- Email: [email protected]
[12] Large-scale production, concentration, and partial purification of human immune interferon
[12]
NATURALIFN-y PURIFICATION
83
Finally, the method presented consists of procedures which represent no significant barriers to large-scale operations. This method is recommended for general applications in both small and large-scale cases where minimal cost and maximal yield and purity of crude immune interferon are important considerations.
[12] L a r g e - S c a l e P r o d u c t i o n , C o n c e n t r a t i o n , a n d P a r t i a l Purification of Human Immune Interferon
By JERZY A. GEORGIADES Induction of the synthesis of gamma interferon by lymphocytes is connected with the activation of a variety of peripheral white blood cells (PWBC) with nonspecific mitogens (SEA, SEB, PHA, or Con A). Normally PWBC from a large number of donors are employed for large-scale production. In such situations, a mixed lymphocyte reaction occurs in addition to mitogen activation. As a result of the combined activation, different types of PWBC release into the media gamma interferon plus a variety of enzymes (proteases, phosphatases, oxygenases, phosphorylases, lipases, etc.) and other lymphokines (interleukins 1,2, and 3, inhibitors, other molecules, and growth factors). In media from stimulated PWBC, more than 100 different types of activities have been described. It is not clear how many distinct molecules account for all these different activities. Isolation of gamma interferon from the products of activated PWBC and the proteins and mitogens introduced in the incubation medium creates formidable purification problems. In addition, released proteases (J. A. Georgiades, unpublished observation) present in this mixture cause constant degradation of gamma interferon. At the time of harvest, the medium contains intact gamma interferon molecules as well as partially degraded interferon products which may have biological activity but differ physicochemically and in stability from the native molecules 2 (J. A. Georgiades, unpublished observation). In earlier studies, we have observed such heterogeneity in purified gamma interferon preparations. 3.4 l j. Georgiades, S. Baron, W. Fleichmann, Jr., M. Langford, D. Weigent, and G. Stanton, in "Interferons" (P. E. Came and W. A. Carter, eds.), p. 305. Springer-Verlag, Berlin and New York. z j. Sims, Proc. 1984 TNO-IS1R Meet. Interferon Syst., 1984 (1984). 3 j. Georgiades, in "Texas Reports on Biology and Medicine" (S. Baron, F. Dianziani, and G. Stanton, eds.), p. 260. University of Texas Medical Branch, Galveston, 1982. 4 j. Friedlander, D. Fischer, and M. Rubinstein, Anal. Biochem. 137, 115 (1984).
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
84
INDUCTION OF INTERFERONS
[12]
The properties of gamma interferon, especially its pH and heat sensitivity, its destruction by organic solvents and ionic detergents are additional factors which make the purification procedure complex. The observation of Pestka e t al. 5 that the functional unit of gamma interferon is trimeric or tetrameric adds another dimension of difficulty for purification. Use of any drastic separation methodology leads to disassociation and appearance of monomeric forms of gamma interferon. 6 Thus, it is desirable to purify gamma interferon in a manner designed to produce the native form. Existing purification methods utilize adsorption to controlled pore glass beads. 7-~° Eluted with ethylene glycol or TMAC, gamma interferon is further purified by partition chromatography, by affinity chromatography columns 6-8 or by ion-exchange matrices: Recently, partially purified material was also purified on an immunoaffinity column prepared from monoclonal antibodies and successfully eluted at high pH. H Though these methods are successful on a laboratory or analytical scale, they are laborious and frequently require intermediate steps like dialysis. Frequently end products, although very highly purified, show lack of stability under conditions of clinical use and appear to be in the monomeric form. 8 Furthermore, use of such methods (ethylene glycol or high pH) for prolonged periods of time during purification may cause irreversible losses of other biologically active molecules. ~ These could also be of clinical interest and could be recovered during gamma interferon purification should the process be gentle. Taking all of the above into consideration, we developed a purification procedure for gamma interferon which would satisfy the following criteria: 1. The method should be applicable for large-scale production. 2. It should be possible to purify 100-500 liters of crude material without difficulty. 5 S. Pestka, B. Kelder, P. Familletti, J. Moschera, R. Crowl, and E. Kempner, J. Biol. Chem. 258, 9706 (1983). 6 y. Yip, B. Barrowclough, C. Urban, and J. Vilgek, Science 215, 411 (1982). 7 j. Georgiades, M. Langford, G. Stanton, and H. Johnson, IRCS Med. Sci.: Libr. Compend. 7, 559 (1979). 8 I. A. Braude, U.S. Patent 4,440,675 (1984). 9 M. Wiranowska-Stewart, L. Lin, I. A. Braude, and W. Stewart, II. Mol. Immunol. 17, 625 (1980). 10 V. Papermaster and S. Baron, in "Texas Reports on Biology and Medicine" (S. Baron, F. Dianzani, and G. Stanton, eds.), p. 672. University of Texas Medical Branch, Galveston, 1982. ii D. Novick, Z. Eshhar, D. Fischer, J. Friedlander, and M. Rubinstein, EMBO J. 2, 1527 0983).
[12]
NATURAL
IFN-3, PURIFICATION
85
3. The procedure must be fast enough to accomplish the process within 36-48 hr. 4. Each step must be carried out under mild chromatographic conditions so that fractions which do not contain gamma interferon may be used for further purification of other biologically active molecules. 5. The purified interferon must be free from other commonly known lymphokines. 6. Purified interferon must be stable for at least 12 weeks at 2-8 ° . 7. The resulting product should be in its native form. In developing the purification procedure described below, ~2we tried to take into consideration all the above mentioned factors. This method of purification has now been in routine use for more than two years. The procedure gives a reproducible, consistent product. Interferon Gamma Induction To achieve success in purification, it was necessary to achieve adequate induction of gamma interferon by the PWBC. Gamma interferon batches which gave us an initial titer below 300 units/m[ usually gave poor results during purification.13 For that reason, we prepared crude gamma interferon from cultures of PWBC stimulated for 4 days with SEB (early induction), followed by 2 days stimulation with PHA (late induction). Purification procedures described herein are applicable to both types of induction of crude gamma interferon. For our purposes, the induction media from which the PWBC have been removed is called "crude material." Purification Procedure Step I O n e h u n d r e d (100) liters o f c r u d e m a t e r i a l with an a v e r a g e titer o f 103 u n i t s / m l a n d c o n t a i n i n g I m M E D T A t e t r a s o d i u m salt w a s clarified t h r o u g h a 0 . 5 - ~ m M i l l i p o r e m e m b r a n e at r o o m t e m p e r a t u r e b y m e a n s o f a P r o c o n p u m p . T h e s t a r t i n g m a t e r i a l t y p i c a l l y c o n t a i n e d a total o f ! x l0 w units o f g a m m a i n t e r f e r o n a n d 180 g o f p r o t e i n (specific a c t i v i t y 600 u n i t s / mg protein). ~2j. Georgiades, Gumulka, and Sulkowski, in preparation. ~3j. Georgiades, in "Texas Reports on Biology and Medicine" (S. Baron, F. Dianzani, and G. Stanton, eds.), p. 179. University of Texas Medical Branch, Galveston, 1982.
86
INDUCTION OF INTERFERONS
[12]
Retained material, approximateJy 2000 ml, contained about 1.4 mg/ml of protein and was not used. The ultrafiltrate from the 0.5-/xm cassette filter (containing 1 x 108 units of gamma interferon) was directly concentrated five times (to 20 liters) on a 10,000 molecular weight cut-off membrane. Five separate times, 20 liters of deionized water was added and the mixture concentrated to the original volume of 20 liters. The ultrafiltrate did not contain a detectable level of gamma interferon activity. With this step, specific activity of gamma interferon in the retained material increased to 900 units/mg of protein. Decrease of the ionic strength on the cassette system following addition of deionized water did not affect the interferon titer. This step gave approximately 1.5-fold purification.
Step H The concentrated material was acidified to pH 6.5 to 6.7 with 50% acetic acid while being cooled quickly to 10 to 12°, then mixed with two liters of activated CM-Sepharose C1-6B. After mixing for one hr at 4°, the supernatant was separated from the beads by means of filtration. CMSepharose with adsorbed interferon was loaded into a column. The matrix was washed with 0.02 M sodium acetate buffer, pH 6.3, until all unadsorbed proteins were removed, then washed with 0.02 M sodium phosphate buffer, pH 7.0 to 7.05, to remove proteins of noninterest. Proteins released at this pH were discarded. Gamma interferon was eluted with phosphate buffer containing 1.0 M sodium chloride, pH 7.2 to 7.4, at a linear flow rate of 10 cm/hr. Approximately 90% of the applied interferon was found in the major fraction. The collected fraction of 1100 ml contained 605 mg (i.e., 0.55 mg/ml) of protein and l06 units/ml of interferon (specific activity 1.8 z 106 units/mg protein). Purification was 300-fold; recovery was 200%.
Step III The interferon fraction from the CM column was directly applied to a zinc-chelate column ~4,15 equilibrated with 0.02 m sodium phosphate/1 M NaC1, pH 7.4, at a temperature of 4°. Material not adsorbed to the column contained 90% of the applied interferon activity. The phosphate buffer with 1.0 M NaCI wash removed the remaining 10% of activity. This step gave total recovery of applied interferon and threefold decrease in protein
14 K. J. W. Heine and A. Billiau, this series, Vol. 78, p. 448. ~5V. G. Edy, A. Billiau, and P. DeSomer, J. Biol. Chem. 252, 5934 (1977).
[12]
NATURALIFN-7 PURIFICATION
concentration. Specific activity increased from 1.8 × 106 to 5.4 units/rag.
87 x
10 6
Step IV The fraction which was unadsorbed to the zinc chelate column and wash fraction were pooled and applied directly onto a Con A-agarose column at 4°. This breakthrough fraction contained 1% of the interferon activity and 75% of applied proteins. After washing with PBS, interferon was eluted with 0.1 M methyl-D-a-mannopyranoside containing 1.0 M NaCI. The major interferon fraction contains 80% of applied activity with specific activity of 1 x 10 7 units/mg of protein. The final gamma interferon product was free of inducers, IL-I, IL-2, IL-3, and had an undetectable level of lymphotoxins, MIF, and chemotactic factors. Levels of other types of interferons were undetectable as shown by absence of interferon activity on bovine embryo kidney cells (BEKC) and its sensitivity to heating, and pH 2. In addition, it was not neutralized by specific antibodies to Hu-IFN-a, Hu-IFN-fl, or a mixture of antibodies to IFN-a and IFN-fl, but was neutralized by a specific antibody against Hu-IFN-y. Conclusions The above purification method is simple, effective, and relatively fast (purification time does not exceed 48 hr). In addition, this method can be applied to large quantities and can be totally automated without need for intermediate steps which would otherwise require adjustment of the material between steps of purification. Moreover, this method minimizes the possibility of bacterial and fungal contamination, is reproducible, and IFN-y appears to be in tetrameric form. Because of the very mild conditions of the purification, protein denaturation or alteration is reduced. Intermediate fractions obtained during IFN-y purification can be utilized for purification of other lymphokines (IL-2 for example).
NATURALIFN-y PURIFICATION
83
Finally, the method presented consists of procedures which represent no significant barriers to large-scale operations. This method is recommended for general applications in both small and large-scale cases where minimal cost and maximal yield and purity of crude immune interferon are important considerations.
[12] L a r g e - S c a l e P r o d u c t i o n , C o n c e n t r a t i o n , a n d P a r t i a l Purification of Human Immune Interferon
By JERZY A. GEORGIADES Induction of the synthesis of gamma interferon by lymphocytes is connected with the activation of a variety of peripheral white blood cells (PWBC) with nonspecific mitogens (SEA, SEB, PHA, or Con A). Normally PWBC from a large number of donors are employed for large-scale production. In such situations, a mixed lymphocyte reaction occurs in addition to mitogen activation. As a result of the combined activation, different types of PWBC release into the media gamma interferon plus a variety of enzymes (proteases, phosphatases, oxygenases, phosphorylases, lipases, etc.) and other lymphokines (interleukins 1,2, and 3, inhibitors, other molecules, and growth factors). In media from stimulated PWBC, more than 100 different types of activities have been described. It is not clear how many distinct molecules account for all these different activities. Isolation of gamma interferon from the products of activated PWBC and the proteins and mitogens introduced in the incubation medium creates formidable purification problems. In addition, released proteases (J. A. Georgiades, unpublished observation) present in this mixture cause constant degradation of gamma interferon. At the time of harvest, the medium contains intact gamma interferon molecules as well as partially degraded interferon products which may have biological activity but differ physicochemically and in stability from the native molecules 2 (J. A. Georgiades, unpublished observation). In earlier studies, we have observed such heterogeneity in purified gamma interferon preparations. 3.4 l j. Georgiades, S. Baron, W. Fleichmann, Jr., M. Langford, D. Weigent, and G. Stanton, in "Interferons" (P. E. Came and W. A. Carter, eds.), p. 305. Springer-Verlag, Berlin and New York. z j. Sims, Proc. 1984 TNO-IS1R Meet. Interferon Syst., 1984 (1984). 3 j. Georgiades, in "Texas Reports on Biology and Medicine" (S. Baron, F. Dianziani, and G. Stanton, eds.), p. 260. University of Texas Medical Branch, Galveston, 1982. 4 j. Friedlander, D. Fischer, and M. Rubinstein, Anal. Biochem. 137, 115 (1984).
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
84
INDUCTION OF INTERFERONS
[12]
The properties of gamma interferon, especially its pH and heat sensitivity, its destruction by organic solvents and ionic detergents are additional factors which make the purification procedure complex. The observation of Pestka e t al. 5 that the functional unit of gamma interferon is trimeric or tetrameric adds another dimension of difficulty for purification. Use of any drastic separation methodology leads to disassociation and appearance of monomeric forms of gamma interferon. 6 Thus, it is desirable to purify gamma interferon in a manner designed to produce the native form. Existing purification methods utilize adsorption to controlled pore glass beads. 7-~° Eluted with ethylene glycol or TMAC, gamma interferon is further purified by partition chromatography, by affinity chromatography columns 6-8 or by ion-exchange matrices: Recently, partially purified material was also purified on an immunoaffinity column prepared from monoclonal antibodies and successfully eluted at high pH. H Though these methods are successful on a laboratory or analytical scale, they are laborious and frequently require intermediate steps like dialysis. Frequently end products, although very highly purified, show lack of stability under conditions of clinical use and appear to be in the monomeric form. 8 Furthermore, use of such methods (ethylene glycol or high pH) for prolonged periods of time during purification may cause irreversible losses of other biologically active molecules. ~ These could also be of clinical interest and could be recovered during gamma interferon purification should the process be gentle. Taking all of the above into consideration, we developed a purification procedure for gamma interferon which would satisfy the following criteria: 1. The method should be applicable for large-scale production. 2. It should be possible to purify 100-500 liters of crude material without difficulty. 5 S. Pestka, B. Kelder, P. Familletti, J. Moschera, R. Crowl, and E. Kempner, J. Biol. Chem. 258, 9706 (1983). 6 y. Yip, B. Barrowclough, C. Urban, and J. Vilgek, Science 215, 411 (1982). 7 j. Georgiades, M. Langford, G. Stanton, and H. Johnson, IRCS Med. Sci.: Libr. Compend. 7, 559 (1979). 8 I. A. Braude, U.S. Patent 4,440,675 (1984). 9 M. Wiranowska-Stewart, L. Lin, I. A. Braude, and W. Stewart, II. Mol. Immunol. 17, 625 (1980). 10 V. Papermaster and S. Baron, in "Texas Reports on Biology and Medicine" (S. Baron, F. Dianzani, and G. Stanton, eds.), p. 672. University of Texas Medical Branch, Galveston, 1982. ii D. Novick, Z. Eshhar, D. Fischer, J. Friedlander, and M. Rubinstein, EMBO J. 2, 1527 0983).
[12]
NATURAL
IFN-3, PURIFICATION
85
3. The procedure must be fast enough to accomplish the process within 36-48 hr. 4. Each step must be carried out under mild chromatographic conditions so that fractions which do not contain gamma interferon may be used for further purification of other biologically active molecules. 5. The purified interferon must be free from other commonly known lymphokines. 6. Purified interferon must be stable for at least 12 weeks at 2-8 ° . 7. The resulting product should be in its native form. In developing the purification procedure described below, ~2we tried to take into consideration all the above mentioned factors. This method of purification has now been in routine use for more than two years. The procedure gives a reproducible, consistent product. Interferon Gamma Induction To achieve success in purification, it was necessary to achieve adequate induction of gamma interferon by the PWBC. Gamma interferon batches which gave us an initial titer below 300 units/m[ usually gave poor results during purification.13 For that reason, we prepared crude gamma interferon from cultures of PWBC stimulated for 4 days with SEB (early induction), followed by 2 days stimulation with PHA (late induction). Purification procedures described herein are applicable to both types of induction of crude gamma interferon. For our purposes, the induction media from which the PWBC have been removed is called "crude material." Purification Procedure Step I O n e h u n d r e d (100) liters o f c r u d e m a t e r i a l with an a v e r a g e titer o f 103 u n i t s / m l a n d c o n t a i n i n g I m M E D T A t e t r a s o d i u m salt w a s clarified t h r o u g h a 0 . 5 - ~ m M i l l i p o r e m e m b r a n e at r o o m t e m p e r a t u r e b y m e a n s o f a P r o c o n p u m p . T h e s t a r t i n g m a t e r i a l t y p i c a l l y c o n t a i n e d a total o f ! x l0 w units o f g a m m a i n t e r f e r o n a n d 180 g o f p r o t e i n (specific a c t i v i t y 600 u n i t s / mg protein). ~2j. Georgiades, Gumulka, and Sulkowski, in preparation. ~3j. Georgiades, in "Texas Reports on Biology and Medicine" (S. Baron, F. Dianzani, and G. Stanton, eds.), p. 179. University of Texas Medical Branch, Galveston, 1982.
86
INDUCTION OF INTERFERONS
[12]
Retained material, approximateJy 2000 ml, contained about 1.4 mg/ml of protein and was not used. The ultrafiltrate from the 0.5-/xm cassette filter (containing 1 x 108 units of gamma interferon) was directly concentrated five times (to 20 liters) on a 10,000 molecular weight cut-off membrane. Five separate times, 20 liters of deionized water was added and the mixture concentrated to the original volume of 20 liters. The ultrafiltrate did not contain a detectable level of gamma interferon activity. With this step, specific activity of gamma interferon in the retained material increased to 900 units/mg of protein. Decrease of the ionic strength on the cassette system following addition of deionized water did not affect the interferon titer. This step gave approximately 1.5-fold purification.
Step H The concentrated material was acidified to pH 6.5 to 6.7 with 50% acetic acid while being cooled quickly to 10 to 12°, then mixed with two liters of activated CM-Sepharose C1-6B. After mixing for one hr at 4°, the supernatant was separated from the beads by means of filtration. CMSepharose with adsorbed interferon was loaded into a column. The matrix was washed with 0.02 M sodium acetate buffer, pH 6.3, until all unadsorbed proteins were removed, then washed with 0.02 M sodium phosphate buffer, pH 7.0 to 7.05, to remove proteins of noninterest. Proteins released at this pH were discarded. Gamma interferon was eluted with phosphate buffer containing 1.0 M sodium chloride, pH 7.2 to 7.4, at a linear flow rate of 10 cm/hr. Approximately 90% of the applied interferon was found in the major fraction. The collected fraction of 1100 ml contained 605 mg (i.e., 0.55 mg/ml) of protein and l06 units/ml of interferon (specific activity 1.8 z 106 units/mg protein). Purification was 300-fold; recovery was 200%.
Step III The interferon fraction from the CM column was directly applied to a zinc-chelate column ~4,15 equilibrated with 0.02 m sodium phosphate/1 M NaC1, pH 7.4, at a temperature of 4°. Material not adsorbed to the column contained 90% of the applied interferon activity. The phosphate buffer with 1.0 M NaCI wash removed the remaining 10% of activity. This step gave total recovery of applied interferon and threefold decrease in protein
14 K. J. W. Heine and A. Billiau, this series, Vol. 78, p. 448. ~5V. G. Edy, A. Billiau, and P. DeSomer, J. Biol. Chem. 252, 5934 (1977).
[12]
NATURALIFN-7 PURIFICATION
concentration. Specific activity increased from 1.8 × 106 to 5.4 units/rag.
87 x
10 6
Step IV The fraction which was unadsorbed to the zinc chelate column and wash fraction were pooled and applied directly onto a Con A-agarose column at 4°. This breakthrough fraction contained 1% of the interferon activity and 75% of applied proteins. After washing with PBS, interferon was eluted with 0.1 M methyl-D-a-mannopyranoside containing 1.0 M NaCI. The major interferon fraction contains 80% of applied activity with specific activity of 1 x 10 7 units/mg of protein. The final gamma interferon product was free of inducers, IL-I, IL-2, IL-3, and had an undetectable level of lymphotoxins, MIF, and chemotactic factors. Levels of other types of interferons were undetectable as shown by absence of interferon activity on bovine embryo kidney cells (BEKC) and its sensitivity to heating, and pH 2. In addition, it was not neutralized by specific antibodies to Hu-IFN-a, Hu-IFN-fl, or a mixture of antibodies to IFN-a and IFN-fl, but was neutralized by a specific antibody against Hu-IFN-y. Conclusions The above purification method is simple, effective, and relatively fast (purification time does not exceed 48 hr). In addition, this method can be applied to large quantities and can be totally automated without need for intermediate steps which would otherwise require adjustment of the material between steps of purification. Moreover, this method minimizes the possibility of bacterial and fungal contamination, is reproducible, and IFN-y appears to be in tetrameric form. Because of the very mild conditions of the purification, protein denaturation or alteration is reduced. Intermediate fractions obtained during IFN-y purification can be utilized for purification of other lymphokines (IL-2 for example).