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Recombinant DNA technology also facilitated the production of IFNs in quantities large enough to satisfy potential medical needs. The 1980s witnessed the cloning and expression of most IFN genes in a variety of expression systems. The expression of specific genes obviously yielded a product containing a single IFN (sub)-type.
Most IFNs have now been produced in a variety of expression systems including E. coli, fungi, yeast and also some mammalian cell lines such as Chinese hamster ovary (CHO) cell lines and monkey kidney cell lines. Most IFNs currently in medical use are recombinant human (rh) products produced in E. coli. The inability of E. coli to carry out post-translational modifications is in most instances irrelevant, as the majority of human IFN-as, as well as IFN-b, are not normally glycosylated. While IFN-g is glycosylated, the E. coli-derived unglycosylated form displays a biological activity identical to the native human protein.
The production of IFN in recombinant microbial systems obviously means that any final product contaminants will be microbial in nature. A high degree of purification is thus required to minimize the presence of such non-human substances. Most IFN final product preparations are in the region of 99% pure. Such purity levels are achieved by extensive chromatographic purification. While standard techniques such as gel filtration and ion-exchange are extensively used, reported IFN purification protocols have also entailed the use of various affinity techniques, e.g. using anti-IFN monoclonal antibodies, reactive dyes or lectins (for glycosylated IFNs). Hydroxyapatite, metal-affinity and hydrophobic interaction chromatography have also been employed in purification protocols. Many production columns are run in fast protein or high-performance liquid chromotography (FPLC or HPLC) format, yielding improved and faster resolution. Immunoassays are used to detect and quantify the IFNs during downstream processing, although the product (particularly the finished product) is also usually subjected to a relevant bioassay. The production and medical uses of selected IFNs are summarized in the sections below.
Production and medical uses of IFN-a
Clinical studies undertaken in the late 1970s, with multi-component, impure IFN-a preparations, clearly illustrated the therapeutic potential of such interferons as an anti-cancer agent. These studies found that IFN-a could induce regression of tumours in significant numbers of patients suffering from breast cancer, certain lymphomas (malignant tumour of the lymph nodes) and multiple myeloma (malignant disease of the bone marrow). The IFN preparations could also delay recurrence of tumour growth after surgery in patients suffering from osteogenic sarcoma (cancer of connective tissue involved in bone formation).
The first recombinant IFN to become available for clinical studies was IFN-a2A, in 1980. Shortly afterwards the genes coding for additional IFN-as were cloned and expressed, allowing additional clinical studies. The anti-viral, anti-tumour and immuno-modulatory properties of these IFNs assured their approval for a variety of medical uses. rhIFN-as manufactured/ marketed by a number of companies (Table 4.8) are generally produced in E. coli.
Clinical trials have shown the recombinant IFNs to be effective in the treatment of various cancer types, with rhIFN-a2A and -a2B both approved for treatment of hairy cell leukaemia. This is a rare B lymphocyte neoplasm for which few effective treatments were previously available. Administration of the rIFNs promotes significant regression of the cancer in up to 90% of patients.
THE CYTOKINES—THE INTERFERON FAMILY 211
Table 4.9. Some of the indications (i.e. medical conditions) for which intron A is approved. Note that the vast majority are either forms of cancer or viral infections
Hairy cell leukaemia Renal cell carcinoma Basal cell carcinoma Malignant melanoma
Laryngeal papillomatosis* Mycosis fungoides** Condyloma acuminata *** Chronic hepatitis B Hepatitis C Chronic hepatitis D Chronic hepatitis, non-A, non-B/C hepatitis
AIDS-related Kaposi’s sarcoma Multiple myeloma Chronic myelogenous leukaemia Non-Hodgkin’s lymphoma
*Benign growths (papillomas) in the larynx.
**A fungal disease.
Schering Plough’s rhIFN a-2B (Intron A) was first approved in the USA in 1986 for treatment of hairy cell leukaemia, but is now approved for use in more than 50 countries for up to 16 indications (Table 4.9). The producer microorganism is E. coli, which harbours a cytoplasmic expression vector (KMAC-43) containing the IFN gene. The gene product is expressed intracellularly and in soluble form. Intron A manufacturing facilities are located in New Jersey and Brinny, County Cork, Ireland.
Upstream processing (fermentation) and downstream processing (purification and formulation) are physically separated by being undertaken in separate buildings. Fermentation is generally undertaken in specially designed 42000 litre stainless steel vessels. After recovery of the product from the cells, a number of chromatographic purification steps are undertaken, essentially within a large cold room adapted to function under clean room conditions. Crystallization of the IFN-a-2B is then undertaken as a final purification step. The crystalline product is redissolved in phosphate buffer, containing glycine and human albumin as excipients. After aseptic filling, the product is normally freeze-dried. Intron A is usually sold at five commercial strengths (3, 5, 10, 25 and 50 million IU/vial).