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[65] Purification of human fibroblast interferon by adsorption to controlled-pore glass and zinc-chelate chromatography
448
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CHARACTERIZATION
[65]
[65] Purification of Human Fibroblast Interferon by Adsorption to Controlled-Pore Glass and Zinc-Chelate Chromatography By
K. J. W. HEINE AND A. BILLIAU
Most purification techniques described for human interferons 1-5 yield either small quantities o f high purity, e.g., sufficient for determination o f specific activities, or larger quantities o f low purity. H o w e v e r , methods have been described for purification o f larger quantities o f completely pure interferon. 4,6,~ The procedure described here yields large quantities o f pure human fibroblast interferon in two steps. The overall recovery is at least 60% o f the starting materials, and the specific activity o f the end product varies from 1.5 to 3.1 x 109 reference units/mg, averaging about 2 x 109 reference units per milligram o f protein. The uniqueness o f the method is that the two steps yield a pure product when used in combination with each other, but not when either step is used in combination with other purification procedures, such as concanavalin A-Sepharose 8 or phenyl-Sepharose chromatography. The first step involves the partial purification o f crude human fibroblast interferon by controlled-pore glass (CPG) adsorption as described b y Billiau et al. 9 The second step consists of a modified and large-scale adaptation o f the zinc-chelate chromatographic purification m e t h o d originally designed by E d y et al. lo as a first step in the purification o f crude interferon. Materials Equipment
Cold room + 4 ° Laminar-flow hood 1 w. Berthoid, C. Tan, and Y. H. Tan, J. Biol. Chem. 253, 5206 (1978). E. Knight, Jr., J. Gen. Virol. 40, 681 (1978). a E. Knight, Jr., Proc. Natl. Acad, Sci. U.S.A. 73, 520 (1976). 4 M. Rubinstein, S. Rubinstein, P. G. Familletti, R. S. Miller, A. A. Waldman, and S. Pest_k_a,Proc. Natl. Acad. Sci. U.S.A. 76, 640 (1979). A. J. Mikulski, J. W. Heine, H. V. Le, and E. Sulkowski, Prep. Biochem. 10, 103 (1980). J. W. Heine, M. De Ley, J. Van Damme, A. Billiau, and P. De Somer, Ann. N. Y. Acad. Sci. 350, 364 (1980). E. Knight, Jr., Science 207, 525 (1980). 8 K. C. Chadha, P. M. Grob, A. J. Mikulski, L. R. Davis, Jr., and E. Sulkowski, J. Gen. Virol. 43, 701 (1979). A. Billiau, J. Van Damme, F, Van Leuven, V. G. Edy, M. De Ley, J. J. Cassiman, H. Van den Berghe, and P. De Somer, Antimicrob. Agents Chemother. 16, 49 (1979). 1oV. G. Edy, A. Billiau, and P. De Somer, J. Biol. Chem. 252, 5934 (1977). METHODS IN ENZYMOLOGY, VOL. 78
Copyright© 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7
[65]
PURIFICATION OF HUMAN FIBROBLAST INTERFERON
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Spectrofluorometer Micropipetters (5-200 ~1), sterile pipette tips Fraction collector, peristaltic pump Gradient maker, prepared by connecting two 50-ml plastic syringes with epoxy glue Magnetic stirrer pH meter with long slender electrode Shaking water bath, 37° to 80° Controlled-pore glass beads (CPG), CPG 10-350 (Serva Feinbiochemica, P.O. Box 105260, D-6400 Heidelberg, West Germany) Spinner culture flasks (Wheaton Scientific, 1000 North Tenth St., Millville, NJ 08332) Sintered-glass filter, suction flask (1 liter) Dialyzing tubing, large 6 ~ inch, med. 3 ~ inch, small 1~ inch with a cutoff of 12,000-15,000 MW (Medical International Ltd., 49 Queen Victoria St., London EC4N 4SA, England) Columns K9/15, K15/30, or larger (Pharmacia Fine Chemicals, Uppsala, Sweden) Membrane filtration apparatus and 220 nm Millipore membranes (Millipore Corporation, Bedford, MA 01730) Plastic tissue culture flasks 75 cma, Falson 3024 F; polypropylene tubes, 17 × 100 mm, Falcon 2050; and 12 × 75 mm, Falcon 2063 Assortment of sterile glassware, flasks, beakers, graduated cylinders, bottles, pipettes, etc. Limulus amebocyte lysate (LAL) test kit (Microbiological Associates, Bethesda, MD) Chemicals
All chemicals employed are of highest available quality if the source is not given. Crude human fibroblast interferon, as described by Van Damme and BiUiau, this volume [13] (see section on partial purification of interferon) Epoxy-activated Sepharose 6B, Ficoll 400 (Pharmacia Fine Chemicals, Uppsala, Sweden) Iminodiacetic acid, disodium salt (Aldrich Europe, Beerse, Belgium) Ethanolamine Fluram, 1,4-dioxane Sodium carbonate (Na~CO3) Sodium chloride (NaCI) Zinc chloride (ZnCI~) Sodium phosphate, monobasic (NaH2PO4) Sodium phosphate, dibasic (Na2HPO4)
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PURIFICATION AND CHARACTERIZATION
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Sodium acetate Glacial acetic acid Sodium azide Glycine Hydrochloric acid (conc. HCI) Ethylenediaminetetraacetic acid (EDTA) Stabilizing proteins, such as 4.5% human plasma protein fraction (HPPF) Belgian Red Cross, National Blood Transfusion Service) Triple-glass-distilled water Pyrogen-free water (clinical grade)
Buffers Glycine.HCl buffers: 0.01 M, pH 3.5, and 0.3 M, pH 2.0, containing 0.10 mg/ml of HPPF. Make up molar glycine solution and adjust pH with conc. HC1. Phosphate-buffered saline (PBS): Dulbecco's formula (modified) without magnesium or calcium (Flow Laboratories, Irvine, Scotland) PB-NaCI buffer: 0.02 M phosphate buffer-1 M NaC1, pH 7.4 1. Make up 10x stock, pH 7.9, by dissolving 1.93 g of NaH~PO4.H20 (MW 138) and 26.4 g:of,Na~HPO4 (MW 142) in 1 liter of distilled H20. Sterilize by membrane filtration (220 nm) and store at 4 ° 2. Make up 5 × NaCI by dissolving 292.20 g of NaCI (MW 58.44) in I liter of distilled HzO. Sterilize by filtration and store at 4 °. 3. Before using, mix under sterile conditions: 100 ml of 10x PB, pH 7.9; 200 ml of 5 x NaCI; and 700 ml of pyrogen-free H20. Sodium acetate-NaC1 buffer: 0.1 M sodium acetate- 1 M NaC1, pH 4.0, 4.2, and 5.9: 1. Make up 10x stock of sodium acetate by dissolving 136.1 g of sodium acetate.3 H20 (MW 136.1) in 1 liter of distilled H20. Sterilize by filtration and store at 4 °. 2. At time of use mix under sterile condition 1 M acetate diluting it 1:10 and 5 M NaCI diluting it 1:5, adding some pyrogen-free H20; adjust to desired pH (5.9, 4.2, and 4.0) with glacial acetic acid. Then bring to final volume with pyrogen-free H~O. The pH electrode is sterilized by rinsing with 70% EtOH and pyrogenfree water. EDTA buffer 1. Make up 0.2 M EDTA (MW 372.24) by dissolving 37.22 g in 500 ml of distilled H20 and adjust pH 7.2 with saturated NaOH. Sterilize by filtration and store at 4°. 2. Mix aseptically: 100 ml of 0.2 M EDTA, pH 7.2; 40 ml of 0.2 M PB, pH 7.9; 80 ml of 5 M NaC1; and 180 ml ofpyrogen-free water
[65]
PURIFICATION OF HUMAN FIBROBLAST INTERFERON
451
Preparation of Chromatographic Materials Charging and Regeneration of Controlled-Pore Glass Beads. The procedure is carried out as described by Van Damme and Billiau in this volume [13] (see section on partial purification of interferon). Preparation of Zinc-Chelate Chromatography. The procedure is cartied out as described by Porath et al. 11 and Chadha et al. 8 Immobilization of iminodiacetic acid to epoxy-activated Sepharose 6B: I. Swell 15 g (one package) of epoxy-activated Sepharose 6B for 2 hr in 250 ml of distilled H20. Wash with 2000 ml of distilled H20 employing a suitable size sintered-glass filter, fitted to a l-liter suction flask. 2. Suspend in 50 ml of 2 M NaCO3 containing l0 g of iminodiacetic acid and place in a shaking water bath for 20 hr at 65 °. 3. Wash as in step 1 with 2000 ml of distilled H20 followed by resuspending in 200 ml of I N ethanolamine for ! hr at 65°, as above, to neutralize the unreacted sites. 4. Wash extensively with distilled H20 and store as a suspension with azide at 4°. Column preparation I. Choose a column size to yield a minimum 10-fold concentration of applied sample; i.e., with a sample of 50 ml on a 5 ml bed volume the interferon will be eluted in 3-4 ml, greater than 10-fold concentration. Thus, for samples of 5 to 50 ml, use a column of 0.9 x 8 cm (K9/15); and for 100 to 400 ml, use a column of 1.5 × 16 cm (K15/30). 2. Develop column under gravity at room temperature with degassed matrix. 3. Then place column in the cold room and wash with 10 bed volumes of cold distilled H20. 4. Wash with 5 bed volumes of cold E D T A - P B - N a C I buffer. 5. Wash with 5 bed volumes of cold PB-NaCI buffer. 6. Wash with 5 bed volumes of cold sodium acetate-NaC1, pH 4.0. 7. Wash with 2 bed volumes of cold sodium acetate-NaCl, pH 4.0, containing 5 mg/ml of ZnCI2. This is prepared fresh each time just before use and is also sterilized by filtration through a syringemounted membrane filter (220 nm). At completion of the zinc chelation, check last eluent (one drop) in 0.5 ml of CaCO3 solution for formation of precipitate. 8. If zinc saturation as tested above has been achieved, remove ex11 j. Porath, J. Carlsson, I. Olsson, and G. Belfson, Nature (London) 251t, 598 (1975).
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PURIFICATION AND CHARACTERIZATION
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cess by washing with 5 bed volumes of sodium acetate-NaC1, pH 4.0. Equilibrate the column for sample application by washing with 5 bed volumes of PB-NaC1 buffer, pH 7.4. 10. The column is regenerated by starting with step 4. The number of times of possible regeneration has not been determined, After frequent use (10×) the top of the column becomes clogged with debris from applied samples. To overcome this the top of the bed is removed and replaced by fresh material to its orginal height. Furthermore an observation that might suggest replacement of columns is tailing of interferon upon elution from the column after 20 or more regenerations. .
Fluorometric Protein Assay x2 1. Add 10-100/zl of sample to 2 ml of 0.01 M PB (pH 7.4) and mix on mechanical mixer. 2. Prepare a fresh solution of 6 mg of Fluram in 20 ml of 1,4-dioxane, add 0.7 ml to step 1 preparation, and mix immediately. 3. Allow to stand for 15 min and read on a spectrofluorometer at 480 nm using bovine serum albumin as a standard.
Limulus Assay Follow test as described in Instruction Booklet accompanying the L A L test kit.
Interferon Assay Carry out as described by Van Damme and Billiau in this volume [13] (section on Interferon Assay) and by Finter. 13
Purification
First Purification Step: Adsorption and Desorption from CPG This step involves a batch-type adsorption of crude interferon onto CPG beads. Mix 1 volume of CPG beads with 30 volumes of crude interferon (clarified cell culture fluid) in a suitable-sized spinner culture flask 1~ p. BOhlen, J. Stein, W. Dairman, and S. Udenfriend, Arch. Biochem. Biophys. 155, 213 (1973). la N. B. Finter, J. Gen. Virol. 5, 419 (1969).
[65]
PURIFICATION OF HUMAN FIBROBLAST INTERFERON
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and stir gently (enough to keep beads in suspension) for 2 hr at 4 °. The crude interferon should be freshly harvested or could have been stored at 4 ° for a few days, but not stored frozen prior to purification. If stored at 4 ° for a few days care should be taken not to allow the pH of the culture fluid to rise above pH 7.6, as this would affect the stability of the interferon. With the settling of the glass beads the supernatant is decanted. The beads are washed twice with 10 volumes of PBS followed by a single wash at the same ratio with 0.01 M glycerine.HC1 buffer, pH 3.5, to remove any loosely bound protein. With the glycine wash, the beads are quantitatively transferred to a smaller receptacle. The interferon is then eluted by twice gently stirring for 5 min 1 volume of beads in 1 volume of 0.3 M glycerine.HCl buffer, pH 2.0, containing 0.1 mg/ml of HPPF. This is followed by twice elucting the beads with the same buffer, but with an interaction time of 30 min each. All elution fluids are separated from the beads by filtration on a sintered-glass filter under gentle suction to recover all supernatant. The glass filter is pretreated with human plasma protein in the glycerine buffer to avoid adsorption of the interferon during filtration. The eluates are pooled. If it is not possible to prepare the sample for zinc-chelate chromatography at once, the eluate can be stored for a few days at + 4 ° in plastic tissue culture flasks (Falcon 3024F) without affecting the stability of the interferon. The CPG purification results in better than 60% recovery with a 10fold concentration and removal of about 90% of the impurities as shown in the table.
Zinc-Chelate Chromatography The material from the CPG purification is dialyzed extensively against PB-NaC1, pH 7.4, to reduce the 0.3 M glycerine concentration to approximately 0.1 t~M. This amounts to dialzying a 100-ml sample against four changes of 4 liters each. To ensure stability of the interferon during this dialysis, additional HPPF may be added. The addition of this stabilizer has no effect on the resulting purity of the interferon, as it is not retained on the zinc-chelated column. Furthermore, the CPG-purified material to be purified by zinc-chelate chromatography should never have been frozen or lyophilized. It appears from experience that with long-term storage at either 4°, or under frozen condition or, more so, in the lyophilized state, the albumin from the culture medium that copurified on CPG becomes inseparably associated with the interferon and thus influences the purity of the final product. The dialyzed sample is applied to the column at a rate of 15-20 ml/hr
454
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[65]
LARGE-SCALE PURIFICATION OF HUMAN FIBROBLAST INTERFERON IN T w o STEPS:
BY CONTROLLED-POREGLASS(CPG) ANO ZINC-CHELATECrmOMATOGRAPHY
Designation CPG purification Crude Unabsorbed Wash Eluate I Eluate II
Volume (ml) 2100 2100 2100 140~ 140 )
Total protein (mg) 705 --140a
Recovery Zinc-chelate purification Dialyzed fraction of eluate pool Void + pH 7.4 wash pH 5.9 wash Purified interferon Recovery Overall recovery
Total activity (units) 6.6 x 0.24 x 0.06 x 4.9 x
10r 10r 10r 107
Specific activity (units/mg) 9.4 x lift --3.5 x 105
74.2% 280 350 150 10-12
140~ 132 3.80 0.025
4.9 x 10r
3.5 x 10~
<200 0.21 × 10r 4.58 x 107
0.55 × 106 1.8 x 109
93.5% 69.4%
Protein accounted for by addition of human plasma protein fraction has been subtracted from this value.
with the aid o f a peristaltic pump. This is followed by washing with one bed volume o f P B - N a C I to r e m o v e any remaining unbound proteins. To remove additional weakly bound contaminating proteins, the column is washed with 5 or more bed volumes o f sodium a c e t a t e - N a C l buffer, p H 5.9. This will also r e m o v e a small fraction ( < 10%) o f the interferon. This may be as high as 50% if the quality o f the interferon is p o o r - - t h a t is, interferon p r o d u c e d b y aging cells or interferon from low-yielding cell cultures. F o r small samples ( 5 - 5 0 ml, employing a 0.9 × 8 cm column) the interferon was eluted by developing a p H gradient o f sodium a c e t a t e - N a C 1 o f 20 ml o f buffer at p H 5.9 and 20 ml at p H 4.0 using the gradient maker described in the Materials section. F o r large-scale purification ( > 100 ml) the interferon is eluted with sodium a c e t a t e - N a C l , p H 4.2. U p to the final elution, I0 ml-fractions are collected in 15-ml polypropylene tubes (12 × 100 ram). With the approach o f the expected p H range (pH 5.6 to 4.2) where 98% o f the interferon elutes, 1-ml fractions are collected in
[65]
PURIFICATION OF HUMAN FIBROBLAST INTERFERON
455
12 x 75-mm polypropylene tubes. Polypropylene tubes are employed for reasons of stability of the interferon in these tubes. 14 Immediately upon completion of the elution of the interferon, 0.1 ml of each of the fractions is removed, employing a micropipetter and a fresh sterile tip for each fraction, for protein determination by the fluorometric method. H These and the following manipulations are all done in a laminarflow hood to minimize the risks of contamination. Additional 10-/~1 aliquots are removed from each fraction and diluted 1 : 10 into complete culture medium for interferon titration. The pH of the fractions is determined by insertion of a slender electrode starting with the last collected fraction. At this point 0.1 ml of 5% protein stabilizer is added for the preservation of biological activity during handling and storage. The zinc-chelate chromatography further concentrates the sample by 10-fold with a recovery of greater than 90%, usually averaging around 95% and yielding an interferon preparation averaging 2 x 109 reference units per milligram of protein (see the table). For future use fractions collected within the pH 5.6 to 4.2 range are pooled, placed in a dialysis bag, and concentrated by submersion in viscous PBS-Ficoll solution to one-third or less of its original volume. The material is further dialyzed against PBS to lower the salt concentration and neutralize the pH, then the sample is transferred to a lyophilization vial and lyophilized for storage. At this point small samples are taken for titration, sterility, and LAL test. All the samples tested as yet passed both sterility and LAL tests. The sample can be reconstituted without loss of activity. Also no loss of biological activity has been observed during this last process of concentration, dialysis, and lyophilization. Discussion Initial studies on the purification of interferon by the combination of the CPG and zinc-chelate methods showed that it was an advantage to follow the CPG by the zinc-chelate method, rather than the reverse. Further, if lyophilized CPG-purified material was utilized for purification on zincchelate columns, a large amount of protein was found to copurify with the interferon. This was observed also in combinations with other purification procedures (unpublished observations). However, when the interferon was employed as a fresh eluate from the CPG beads, no protein could be detected with the sensitive fluorometric protein assay. Upon purification of larger quantities of interferon by the combined CPG-zinc-chelate method, an average specific activity in the end product of 2 x l& refer~4 j. W. Heine, A. J. Mikulski, E, Sulkowski, and W. A. Carter, Arch. Viro[. 57, 185 (1978).
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[65]
ence units per miligram of protein was observed. This value is in agreement with the theoretical estimation of Ng and Vil~ek 15 and is slightly higher than that reported by Berthold et al. i and K n i g h t Y In view of the lower recoveries found by these workers, l'~'r it is possible that the specific activity was lower owing to the presence of inactivated interferon. Furthermore, our value reported here is more realistic in view of the specific activity of 108.3 units per milligram of partially purified material reported by Edy e t al. lo The indication that interferon was purified to homogeneity by this two-step procedure needed further evidence, and this was sought by radiolabeling experiments. As reported and described by Heine et al.,6 radioiodination of the purified interferon employing the Bolton-Hunter reagent 2 demonstrated the presence of a major radiolabeled protein of 22,000 daltons corresponding to the biological activity. By using different proteins (HPPF and OVA) as stabilizers, it was found that extensive transfer of label to stabilizers occurred after completion and neutralization of the radioiodination reaction. Therefore, the complete purity of the interferon preparation prior to addition of stabilizer could not be demonstrated with this technique. However, the purity of the interferon was demonstrated by the presence of only one band (at 22,000 daltons) in the autoradiogram prepared from the purified interferon labeled in vivo with 14C-labeled amino acids. 6 The possibility that minute impurities copurified with the interferon and are not detectable with the in vivo label is not ruled out, but owing to the high specific activity of the interferon preparation this is considered less likely. Additional in vitro labeling experiments employing 14C-labeled formaldehyde and analysis of the same by two-dimensional polyacrylamide gel electrophoresis are in progress and should resolve the issue. The purification method described here h a s several advantages over those available until now. First, the method is simple in that it involves only two steps, one of which is a batch procedure. Second, it is inexpensive in that the reagents can indefinitely be regenerated. Third, the method yields a pure end product with an average recovery of over 60% of the original biological activity. Acknowledgments This investigationwas supportedby a grant fromthe CancerResearchFoundationof the BelgianGeneralSavingsand RetirementFund (ASLK-CGER).The productionof interferon was madepossible by a research contract (No. 76/81-10) obtainedfrom the Geconcerteerde Onderzoeksacties. The technicalassistance of Francine Cornette, Chris Neuckermans-Dillen, Isabelle Ronsse,R. Conings,W. Put, and J. P. Lenaertsis acknowledged.The authors thank Jane Putzeysfor excellenteditorial help. 15M. H. Ng and J. Vil~ek,Adv. Protein Chem. 26, 179 (1972).
PURIFICATION
AND
CHARACTERIZATION
[65]
[65] Purification of Human Fibroblast Interferon by Adsorption to Controlled-Pore Glass and Zinc-Chelate Chromatography By
K. J. W. HEINE AND A. BILLIAU
Most purification techniques described for human interferons 1-5 yield either small quantities o f high purity, e.g., sufficient for determination o f specific activities, or larger quantities o f low purity. H o w e v e r , methods have been described for purification o f larger quantities o f completely pure interferon. 4,6,~ The procedure described here yields large quantities o f pure human fibroblast interferon in two steps. The overall recovery is at least 60% o f the starting materials, and the specific activity o f the end product varies from 1.5 to 3.1 x 109 reference units/mg, averaging about 2 x 109 reference units per milligram o f protein. The uniqueness o f the method is that the two steps yield a pure product when used in combination with each other, but not when either step is used in combination with other purification procedures, such as concanavalin A-Sepharose 8 or phenyl-Sepharose chromatography. The first step involves the partial purification o f crude human fibroblast interferon by controlled-pore glass (CPG) adsorption as described b y Billiau et al. 9 The second step consists of a modified and large-scale adaptation o f the zinc-chelate chromatographic purification m e t h o d originally designed by E d y et al. lo as a first step in the purification o f crude interferon. Materials Equipment
Cold room + 4 ° Laminar-flow hood 1 w. Berthoid, C. Tan, and Y. H. Tan, J. Biol. Chem. 253, 5206 (1978). E. Knight, Jr., J. Gen. Virol. 40, 681 (1978). a E. Knight, Jr., Proc. Natl. Acad, Sci. U.S.A. 73, 520 (1976). 4 M. Rubinstein, S. Rubinstein, P. G. Familletti, R. S. Miller, A. A. Waldman, and S. Pest_k_a,Proc. Natl. Acad. Sci. U.S.A. 76, 640 (1979). A. J. Mikulski, J. W. Heine, H. V. Le, and E. Sulkowski, Prep. Biochem. 10, 103 (1980). J. W. Heine, M. De Ley, J. Van Damme, A. Billiau, and P. De Somer, Ann. N. Y. Acad. Sci. 350, 364 (1980). E. Knight, Jr., Science 207, 525 (1980). 8 K. C. Chadha, P. M. Grob, A. J. Mikulski, L. R. Davis, Jr., and E. Sulkowski, J. Gen. Virol. 43, 701 (1979). A. Billiau, J. Van Damme, F, Van Leuven, V. G. Edy, M. De Ley, J. J. Cassiman, H. Van den Berghe, and P. De Somer, Antimicrob. Agents Chemother. 16, 49 (1979). 1oV. G. Edy, A. Billiau, and P. De Somer, J. Biol. Chem. 252, 5934 (1977). METHODS IN ENZYMOLOGY, VOL. 78
Copyright© 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7
[65]
PURIFICATION OF HUMAN FIBROBLAST INTERFERON
449
Spectrofluorometer Micropipetters (5-200 ~1), sterile pipette tips Fraction collector, peristaltic pump Gradient maker, prepared by connecting two 50-ml plastic syringes with epoxy glue Magnetic stirrer pH meter with long slender electrode Shaking water bath, 37° to 80° Controlled-pore glass beads (CPG), CPG 10-350 (Serva Feinbiochemica, P.O. Box 105260, D-6400 Heidelberg, West Germany) Spinner culture flasks (Wheaton Scientific, 1000 North Tenth St., Millville, NJ 08332) Sintered-glass filter, suction flask (1 liter) Dialyzing tubing, large 6 ~ inch, med. 3 ~ inch, small 1~ inch with a cutoff of 12,000-15,000 MW (Medical International Ltd., 49 Queen Victoria St., London EC4N 4SA, England) Columns K9/15, K15/30, or larger (Pharmacia Fine Chemicals, Uppsala, Sweden) Membrane filtration apparatus and 220 nm Millipore membranes (Millipore Corporation, Bedford, MA 01730) Plastic tissue culture flasks 75 cma, Falson 3024 F; polypropylene tubes, 17 × 100 mm, Falcon 2050; and 12 × 75 mm, Falcon 2063 Assortment of sterile glassware, flasks, beakers, graduated cylinders, bottles, pipettes, etc. Limulus amebocyte lysate (LAL) test kit (Microbiological Associates, Bethesda, MD) Chemicals
All chemicals employed are of highest available quality if the source is not given. Crude human fibroblast interferon, as described by Van Damme and BiUiau, this volume [13] (see section on partial purification of interferon) Epoxy-activated Sepharose 6B, Ficoll 400 (Pharmacia Fine Chemicals, Uppsala, Sweden) Iminodiacetic acid, disodium salt (Aldrich Europe, Beerse, Belgium) Ethanolamine Fluram, 1,4-dioxane Sodium carbonate (Na~CO3) Sodium chloride (NaCI) Zinc chloride (ZnCI~) Sodium phosphate, monobasic (NaH2PO4) Sodium phosphate, dibasic (Na2HPO4)
450
PURIFICATION AND CHARACTERIZATION
[65]
Sodium acetate Glacial acetic acid Sodium azide Glycine Hydrochloric acid (conc. HCI) Ethylenediaminetetraacetic acid (EDTA) Stabilizing proteins, such as 4.5% human plasma protein fraction (HPPF) Belgian Red Cross, National Blood Transfusion Service) Triple-glass-distilled water Pyrogen-free water (clinical grade)
Buffers Glycine.HCl buffers: 0.01 M, pH 3.5, and 0.3 M, pH 2.0, containing 0.10 mg/ml of HPPF. Make up molar glycine solution and adjust pH with conc. HC1. Phosphate-buffered saline (PBS): Dulbecco's formula (modified) without magnesium or calcium (Flow Laboratories, Irvine, Scotland) PB-NaCI buffer: 0.02 M phosphate buffer-1 M NaC1, pH 7.4 1. Make up 10x stock, pH 7.9, by dissolving 1.93 g of NaH~PO4.H20 (MW 138) and 26.4 g:of,Na~HPO4 (MW 142) in 1 liter of distilled H20. Sterilize by membrane filtration (220 nm) and store at 4 ° 2. Make up 5 × NaCI by dissolving 292.20 g of NaCI (MW 58.44) in I liter of distilled HzO. Sterilize by filtration and store at 4 °. 3. Before using, mix under sterile conditions: 100 ml of 10x PB, pH 7.9; 200 ml of 5 x NaCI; and 700 ml of pyrogen-free H20. Sodium acetate-NaC1 buffer: 0.1 M sodium acetate- 1 M NaC1, pH 4.0, 4.2, and 5.9: 1. Make up 10x stock of sodium acetate by dissolving 136.1 g of sodium acetate.3 H20 (MW 136.1) in 1 liter of distilled H20. Sterilize by filtration and store at 4 °. 2. At time of use mix under sterile condition 1 M acetate diluting it 1:10 and 5 M NaCI diluting it 1:5, adding some pyrogen-free H20; adjust to desired pH (5.9, 4.2, and 4.0) with glacial acetic acid. Then bring to final volume with pyrogen-free H~O. The pH electrode is sterilized by rinsing with 70% EtOH and pyrogenfree water. EDTA buffer 1. Make up 0.2 M EDTA (MW 372.24) by dissolving 37.22 g in 500 ml of distilled H20 and adjust pH 7.2 with saturated NaOH. Sterilize by filtration and store at 4°. 2. Mix aseptically: 100 ml of 0.2 M EDTA, pH 7.2; 40 ml of 0.2 M PB, pH 7.9; 80 ml of 5 M NaC1; and 180 ml ofpyrogen-free water
[65]
PURIFICATION OF HUMAN FIBROBLAST INTERFERON
451
Preparation of Chromatographic Materials Charging and Regeneration of Controlled-Pore Glass Beads. The procedure is carried out as described by Van Damme and Billiau in this volume [13] (see section on partial purification of interferon). Preparation of Zinc-Chelate Chromatography. The procedure is cartied out as described by Porath et al. 11 and Chadha et al. 8 Immobilization of iminodiacetic acid to epoxy-activated Sepharose 6B: I. Swell 15 g (one package) of epoxy-activated Sepharose 6B for 2 hr in 250 ml of distilled H20. Wash with 2000 ml of distilled H20 employing a suitable size sintered-glass filter, fitted to a l-liter suction flask. 2. Suspend in 50 ml of 2 M NaCO3 containing l0 g of iminodiacetic acid and place in a shaking water bath for 20 hr at 65 °. 3. Wash as in step 1 with 2000 ml of distilled H20 followed by resuspending in 200 ml of I N ethanolamine for ! hr at 65°, as above, to neutralize the unreacted sites. 4. Wash extensively with distilled H20 and store as a suspension with azide at 4°. Column preparation I. Choose a column size to yield a minimum 10-fold concentration of applied sample; i.e., with a sample of 50 ml on a 5 ml bed volume the interferon will be eluted in 3-4 ml, greater than 10-fold concentration. Thus, for samples of 5 to 50 ml, use a column of 0.9 x 8 cm (K9/15); and for 100 to 400 ml, use a column of 1.5 × 16 cm (K15/30). 2. Develop column under gravity at room temperature with degassed matrix. 3. Then place column in the cold room and wash with 10 bed volumes of cold distilled H20. 4. Wash with 5 bed volumes of cold E D T A - P B - N a C I buffer. 5. Wash with 5 bed volumes of cold PB-NaCI buffer. 6. Wash with 5 bed volumes of cold sodium acetate-NaC1, pH 4.0. 7. Wash with 2 bed volumes of cold sodium acetate-NaCl, pH 4.0, containing 5 mg/ml of ZnCI2. This is prepared fresh each time just before use and is also sterilized by filtration through a syringemounted membrane filter (220 nm). At completion of the zinc chelation, check last eluent (one drop) in 0.5 ml of CaCO3 solution for formation of precipitate. 8. If zinc saturation as tested above has been achieved, remove ex11 j. Porath, J. Carlsson, I. Olsson, and G. Belfson, Nature (London) 251t, 598 (1975).
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cess by washing with 5 bed volumes of sodium acetate-NaC1, pH 4.0. Equilibrate the column for sample application by washing with 5 bed volumes of PB-NaC1 buffer, pH 7.4. 10. The column is regenerated by starting with step 4. The number of times of possible regeneration has not been determined, After frequent use (10×) the top of the column becomes clogged with debris from applied samples. To overcome this the top of the bed is removed and replaced by fresh material to its orginal height. Furthermore an observation that might suggest replacement of columns is tailing of interferon upon elution from the column after 20 or more regenerations. .
Fluorometric Protein Assay x2 1. Add 10-100/zl of sample to 2 ml of 0.01 M PB (pH 7.4) and mix on mechanical mixer. 2. Prepare a fresh solution of 6 mg of Fluram in 20 ml of 1,4-dioxane, add 0.7 ml to step 1 preparation, and mix immediately. 3. Allow to stand for 15 min and read on a spectrofluorometer at 480 nm using bovine serum albumin as a standard.
Limulus Assay Follow test as described in Instruction Booklet accompanying the L A L test kit.
Interferon Assay Carry out as described by Van Damme and Billiau in this volume [13] (section on Interferon Assay) and by Finter. 13
Purification
First Purification Step: Adsorption and Desorption from CPG This step involves a batch-type adsorption of crude interferon onto CPG beads. Mix 1 volume of CPG beads with 30 volumes of crude interferon (clarified cell culture fluid) in a suitable-sized spinner culture flask 1~ p. BOhlen, J. Stein, W. Dairman, and S. Udenfriend, Arch. Biochem. Biophys. 155, 213 (1973). la N. B. Finter, J. Gen. Virol. 5, 419 (1969).
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and stir gently (enough to keep beads in suspension) for 2 hr at 4 °. The crude interferon should be freshly harvested or could have been stored at 4 ° for a few days, but not stored frozen prior to purification. If stored at 4 ° for a few days care should be taken not to allow the pH of the culture fluid to rise above pH 7.6, as this would affect the stability of the interferon. With the settling of the glass beads the supernatant is decanted. The beads are washed twice with 10 volumes of PBS followed by a single wash at the same ratio with 0.01 M glycerine.HC1 buffer, pH 3.5, to remove any loosely bound protein. With the glycine wash, the beads are quantitatively transferred to a smaller receptacle. The interferon is then eluted by twice gently stirring for 5 min 1 volume of beads in 1 volume of 0.3 M glycerine.HCl buffer, pH 2.0, containing 0.1 mg/ml of HPPF. This is followed by twice elucting the beads with the same buffer, but with an interaction time of 30 min each. All elution fluids are separated from the beads by filtration on a sintered-glass filter under gentle suction to recover all supernatant. The glass filter is pretreated with human plasma protein in the glycerine buffer to avoid adsorption of the interferon during filtration. The eluates are pooled. If it is not possible to prepare the sample for zinc-chelate chromatography at once, the eluate can be stored for a few days at + 4 ° in plastic tissue culture flasks (Falcon 3024F) without affecting the stability of the interferon. The CPG purification results in better than 60% recovery with a 10fold concentration and removal of about 90% of the impurities as shown in the table.
Zinc-Chelate Chromatography The material from the CPG purification is dialyzed extensively against PB-NaC1, pH 7.4, to reduce the 0.3 M glycerine concentration to approximately 0.1 t~M. This amounts to dialzying a 100-ml sample against four changes of 4 liters each. To ensure stability of the interferon during this dialysis, additional HPPF may be added. The addition of this stabilizer has no effect on the resulting purity of the interferon, as it is not retained on the zinc-chelated column. Furthermore, the CPG-purified material to be purified by zinc-chelate chromatography should never have been frozen or lyophilized. It appears from experience that with long-term storage at either 4°, or under frozen condition or, more so, in the lyophilized state, the albumin from the culture medium that copurified on CPG becomes inseparably associated with the interferon and thus influences the purity of the final product. The dialyzed sample is applied to the column at a rate of 15-20 ml/hr
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LARGE-SCALE PURIFICATION OF HUMAN FIBROBLAST INTERFERON IN T w o STEPS:
BY CONTROLLED-POREGLASS(CPG) ANO ZINC-CHELATECrmOMATOGRAPHY
Designation CPG purification Crude Unabsorbed Wash Eluate I Eluate II
Volume (ml) 2100 2100 2100 140~ 140 )
Total protein (mg) 705 --140a
Recovery Zinc-chelate purification Dialyzed fraction of eluate pool Void + pH 7.4 wash pH 5.9 wash Purified interferon Recovery Overall recovery
Total activity (units) 6.6 x 0.24 x 0.06 x 4.9 x
10r 10r 10r 107
Specific activity (units/mg) 9.4 x lift --3.5 x 105
74.2% 280 350 150 10-12
140~ 132 3.80 0.025
4.9 x 10r
3.5 x 10~
<200 0.21 × 10r 4.58 x 107
0.55 × 106 1.8 x 109
93.5% 69.4%
Protein accounted for by addition of human plasma protein fraction has been subtracted from this value.
with the aid o f a peristaltic pump. This is followed by washing with one bed volume o f P B - N a C I to r e m o v e any remaining unbound proteins. To remove additional weakly bound contaminating proteins, the column is washed with 5 or more bed volumes o f sodium a c e t a t e - N a C l buffer, p H 5.9. This will also r e m o v e a small fraction ( < 10%) o f the interferon. This may be as high as 50% if the quality o f the interferon is p o o r - - t h a t is, interferon p r o d u c e d b y aging cells or interferon from low-yielding cell cultures. F o r small samples ( 5 - 5 0 ml, employing a 0.9 × 8 cm column) the interferon was eluted by developing a p H gradient o f sodium a c e t a t e - N a C 1 o f 20 ml o f buffer at p H 5.9 and 20 ml at p H 4.0 using the gradient maker described in the Materials section. F o r large-scale purification ( > 100 ml) the interferon is eluted with sodium a c e t a t e - N a C l , p H 4.2. U p to the final elution, I0 ml-fractions are collected in 15-ml polypropylene tubes (12 × 100 ram). With the approach o f the expected p H range (pH 5.6 to 4.2) where 98% o f the interferon elutes, 1-ml fractions are collected in
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12 x 75-mm polypropylene tubes. Polypropylene tubes are employed for reasons of stability of the interferon in these tubes. 14 Immediately upon completion of the elution of the interferon, 0.1 ml of each of the fractions is removed, employing a micropipetter and a fresh sterile tip for each fraction, for protein determination by the fluorometric method. H These and the following manipulations are all done in a laminarflow hood to minimize the risks of contamination. Additional 10-/~1 aliquots are removed from each fraction and diluted 1 : 10 into complete culture medium for interferon titration. The pH of the fractions is determined by insertion of a slender electrode starting with the last collected fraction. At this point 0.1 ml of 5% protein stabilizer is added for the preservation of biological activity during handling and storage. The zinc-chelate chromatography further concentrates the sample by 10-fold with a recovery of greater than 90%, usually averaging around 95% and yielding an interferon preparation averaging 2 x 109 reference units per milligram of protein (see the table). For future use fractions collected within the pH 5.6 to 4.2 range are pooled, placed in a dialysis bag, and concentrated by submersion in viscous PBS-Ficoll solution to one-third or less of its original volume. The material is further dialyzed against PBS to lower the salt concentration and neutralize the pH, then the sample is transferred to a lyophilization vial and lyophilized for storage. At this point small samples are taken for titration, sterility, and LAL test. All the samples tested as yet passed both sterility and LAL tests. The sample can be reconstituted without loss of activity. Also no loss of biological activity has been observed during this last process of concentration, dialysis, and lyophilization. Discussion Initial studies on the purification of interferon by the combination of the CPG and zinc-chelate methods showed that it was an advantage to follow the CPG by the zinc-chelate method, rather than the reverse. Further, if lyophilized CPG-purified material was utilized for purification on zincchelate columns, a large amount of protein was found to copurify with the interferon. This was observed also in combinations with other purification procedures (unpublished observations). However, when the interferon was employed as a fresh eluate from the CPG beads, no protein could be detected with the sensitive fluorometric protein assay. Upon purification of larger quantities of interferon by the combined CPG-zinc-chelate method, an average specific activity in the end product of 2 x l& refer~4 j. W. Heine, A. J. Mikulski, E, Sulkowski, and W. A. Carter, Arch. Viro[. 57, 185 (1978).
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ence units per miligram of protein was observed. This value is in agreement with the theoretical estimation of Ng and Vil~ek 15 and is slightly higher than that reported by Berthold et al. i and K n i g h t Y In view of the lower recoveries found by these workers, l'~'r it is possible that the specific activity was lower owing to the presence of inactivated interferon. Furthermore, our value reported here is more realistic in view of the specific activity of 108.3 units per milligram of partially purified material reported by Edy e t al. lo The indication that interferon was purified to homogeneity by this two-step procedure needed further evidence, and this was sought by radiolabeling experiments. As reported and described by Heine et al.,6 radioiodination of the purified interferon employing the Bolton-Hunter reagent 2 demonstrated the presence of a major radiolabeled protein of 22,000 daltons corresponding to the biological activity. By using different proteins (HPPF and OVA) as stabilizers, it was found that extensive transfer of label to stabilizers occurred after completion and neutralization of the radioiodination reaction. Therefore, the complete purity of the interferon preparation prior to addition of stabilizer could not be demonstrated with this technique. However, the purity of the interferon was demonstrated by the presence of only one band (at 22,000 daltons) in the autoradiogram prepared from the purified interferon labeled in vivo with 14C-labeled amino acids. 6 The possibility that minute impurities copurified with the interferon and are not detectable with the in vivo label is not ruled out, but owing to the high specific activity of the interferon preparation this is considered less likely. Additional in vitro labeling experiments employing 14C-labeled formaldehyde and analysis of the same by two-dimensional polyacrylamide gel electrophoresis are in progress and should resolve the issue. The purification method described here h a s several advantages over those available until now. First, the method is simple in that it involves only two steps, one of which is a batch procedure. Second, it is inexpensive in that the reagents can indefinitely be regenerated. Third, the method yields a pure end product with an average recovery of over 60% of the original biological activity. Acknowledgments This investigationwas supportedby a grant fromthe CancerResearchFoundationof the BelgianGeneralSavingsand RetirementFund (ASLK-CGER).The productionof interferon was madepossible by a research contract (No. 76/81-10) obtainedfrom the Geconcerteerde Onderzoeksacties. The technicalassistance of Francine Cornette, Chris Neuckermans-Dillen, Isabelle Ronsse,R. Conings,W. Put, and J. P. Lenaertsis acknowledged.The authors thank Jane Putzeysfor excellenteditorial help. 15M. H. Ng and J. Vil~ek,Adv. Protein Chem. 26, 179 (1972).