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