averaged 750 units per cm2 of culture area. .... consist ofa polystyrene sheet (370 by 23 cm) rolled up .... (1) Replace serum-containing medium by 50 ml of.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1979, p. 49-55 0066-4804/79/07-0049/07$02.00/0
Vol. 16, No. 1
Human Fibroblast Interferon for Clinical Trials: Production, Partial Purification, and Characterization ALFONS BILLIAU,'* JO VAN DAMME,1 FRED VAN LEUVEN,2 VICTOR G. EDY,t MARC DE LEY,' JEAN-JACQUES CASSIMAN,2 HERMAN VAN DEN BERGHE,2 AND PIET DE SOMER' Divisions of Microbiology (Rega Institute)' and of Human Genetics,2 Department of Human Biology, University of Leuven, B-3000 Leuven, Belgium Received for publication 30 April 1979
The production and partial purification of human fibroblast interferon for performing clinical trials is described. The interferon was produced by superinduction (exposure to riboinosinic-ribocytidylic acid, cycloheximide, and actinomycin D) of large numbers of human diploid fibroblast cultures. The yield averaged 750 units per cm2 of culture area. The interferon was concentrated and purified by a two-step procedure involving acid desorption from controlled-pore glass beads and dialysis against polyethylene glycol. Human plasma protein was added as a stabilizer. The lyophilized end product had a specific activity of 0.5 x 106 to 1 x 106 units/mg of protein; it could be reconstituted for injection at a concentration of 2 x 106 units/ml. The composition of this interferon was characterized by crossed immunoelectrophoresis with polyspecific antibodies prepared against the principal sources of potential contaminants: human serum, calf serum, and normal human fibroblasts. Several components of each source were detected. Although the major component of calf serum, bovine serum albumin, was absent, other minor components were retained by the production and purification sequence. One of the main contaminants of fibroblast origin was found to be fibronectin.
superinduction by sequential exposure to riboinosinic-ribocytidylic acid [poly(I:C)], cycloheximide, and actinomycin D) (3, 11). Moreover, Finterferon seems to lose activity more easily during the concentration and purification procedures (7). However, F-interferon has the theoretical advantage that it is not limited by the supply of fresh leukocytes. Furthernore, in certain clinical applications F-interferon may have advantages over L-interferon. In our laboratory F-interferon was produced for parenteral administration in humans at dosages of up to 20 x 106 units per injection. In the present communication we describe the technique and results of routine production and puover, this type of interferon is stable and rela- rification, as well as an immunochemical chartively easy to concentrate and purify. As a result acterization of the final preparations. of the efforts of one single laboratory (5), LMATERIALS AND METHODS interferon has been available in an injectable form for several years, and data from clinical Diploid cell strains (EISM, E,L, E4SM, E4L, E6SM, trials are slowly accumulating. The production E6L) were established from skin and muscle or lung of of F-interferon is more complicated, requiring human embryos (ca. 15 weeks) obtained by induced the cultivation of large numbers of diploid skin abortions. The cells were cultivated by trypsinization 1/2 splits in stationary bottles for one to five fibroblasts, as well as a sequence of delicate and passages. At each passage level part of the cells were manipulations to achieve optimal induction frozen in liquid nitrogen. The cells were (priming with small amounts of interferon and The absence of mycoplasmas was verifiedkaryotyped. (16), and The interferon mechanism is considered as an important element of the natural defense of vertebrates against viruses and cancer (for a review see reference 17). Human interferon exists in at least three molecular variants: L- (leukocyte), F(fibroblast), and immune interferons (8, 12, 15, 18, 19). To test the clinical applicability of these interferons, systems for mass production are needed. Furthermore, the crude preparations must be concentrated and purified to the extent that no harmful contaminants are left. The production of L-interferon is relatively simple (5): fresh leukocyte cells from blood donors, when exposed to certain paramyxoviruses, release relatively large quantities of L-interferon. More-
the strains were screened for interferon-producing potential as described earlier (2). Karyotyping and my-
t Present address: Theodor Kocher Institute, Postfach 99, CH-3000 Bern 9, Switzerland.
BILLIAU ET AL.
coplasma tests were repeated at several later passage levels. The culture medium was Eagle minimum essential medium with added nonessential amino acids, supplemented with either fetal or newborn calf serum as indicated in the text. Antibiotics, when present, were penicillin at 100 ug/ml and streptomycin at 100 ,ug/mi. All culture reagents were purchased from Gibco-BioCult, Ltd. (Paisley, Scotland). Mass cell propagation was done in plastic roller bottles (11-cm diameter, Corning Glass Works, Oneonta, N.Y.). Initially only bottles of 490-cm2 surface area (180 ml of growth medium) were used; later an 850-cm2-size bottle (250 ml of medium) became available. The roller machines were adjusted to obtain a linear speed of the culture surface of 8.5 cm/min. Bulk culture vessels (Sterilin, Teddington, England) were also used for cell propagation. These vessels consist of a polystyrene sheet (370 by 23 cm) rolled up inside a cylindrical container (25 by 10 cm). They allow growth of human diploid cells in medium supplemented with 0.01% of an antifoam emulsion (supplied by the manufacturer) and under continuous aeration with a mixture of 95% air and 5% CO2. Poly(I:C) was purchased from P-L Biochemicals, Inc. (Milwaukee, Wis.); cycloheximide, actinomycin D, polyethylene glycol 20,000, and controlled-pore gla beads (CPG; mesh size 120 to 200; particle size 75 to 125 pm; mean pore size 35 nm) were purchased from Serva, GmbH & Co., Heidelberg, Germany. Human plasma protein fraction (Cohn fraction V) was obtained as a sterile pyrogen-free solution containing 45 mg of protein per ml (Belgian Red Cross National Blood Transfusion Service). Sterility controls were done at the different stages of production and on the crude interferon harvest: 10ml aliquots of fluids were filtered through 220-nm membrane filters, which were incubated on blood agar plates. F-interferon was titrated by inhibition of vesicular stomatitis or Semliki Forest virus replication in human diploid cells using a neutral red uptake method (10). The samples were calibrated against an internal laboratory standard of human F-interferon. The unitage assigned to this internal standard was found to be concordant with that of the research reference preparation of F-interferon (G-023-902-527) recently made by the National Institutes of Health, Bethesda, Md. The general procedures used for crossed immunoelectrophoresis (CIE) were as described (20). The gels contained 1% agarose in sodium Veronal buffer (75 mM, pH 8.6), with 2 mM calcium lactate and 15 mM sodium azide. The gels also contained 1% of the nonionic detergent Berol 043 (Berol Kemi AB, Stenungsund, Sweden). All gels were stained with Coomassie brilliant blue after removal by pressing and soaking of non-precipitated proteins. Rabbit anti-human serum antibodies were purchased from Dakopatts (Copenhagen, Denmark). Anti-newborn calf serum antibodies were prepared as described (21). The procedure for the preparation of anti-normal human fibroblast antibodies will be described in detail elsewhere (F. Van Leuven, J. Verlinden, J. J. Cassiman, and H. Van den Berghe, submitted for publication). Briefly, rabbits were injected with
ANTIMICROB. AGENTS CHEMOTHER. whole cultured cells, together with complete Freund adjuvant. After four injections at 2-week intervals, animals were bled by heart puncture. Immunoglobulin G was isolated from the serum and used as such.
RESULTS Production of F-interferon. F-interferon was produced by superinduction of human diploid embryonic skin muscle cells, according to the protocol outlined in Table 1. This protocol differs only slightly from that devised earlier for production of F-interferon on a smaller scale (3, 11). Human plasma protein fraction was added before superinduction to help remove calf serum proteins by a diluting effect. A total Cohn fraction V was chosen rather than purified albumin, because it was readily available as a sterile and pyrogen-free solution, acceptable for parenteral administration in humans. Furthermore, the timing of additions of poly(I:C), cycloheximide, and actinomycin D was modified for convenience, after it was found that these modifications still favored optimal yields of interferon. TABLE 1. Production protocol of F-interferon A. Preparation of cell cultures: (1) Start from liquid nitrogen-frozen stock (passage level 1 to 5). (2) Cultivate cells by weekly 1:2 splits in roller bottles with antibiotic-free medium, containing 10% fetal bovine serum (passage level 7 to 22); incubation at 36.4°C. (3) Propagate cells for one passage in bulk culture vessels by 1:3 split in medium containing 10% newborn calf serum and antibiotics. (4) Twice weekly, prepare batches of 100 to 120 roller bottles from four to six bulk culture vessels, using same medium as (3) above; incubate until confluent (ca. 1 week). (5) Refeed with fresh medium [same formula as (3)
above]. (6) Age for 11 days. B. Induction (clusters of eight roller bottles): (1) Replace serum-containing medium by 50 ml of priming solution: F-interferon (100 units/ml) in medium containing antibiotics and human plasma protein (4.5 mg/ml); incubate for 20 h. (2) Replace priming solution by 20 ml of poly(I:C) (50 ,ug/mi) and cycloheximide (10 Ag/mi) in serum-free medium; incubate for 4 h. (3) Add actinomycin D (1 pAg/ml); incubate for 2 h. (4) Wash cultures twice with 50 ml of serum-free medium and refeed with 20 ml of medium containing human plasma protein (0.45 mg/ml); incubate for 24 h. (5) Harvest fluids per cluster of eight bottles (- 2to 2.5-liter total of crude F-interferon per batch of 100 to 120 roller bottles); store at 4°C for