Human Lymphoblastoid Interferon LARGE SCALE PRODUCTION AND PARTIAL PURIFICATION*

Human lymphoblastoid interferon was produced on an 800-liter scale (2.6 X 10(9) units) by induction of Namalva cells with Newcastle disease virus, strain B1. The interferon was partially purified by anti-leukocyte interferon affinity chromatography, sulfopropyl Sephadex ion exchange chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recovery of interferon after gel electrophoresis varied from 11 to 33% based on the original crude material, with about 35,000-fold purification. The gel electrophoresis resolved the antiviral activity into two components with apparent molecular weights of 18,000 and 22,000; treatment with glycosidases resulted in all the activity being associated with the lower molecular weight species. Interferon activity could be completely (85 to 113%) recovered from the gels by elution into a buffer containing sodium dodecyl sulfate. The presence of sodium dodecyl sulfate did not appear to affect the assay of interferon. The protein could also be completely (75 to 106%) eluted from gels stained with coomassie blue, again with no loss in activity.


SUMMARY
Human lymphoblastoid interferon was produced on an 800~liter scale (2.6 x 10y units) by induction of Namalva cells with Newcastle disease virus, strain Bl. The interferon was partially purified by anti-leukocyte interferon affinity chromatography, sulfopropyl Sephadex ion exchange chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recovery of interferon after gel electrophoresis varied from 11 to 33% based on the original crude material, with about 35,000-fold purification. The gel electrophoresis resolved the antiviral activity into two components with apparent molecular weights of 18,000 and 22,000; treatment with glycosidases resulted in all the activity being associated with the lower molecular weight species. Interferon activity could be completely (85 to 113%) recovered from the gels by elution into a buffer containing sodium dodecyl sulfate. The presence of sodium dodecyl sulfate did not appear to affect the assay of interferon. The protein could also be completely (75 to 106%) eluted from gels stained with Coomasiie blue, again with no loss in activity.

Interferons
are antiviral glycoproteins (l-4) which have very high biological specific activity (at least 2 x lo8 units/ mg of protein for human interferon (5)). Only minute amounts of interferon are produced in tissue culture preparations and although much attention has been drawn to the large scale production, purification, and physicochemical characterization (5-S), interferon has not been purified to a level suitable for structural studies. Lymphoblastoid cells can be successfully grown in large suspension cultures and our major effort has therefore been production of interferon in such ce11s.'s2 Our own long range aims involve isolation, sequencing, and synthesis. However, since others may have interest in the possibilities of large scale biological production, conditions for cell growth and interferon production and purification are given here in some detail.
In the experiments presented, lymphoblastoid interferon has been purified greater than 35,000-fold using affinity and ion exchange chromatography and SDS3-polyacrylamide gel electrophoresis, and is now being produced at batch levels of 800 liters (2.6 x log units). Full biological activity has been recovered in various SDS-gel systems both before and after staining with Coomassie brilliant blue. Removal of the carbohydrate portion of the interferon molecule resulted in a single peak of activity using both isoelectric focusing and SDSpolyacrylamide gel electrophoresis. Human lymphoblastoid interferon was produced by the cell line Namalva induced with Newcastle disease virus, strain Bl'*' and cells were removed by centrifugation.4 Precipitation of proteins from the tissue culture fluid and removal of trichloroacetic acid by Sephadex G-25 resulted in approximately a 50-fold concentration of the crude interferon. The solution was applied to an anti-leukocyte interferon affinity column ( Fig. 1) which bound the applied lymphoblastoid interferon.
Sorensen's citrate buffer, pH 2.2, was used to elute the interferon (25 to lOO%), which was purified 250-to 450-fold. We have recently found that prolonged washing of the column with NaCl/P, after application of the sample gives greater purification without loss of interferon activity.
Since the isoelectric point of interferon is approximately 6, SP-Sephadex which has a pH range 2 to 10, was chosen for ion exchange chromatography.
McIlvaine's citrate/phosphate buffer was used because of its wide range of buffering capacity. When applied directly to a SP-Sephadex column equilibrated with McIlvaine's citrate/phosphate buffer, pH 4.5, only 70 to 75% of the interferon was bound. However, prior dialysis of the interferon-containing fraction against the equilibration buffer resulted in complete binding of the interferon. Since dialysis of large volumes is cumbersome, these two purification steps have been modified. After the aflinity column was washed with NaCl/P,, it was further washed with approximately 200 ml of McIlvaine's citrate/phosphate buffer, pH 5.5. Adjusting the pH of this buffer to 2.6 eluted the interferon, which could then be applied directly to a SP-Sephadex column equilibrated with the same buffer. Recovery of the interferon (40 to 96%) by means of a pH gradient (Fig. 2) gave a 6-to 13fold purification but a large volume. Smaller volumes of eluate (about 5 times lower) could be achieved with less purification (Bfold) by using a single-step elution at pH 6.8. Since lymphoblastoid interferon elutes from the column at pH 5.5, it must be applied at pH 4.5 or below.
Interferon shows a heterogeneous character (and only 31% ) when subjected to isoelectric focusing (Fig. 3~). Treatment with glycosidases and cu-mannosidase (3, 12, 13) resulted in a single peak after isoelectric focusing which gave increased recovery (60%) and purification (12-fold). This procedure appears to be a suitable purification step to follow SP-Sephadex chromatography.
The recovery of interferon activity from stained and unstained SDS-polyacrylamide gels is shown in Table I. The recovery, calculated as a percentage of the interferon activity of each sample applied, varied from 85 to 156% for the unstained gels and from 55 to 150% after staining.
Neither the running buffer nor the percentage of acrylamide used in forming the gel made any appreciable difference in the percentage of activity recovered (Table I). Heating the sample for 2 min at 100" prior to application to the gel did not appear to affect the recovery. However, the addition of mercaptoethanol before heating resulted in only 18% recovery of the applied interferon. In all cases the activity was spread over a distance of about 2.5 cm.
All the experiments showed two peaks of activity (Fig. 4). The leading peak, corresponding to a protein of apparent molecular weight 18,000 (? 1000) contained the majority (>70%) of the active interferon.
The smaller peak which corresponded to a protein of apparent molecular weight 22,000 (* 1000) was always present but always contained less than 30% of the total activity. Treatment with glycosidases (3) resulted in 100% recovery of interferon activity in the lower molecular weight species. Crude interferon (3.1 x 10T units) was applied to a column (11 x 4.5 cm). Specific activity x 10T units) was applied to a column (11 x 4.5 cm). Specific activity in citrate peak, 7.8 x 105 unitslmg of protein; 446fold purification. in citrate peak, 7.8 x 105 unitslmg of protein; 446fold purification. PBS, NaCl/P,. PBS, NaCl/P,.
Preparative SDS-polyacrylamide slab gel electrophoresis of partially purified interferon resulted in a 20-fold purification giving a specific activity of 1.1 X lo7 unitslmg of protein in the lower molecular weight peak fraction. Two broad bands of activity were isolated from the gel with the apparent molecular weights 18,000 and 22,000 and no detectable loss of interferon activity. Fibroblast interferon has also been purified by preparative SDS-gel electrophoresis; however, only 60% of the interferon activity was recovered from the gel (5). TGrma and Paucker (6) and Stewart and Desmyter (17) have shown that 70 to 120% leukocyte interferon activity can be recovered from SDS gels. Two peaks of activity having apparent molecular weight of 15,000 to 16,000 and 20,000 to 21,000 were also  found for leukocyte interferon. Staining the gels with Coomassie brilliant blue R-250 did not adversely affect the recovery of lymphoblastoid interferon from the gels. Assuming a minimum specific activity of 2 x 10' unitslmg (51, the amount of interferon applied to each gel would correspond to less than 1 pg. It is unlikely that the interferon could be detected by Coomassie blue staining at this concentration. Thus, we are not able to conclude whether the recovery of protein activity is due to a poor or reversible reaction with the stain, or whether the regions of the molecule to which the stain binds are not essential for activity. Further purification of lympho- blastoid interferon to a level suitable for preliminary structure studies may be achieved by re-electrophoresing isolated peaks of interferon activity on analytical SDS slab gels. This procedure has been used successfully by Knight (5) in the purification of fibroblast interferon.