d. gen. ViroL (1976), 33, 517-521
527
Printed in Great Britain
Purification of Interferon by Adsorption Cbromatography on Controlled Pore Glass (Accepted
21
July 1976)
SUMMARY
Human fibroblast and mouse L929 cell interferons can be purified by adsorption to and subsequent elution from Controlled Pore Glass. Purification of 4o to 9o-fold to specific activities of I to 5 × lO6 units/mg of protein can be achieved in a single step, with good recovery of activity. Human leukocyte interferon does not bind to the glass and cannot be purified in this way. Previous methods for the purification of human and mouse interferons have been based on physicochemical techniques (Cantell et al. 1974; Knight, 1975, 1976; reviewed by Fantes, 1973), the use of immunoadsorbents (Ogburn, Berg & Paucker, 1973; Sipe et al. I973 ; Anfinsen et al. I974; Berg et al. 1975) and on lectin and hydrophobic chromatography (Davey et al. I974; Jankowski et al. 1975; Davey, Sulkowski & Carter, 1976a, b). In this communication we report that human diploid fibroblast and mouse L929 cell interferons can be purified 4o to 9o-fold with recovery of approx. 67 ~ of the activity by adsorption to Controlled Pore Glass (CPG) granules. The adsorption of interferons to glass was first noted several years ago by Lampson et al. (I963), and Davies (1965) achieved a twofold purification of calf interferon by eluting the adsorbed activity. The use of CPG for adsorption chromatography of several proteins has recently been described (Bock et al. I976), but the conditions for adsorption and elution differ significantly from those which we have used for interferons. Human diploid fibroblast interferon, prepared essentially as described by Billiau, Joniau & De Somer (I973), was produced in Eagle's minimal essential medium (EMEM) buffered with 6 mM-N-2-hydroxyethyl piperazine (Hepes) and 13 mu-N-tris-hydroxymethyl-methyl glycine (Tricine); (Havell et al. 1975) and supplemented with I ~ (v/v) human plasma protein Fraction (National Blood Transfusion Service, Belgian Red Cross). The interferon used in these experiments had a titre of 25 ooo units]ml, and asp. act. of 56 ooo units/mg protein. Mouse L929 cell interferon, with a titre of 16ooo units/ml and asp. act. of 25ooo units/mg protein, was produced by a modification of the method of Knight (I975). Crude concentrated human leukocyte interferon was the gift of Dr Karl Cantell, Helsinki, Finland, and as supplied contained I7ooooo units/ml, with a s p . act. of 29ooo units[mg of protein. Before use it was diluted with EMEM to the same protein concentration (o'45 mg/ml) as the human fibroblast interferon. An I I-8 cm x 0"9 cm (7"5 ml) column of CPG Io-35o (Electro-Nucleonics, Inc., Fairfield, N.J., USA) was used for experiments with human fibroblast interferon; for mouse L929 cell and human leukocyte interferons a I2"5 c m x o ' 9 cm (8 ml) column was used. All experiments were performed at 4 °C. Human interferon was assayed by a modified dye uptake method (Finter, 1969); two laboratory reference standard interferon preparations, calibrated against the British Research Standard for Interferon, MRC B69[I9, were included in each assay. All results are expressed in terms of this standard, although it has been shown (Edy, Billiau & De Somer, 34
VIR 33
518
Short communications I
I
I
I
I~
I
I
I
100
I
-
6 -~
_
90 (A
80
r~
70 60 e~ o
50
3
40
30 20
2
10 1"5
527
Printed in Great Britain
Purification of Interferon by Adsorption Cbromatography on Controlled Pore Glass (Accepted
21
July 1976)
SUMMARY
Human fibroblast and mouse L929 cell interferons can be purified by adsorption to and subsequent elution from Controlled Pore Glass. Purification of 4o to 9o-fold to specific activities of I to 5 × lO6 units/mg of protein can be achieved in a single step, with good recovery of activity. Human leukocyte interferon does not bind to the glass and cannot be purified in this way. Previous methods for the purification of human and mouse interferons have been based on physicochemical techniques (Cantell et al. 1974; Knight, 1975, 1976; reviewed by Fantes, 1973), the use of immunoadsorbents (Ogburn, Berg & Paucker, 1973; Sipe et al. I973 ; Anfinsen et al. I974; Berg et al. 1975) and on lectin and hydrophobic chromatography (Davey et al. I974; Jankowski et al. 1975; Davey, Sulkowski & Carter, 1976a, b). In this communication we report that human diploid fibroblast and mouse L929 cell interferons can be purified 4o to 9o-fold with recovery of approx. 67 ~ of the activity by adsorption to Controlled Pore Glass (CPG) granules. The adsorption of interferons to glass was first noted several years ago by Lampson et al. (I963), and Davies (1965) achieved a twofold purification of calf interferon by eluting the adsorbed activity. The use of CPG for adsorption chromatography of several proteins has recently been described (Bock et al. I976), but the conditions for adsorption and elution differ significantly from those which we have used for interferons. Human diploid fibroblast interferon, prepared essentially as described by Billiau, Joniau & De Somer (I973), was produced in Eagle's minimal essential medium (EMEM) buffered with 6 mM-N-2-hydroxyethyl piperazine (Hepes) and 13 mu-N-tris-hydroxymethyl-methyl glycine (Tricine); (Havell et al. 1975) and supplemented with I ~ (v/v) human plasma protein Fraction (National Blood Transfusion Service, Belgian Red Cross). The interferon used in these experiments had a titre of 25 ooo units]ml, and asp. act. of 56 ooo units/mg protein. Mouse L929 cell interferon, with a titre of 16ooo units/ml and asp. act. of 25ooo units/mg protein, was produced by a modification of the method of Knight (I975). Crude concentrated human leukocyte interferon was the gift of Dr Karl Cantell, Helsinki, Finland, and as supplied contained I7ooooo units/ml, with a s p . act. of 29ooo units[mg of protein. Before use it was diluted with EMEM to the same protein concentration (o'45 mg/ml) as the human fibroblast interferon. An I I-8 cm x 0"9 cm (7"5 ml) column of CPG Io-35o (Electro-Nucleonics, Inc., Fairfield, N.J., USA) was used for experiments with human fibroblast interferon; for mouse L929 cell and human leukocyte interferons a I2"5 c m x o ' 9 cm (8 ml) column was used. All experiments were performed at 4 °C. Human interferon was assayed by a modified dye uptake method (Finter, 1969); two laboratory reference standard interferon preparations, calibrated against the British Research Standard for Interferon, MRC B69[I9, were included in each assay. All results are expressed in terms of this standard, although it has been shown (Edy, Billiau & De Somer, 34
VIR 33
518
Short communications I
I
I
I
I~
I
I
I
100
I
-
6 -~
_
90 (A
80
r~
70 60 e~ o
50
3
40
30 20
2
10 1"5
