 
respectively, and in the second step 91.4% and 91.6% respectively, thus 
resulting in an overall recovery of 52.6% and 53.2% respectively. 
The eluates showed a specific interferon activity of about 
1.7.times.10.sup.9 and 2.times.10.sup.9 units/mg which means that the 
product was of high purity. Further, the purity of the end products was 
tested in the same way as in Example I and gave similar results. This 
means that the method of the invention is suitable indeed for large scale 
operation. 
TABLE B 
______________________________________ 
Vol- Total Total Spec. 
ume protein activity activity 
Material ml mg units units/mg 
______________________________________ 
Starting solution 
3200 1075.2 64 .times. 10.sup.7 
5.9 .times. 10.sup.4 
3200 1462.9 1.5 .times. 10.sup.8 
10.3 .times. 10.sup.4 
Glass beads: 
unadsorbed 4700 -- -- -- 
+ washings 4700 -- -- -- 
Eluates 280 124.6 3.68 .times. 10.sup.7 
2.96 .times. 10.sup.5 
280 169.54 8.7 .times. 10.sup.7 
5.13 .times. 10.sup.5 
Dialyzed solution 
100 44.6 1.32 .times. 10.sup.7 
2.96 .times. 10.sup.5 
100 60.55 3.11 .times. 10.sup.7 
5.14 .times. 10.sup.5 
Zinc chelate: 
unadsorbed 130 42.51 0 0 
+ 1st washings 
130 60.01 0 0 
2nd washings 
150 1.60 0.76 .times. 10.sup.6 
0.47 .times. 10.sup.6 
2nd washings 
150 1.60 0.76 .times. 10.sup.6 
0.47 .times. 10.sup.6 
150 0.48 -- -- 
Eluates 10 .about.0.007 
12.02 .times. 10.sup.6 
.about.1.7 .times. 10.sup.9 
10 0.014 2.85 .times. 10.sup.7 
2 .times. 10.sup.9 
______________________________________