pmt. Nat. Acad. Sci. USA
vol. 71, No. 8, pp. 3139-3142, August 1974

partial Purification of Human Interferon by Affinity Chromatography

(antiviral glycoprotein/immunoabsorbance)

c. B. ilNFINSEN, S. BOSE, L. CORLEY, AND D. GURARI-ROTMAN

Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases. National Institutes of Health.
Bethesda, Maryland 20014

Contributed by C. B. Anjimen, May 20, 19Y4

ABSTRACT  Human interferon prepared by challenge
of leukocytes with Sendai virus, or of fibroblasts with
double-stranded poly(inosinic acid).poly(cytidylic acid),
has been studied with respect to purification by affinity
chromatography. Both leukocyte and fibroblast interferons
are removed from crude tissue culture fluids by means of
columns of antibody to leukocyte interferon attached to
SepharossBB. The antibody was prepared in sheep using,
as antigen, material that had been partially purified by gel
filtration through Sephadex G-100 columns. Many of the
impurities in the crude fibroblast interferon were pre-
sumably not recognized by the sheep antibodies induced
by leukocyte interferon. Fibroblast interferon was, there-
fore, much more effectively purified as the result of this
"common denominator" approach. The fibroblast prod-
uct, in contrast to interferon from leukocytes, could only
be harvested efficiently from the crude starting material
when a carrier protein (bovine-serum albumin, and later,
cytochrome c) was added to the eluting buffers to counter-
act losses, presumably due to adsorption on purification
and assay equipment. Both varieties of interferon exhibit
molecular weights of approximately 20,000-25,000, al-
though association with higher molecular weight proteins
occurs.

Studies on a number of species including man have shown
that interferon possesses genus- or family-specific antiviral
activity against a broad spectrum of viruses. Most of the
relevant literature suggests that interferon is a glycoprotein
having a molecular weight of approsimately 25,000 (1,2).

This potentially valuable antiviral agent shows an extremely
high specific activity and it has been estimated that 1 mg of
pure interferon may be equivalent to as much as lo9 inter-
national units (a unit causes 500/, reduction of viral multi-
plication in a standard tissue culture system). The crude
batches of leukocyte material supplied to us for use in the
experiments described below generally contain approximately
5 X 108-10' units in 400 ml. Even if isolation methods avoided
significant losses, enormous volumes would be required to
produce amounts of the agent suitable for definitive clinical
trials in man. For this reason we have undertaken the isola-
tion of human interferon in sufficient quantity for structure
determination and possible eventual synthesis. This paper
on human interferon outlines an immunoabsorbant-affinity
chromatography procedure, a technique which was shown
previously to be of value in the purification of mouse interferon
(3, 4), and reports our findings on some basic physical and
chemical properties, many of which have been investigated
in the past on interferons from other species.

       MATERIALS AND METHODS
Human interferon, prepared by K. Cantell and Smith, Kline
and French Laboratories, was supplied to us by the -4ntiviral

Substances Program of the National Institute for Allergy
and Infectious Diseases. It was prepared by challenge of
human leukocytes with Sendai virus as described by Cantell
and his colleagues (5), and various samples contained between
12,000 and 25,000 international units/ml. Assays, by a modi-
fication of the micro method originally described for rabbit
kidney cells (6), were carried out under contract with the
North American Biological Association, Inc., Rockville,
Md., and reproducibility was controlled by the inclusion of
coded samples of known dilutions of a frozen stock solution.
In this assay the titer of human reference interferon, no. 6919,
is 5000 units/ml.

A second variety of human interferon was prepared by Dr.
J. Vilcek and his colleagues at New York University, using
the FS-4 strain of human foreskin diploid fibroblasts. This
interferon was induced with a double-stranded complex of
poly(inosinic acid). poly(cytidylic acid), and high yields were
obtained by use of pretreatment of cultures with cyclohes-
imide and actinomycin (7). These cult.ures also yielded inter-
feron at a level of approximately 20,000 units/ml before con-
centration.

As others have observed (8,9) in studies on several species,
interferon shows remarkable stability to low pH. We have,
therefore, effected partial purification by gel filtration on
columns of G-100 Sephadex-Fine (Pharmacia Fine Chemicals,
Inc.) using, as solvent, 0.1 .\I acetic acid, 0.15 1\1 NaCl, pH
3.0. Columns were run at room temperature and fractions
were collected in siliconized glass test tubes or plastic test
tubes. Before application to columns, crude interferon was
either thoroughly dialyzed at 4' against 0.1 AI acetic acid
and lyophilized, or concentrated, before dialysis against
0.1 M acetic acid-O.15 11 NaCl, to a concentration of ap-
proximately 25-30 mg/ml by hmicon pressure filtration using
a 65mm UhIlO filter disc (Amicon Ultrafiltration Cell,
model 202). Essentially no losses of interferon activity oc-
curred during concentration by filtration or dialysis and
lyophilization.

Affinity columns were prepared by means of the CNBr
activation method described by Ax& and Porath (10) as
applied to affinity chromatography in general, and to the
preparation of immunoabsorbant columns (11-13). The
affinity chromatography principle was used not only for con-
centration and purification of interferon as described below
in Results, but also for the removal of antibodies in the crude
r-globulin fraction of serum from sheep that had received
partially purified leukocyte interferon. -4ntiserum to the par-
tially purified leukocyte interferon was produced in a sheep
which, after initial immunization, was given 8 bi-weekly
injections. Each injection consisted of approsimntcly 1 mg
oi the :ictivp frncation, hnvin4 :Ibout, lo6 units of intrrfcron


31-10

ljiochemistry :  .Lnfinsen et al.

lOO(
             IO     20     30     40     50
                   TUBE NUMBER
FIG. 1.  Removal of antibodies against proteins known to be
present in crude leukocyte interferon. A solution (ml*As~ =
2220) in PBS of the r-globulin fraction of the serum from sheep
no. 991 was applied to the "impurities"-agarose column described
in hfaterials and Method-s. Unadsorbed protein was washed
through with PBS and bound antibodies were then eluted with
0.1 hl citrate buffer, pH 2.2 (arrow). Additional elution with 8 M
urea in PBS caused the release of only a small additional amount
of protein. y-globulin in peak A, total ml.Arso = 1576; material
in peak B, total rnl.Aao = 202.5.

activity, from a G-100 Sephades column (see Results). The
antigen, in 1 ml of isotonic saline, was emulsified with an
equal volume of complete Freund's adjuvant. Serum from the
final bleeding of this sheep was fractionated with ammonium
sulfate to yield the r-globulin components. Thus, in a typical
preparation, 825 ml of saturated ammonium sulfate (reagent
grade) was slowly added to 1375 ml of serum. The pH was
adjusted to 7.4 with 2 N NaOH and the mixture was slowly
stirred for 30 min at room temperature. The precipitate was
then centrifuged off and redissolved in phosphate-buffered
saline (PBS). This volume was adjusted to 1375 ml with PBS
and the r-globulins were precipitated again by adding 688
ml of saturated ammonium sulfate. The precipitate was centri-
fuged off as before. This step was repeated once again. The
final precipitate was dissolved in PBS and dialyzed against

I       I       I       I       I
IO      20      30     40      5c
     TUBE NUMBER

FIG. 2.  Passage of the unbound fraction of r-globulins shown
in Fig. 1 through the "impurities" -agarose column after thorough
equilibration with PBS. Peak A, total ml.Aao = 1206; peak B,
total ml.Atso = 29.9.

hoc. Xat. Acad. Sci. C;SA 71 (1974)

100       200        loo       200
          TUBE NUMBER

FIG. 3.  Gel filtration of the proteins present in crude leukocyte
interferon through a column (3.5 X 200 cm) of G-100 Sephadex
in 0.1 M acetic acid-O.15 M NaCl. After concentration by ultra-
filtration, the proteins (total rnl.Aao = 1504 and 1350, re-
spectively), were applied to Columns 0 and P, respectively,
Recoveries of interferon activity in the major peaks were ap-
proximately 20% of the activity applied. The low recovery of
activity may be related to the length of time required to develop
the columns (about 4 days at room temperature). Furthermore,
the protein concentration in the major active fractions was quite
low, a factor known to contribute to losses during purification and
assay.

6000 ml of cold PBS, with three changes. The final solution
(1010 ml) had an optical density of 0.67 after 1: 100 dilution
with PBS.

Antibodies against those proteins thought to constitute
the major impurities in the antigen used to immunize the
sheep were removed by passing the r-globulin solution through
an "impurities'`-agarose column. To prepare a typical column
of this type, 100 mg of material derived from huffy coat cells
by passage through a French press with subsequent dialysis
against 0.1 IM acetic acid and lyophilization, 350 mg of serum
proteins (similarly dialyzed and lyophilized), and 10 mg of
mixed allantoic fluid-Sendai virus proteins were taken up to
20 ml of PBS and coupled to 30 ml of CNBr activated agarose
(12, 13). We are grateful to Dr. Sheldon Wolff and Dr.

TABLE 1.  Anti-human intmjeron titer in sheep no. 991

Interferon

     Serum

1. Normal l/10
      l/100
     l/1000
2. After 5th boost l/10
          l/100
          l/1000

3. After 8th boost l/10
         l/100
          l/1000

l/10    l/100    l/1000

3.1      2.1      0.7
2.8      1.6      0.5
3.0      1.7      0.2

2.0 --co      <o
2.8      1.4     <o
3.3      1.7     <o

1.0      0.3     <o
1.4     <o      <o
2.8     <o      <o

Equal volumes of serum and of various dilutions of interferoll
were mixed and incubated at 37' for 1 hr. The mixtures were
assayed (6) for interferon activity and results are expressed as
log,, titers of interferon, Dilutions of both serum and the stock
interferon solution (12,000 units/ml) were made in Eagle's
minimum essential medium with added glutamine and Hepe.<
buffer.


Pm. Nat. Acad. Sci. USA ?`l (1974)

Charles Dinarello for human huffy coat cells and for human
serum, and to Drs. H. Levy and S. Baron for hen's egg allan-
toic fluid containing Sendai virus. Aliquots of approximately
30 ml of the r-globulin solution were passed through the Ym-
purities"-agarose column; after regeneration of the column
with 0.1 &I citrate buffer, pH 2.2, and subsequent thorough
washing with PBS, the protein that passed through this
column, unretarded, on the first run was passed through a
second (and occassionally a third) time. In general, very
little material was bound upon the second passage of the
unretarded PBS fraction through this "scavenger" column.
All manipulations wzre at room temperature except for ex-
tended storage of the affinity column at 5O between runs
when 0.02% sodium azide was present in the final PBS wash.
Figs. 1 and 2 show the elution patterns obtained during two
successive runs on the mixed protein column. Later columns,
bearing larger amounts of the suspected protein impurities,
have yielded anti-interferon globulins which, after only a
single pass, are essentially devoid of further adsorbable mate-
rials.

RESULTS

Fig. 3 illustrates the results of two typical gel filtration ex-
periments designed to yield leukocyte antigen for immuniza-
tion. In some such runs, e.g., column P, a fraction of the inter-
feron activity moved through the column with front-running
material. Since this front-running activity is eluted (with
essentially no loss of activity) from G-100 Sephadex columns
run with 6 M guanidine hydrochloridea. M acetic acid at a
position consistent with the molecular weight of 20,000-
25,000, we suspect that nonspecific adsorption to larger mole-
cules is occurring. Other esperiments on gel filtration under
a variety of environmental conditions support the idea that
suspected oligomeric, or specifically aggregated forms of
interferon, may be simply nonspecific aggregates with other
larger components of the crude tissue culture fluids.
The immunizations described under Xaterials and Methods
employed the pooled protein-containing fractions showing
interferon activity, e.g., the active tubes in the region of frac-
tion 180-200 in column P. Neutralization of leukocyte in-
terferon activity by addition of various dilutions of hyperim-
mune antiserum to various dilutions of stock interferon was
used to give a rough measure of the progress of specific anti-
interferon production (Table 1). Sheep no. 991 reached a high
level of activity after the eighth boost, and its serum (1500 ml)
was collected. A second sheep, no. 903, also reached high titer
levels and has been retained, without further boosting, as an
animal likely to yield a strong anamnestic response when
more highly purified interferon becomes available.
The major innovation in the present work has been the use
of an immunoabsorbent column prepared with the anti-inter-
feron induced by interferon from one cell type (leukocytes,
by the Cantell method) for the purification of interferon de-
rived from another cell type [diploid fibroblasts prepared by
the procedures of Vilcek and Have11 (7)]. Since many im-
purities in the leukocyte cultures are likely to be absent from
the fibroblast cultures, greater purification might be expected
(see also ref. 4). The results shown in Figs. 4 and 5 illustrate
that, although more complete binding of leukocyte interferon
activity is achieved with the anti-leukocyte interferon column,
the amount of protein in the peak eluted with acidic citrate
buffer is considerably greater than with the fibroblast mate-
rial. In a run such as that illustrated in Fig. 5, the PBS frac-

Affinity Chromatography of Interferon

3141

60-

IOOL      I      I      I      I      I

                        J
too    200    300 400    500
  MILLILITERS OF EFFLUENT

FIN. 4. Purification of human leukocyte interferon on an
immunoabsorbant column. The column (2 X 11 cm) was equili-
brated with PBS and, after unabsorbed protein in the applied
sample of crude interferon (570 mg containing approximately
1.6 X 10' units of interferon) had been washed through, bound
activity was eluted with citrate buffer, pH 2.2 (arrow). The
numbers on the ordinate are taken directly from the chart paper
supplied by the LKB Co.

tion that passed through the column unretarded contained
1824 ml. AZW units and the fraction eluted with pH 2.2 citrate
buffer contained only 12.6 rnl.AzBo units due to protein other
than the added serum albumin. Recovery of applied interferon
activity units varied between 50% aid 100%. We suspect that
this variability is due in part to the relatively large error in-
herent in the biological assay method.

Although bovine-serum albumin was used in the experi-
ments illustrated in Fig. 5, it was later found that interferon
and albumin occasionally formed a comples that interfered
with subsequent purification steps. At present, stabilization
and prevention of adsorption to equipment is achieved using
horse heart cytochrome c. Parenthetically, cytochrome c has
the added advantage of possessing an N-acetyl end group, thus

      100
              100    200    400 500    600

              MILLILITERS OF EFFLUENT
FIG. .i.  Purification of human fibroblast interferoll (1160 mg
with 4 X lo6 units of activity). The same column and conditions
described in the legend for Fig. 4 were used in this experiment,
except that the citrate buffer at pH 2.2 contained bovine serum
albumin (1 mg:`ml).


3142

Biochemistry: -4nfinsen et al.

Proc. Nat. Acad. Sci. USA 71 (19Yd)

TUBE NUMBER

FIG. 6. Purification of human leukocyte interferon (2.3 X IO6 units) on an anti-leukocyte interferon column. After loading of the
sample, the column ww washed with PBS and t,hen with McIlvaine phosphate-citrate buffer pH 3.8, containing ,500 pg of cytochrome c
per ml. Interferon was elated with a pH gradient, starting as indicated by arrow. About 80% of the interferon units were recovered in the
two peaks of interferon activity.

being unsusceptible to amino acid sequencing by Edman deg-
radation.

More recently, elution of interferon from the anti-leukocyte
interferon column has been carried out by means of a pH
gradient. A linear gradient of McIlvaine phosphate-citrate
buffer from pH 3.8 to pH 2.2 has been used, with cytochrome
c in the buffer solution3 at a concentration of 500 pg/ml. At
the end of the gradient, the column was washed with 0.1 M
citrate buffer pH 2.2, before re-equilibration with PBS. As
can be seen in Fig. 6, the amount of protein eluted from the
column by the pH gradient is less than that observed on elu-
tion with citrate buffer pH 2.2 alone in the experiment illus-
trated in Fig. 4. This is due to the use, in this esperiment, of
an anti-leukocyte interferon.column using anti-interferon r-
globulins that had been passed through an improved "scav-
enger" column before coupling to Sepharose. The improved
"scavenger" column had a larger amount of each of the im-
purities bound per gram of Sepharose. By means of the gra-
dient elution system, two peaks of interferon were eluted from
the anti-interferon column. It therefore appears that there
are two species of interferon, differing in their affinity to the
anti-interferon r-globulins. Similar results were obtained on
gradient elution of fibroblast interferon from the antileukocyte
interferon column. In the case of the fibroblast material, a
third species of interferon is present (about 40% of the ac-
tivity) which is not retarded by the affinity column; the
major portion of this activity also escapes binding on a second
pass. Whether these differences reflect the presence of a second
genetic locus that codes for interferon in fibroblasts, or anti-
genie differences stemming from incomplete or chemically
different embellishment of the polypeptide chain with carbo-
hydrate and/or other non-aminoacid moieties, is not known.

The affinity chromatography method has been of consider-
able use (unpublished experiments) in the preparation of
highly radioactive fibroblast interferon (prepared with the
kind cooperation of Drs. J. Vilcek and E. Havell). After subse-
quent enrichment by gel filtration (as in Fig. 3) and disc gel
electrophoresis with the riboflavin catalysis described by
Fantes and Furminger (14)) such material may be suitable for
sequencing studies.

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