Interferon biotechnology

The antiviral and anti-proliferative activity of interferons, as well as their ability to modulate the immune and inflammatory response renders obvious their potential medical application. This has culminated in the approval for clinical use of several interferon preparations (Table 8.8). Ongoing clinical trials are likely to expand the medical uses of these regulatory molecules further over the next few years.

While at least some of these potential therapeutic applications were appreciated as far back as the late 1950s, initial therapeutic application was rendered impractical due to the extremely low

Table 8.8 Interferon-based biopharmaceuticals approved to date for general medical use

Product

Company

Indication

Intron A (rIFN-a-2b)

Schering Plough

Cancer, genital warts, hepatitis

PegIntron A (PEGylated rIFN-a-2b)

Schering Plough

Chronic hepatitis C

Viraferon (rIFN-a-2b)

Schering Plough

Chronic hepatitis B and C

ViraferonPeg (PEGylated rIFN-a-2b)

Schering Plough

Chronic hepatitis C

Roferon A (rhIFN-a-2a)

Hoffman-La-Roche

Hairy cell leukaemia

Actimmune (rhIFN-y-1b)

Genentech

Chronic granulomatous disease (CGD)

Betaferon (rIFN-ß-1b, differs from

Schering AG

MS

human protein in that Cys 17 is

replaced by Ser)

Betaseron (rIFN-ß-1b, differs from

Berlex Laboratories and

Relapsing-remitting MS

human protein in that Cys 17 is

Chiron

replaced by Ser)

Avonex (rhIFN-ß-1a)

Biogen

Relapsing MS

Infergen (rIFN-a, synthetic type I

Amgen (USA)

Chronic hepatitis C

interferon)

Yamanouchi Europe (EU)

Rebif (rhIFN-ß-1a)

Ares Serono

Relapsing-remitting MS

Rebetron (combination of ribavirin

Schering Plough

Chronic hepatitis C

and rhIFN-a-2b)

Alfatronol (rhIFN-a-2b)

Schering Plough

Hepatitis B, C, and various cancers

Virtron (rhIFN-a-2b)

Schering Plough

Hepatitis B and C

Pegasys (Peginterferon a-2a)

Hoffman La Roche

Hepatitis C

Vibragen Omega (rFeline interferon

Virbac

Vet. (reduce mortality/clinical signs of

omega)

canine parvovirosis)

levels at which they are normally produced in the body. Large-scale purification from sources such as blood was non-viable. Furthermore, interferons exhibit species preference and, in some cases, strict species specificity. This rendered necessary the clinical use only of human-derived interferons in human medicine.

Up until the 1970s interferon was sourced (in small quantities) directly from human leukocytes obtained from transfused blood supplies. This 'interferon' preparation actually consisted of a mixture of various IFN-as, present in varying amounts, and was only in the regions of 1 per cent pure. However, clinical studies undertaken with such modest quantities of impure interferon preparations produced encouraging results.

The production of interferon in significant quantities first became possible in the late 1970s, by means of mammalian cell culture. Various cancer cell lines were found to secrete interferons in greater than normal quantities, and were amenable to large-scale cell culture due to their transformed nature. Moreover, hybridoma technology facilitated development of sensitive interferon immunoassays. The Namalwa cell line (a specific strain of human lymphoblastoid cells) became the major industrial source of interferon. The cells were propagated in large animal cell fermenters (up to 8000 l), and subsequent addition of an inducing virus (usually the Sendai virus) resulted in production of significant quantities of leukocyte interferon. Subsequent analysis showed this to consist of at least eight distinct IFN-a subtypes.

Wellferon was the tradename given to one of the first such approved products. Produced by large-scale mammalian (lymphoblastoid) cell cultures, the crude preparation undergoes extensive chromatographic purification, including two immunoaffinity steps. The final product contains nine IFN-a subtypes.

Recombinant DNA technology also facilitated the production of interferons in quantities large enough to satisfy potential medical needs. The 1980s witnessed the cloning and expression of most interferon genes in a variety of expression systems. The expression of specific genes obviously yielded a product containing a single interferon (sub)type.

Most interferons have now been produced in a variety of expression systems, including E. coli, fungi, yeast and some mammalian cell lines, such as CHO cell lines and monkey kidney cell lines. Most interferons currently in medical use are recombinant human (rh) products produced in E. coli. E. coli's inability to carry out post-translational modifications is irrelevant in most instances, as the majority of human IFN-as, as well as IFN-P, are not normally glycosylated. Whereas IFN-y is glycosylated, the E. coli-derived unglycosylated form displays a biological activity similiar to the native human protein.

The production of interferon 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 interferon final product preparations are in the region of 99 per cent pure. Such purity levels are achieved by extensive chromatographic purification. While standard techniques such as gel filtration and ion exchange are extensively used, reported interferon purification protocols have also entailed use of various affinity techniques using, for example, anti-interferon monoclonal antibodies, reactive dyes or lectins (for glycosylated interferons). Hydroxyapatite, metal-affinity and hydrophobic interaction chromatography have also been employed in purification protocols. Many production columns are run in HPLC (or FPLC) format, yielding improved and faster resolution. Immunoassays are used to detect and quantify the interferons during downstream processing, although the product (in particular the finished product) is also usually subjected to a relevant bioassay. The production and medical uses of selected interferons are summarized in the sections below.

8.3.1 Production and medical uses of interferon-a

Clinical studies undertaken in the late 1970s with multicomponent, 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 interferon 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 interferon 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 antiviral, anti-tumour and immunomodulatory properties of these interferons assured their approval for a variety of medical uses. rhIFN-as manufactured/marketed by a number of companies (Table 8.8) are generally produced in E. coli.

Clinical trials have shown the recombinant interferons 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 recombinant interferons promotes significant regression of the cancer in up to 90 per cent of patients.

Schering Plough's rhIFN-a2b (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 8.9). The producer microorganism is E. coli, which harbours a cytoplasmic expression vector (KMAC-43) containing the interferon gene. The gene product is expressed intracellulary. Intron A manufacturing facilities are located in New Jersey and in Brinny, Co. 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 42 000 l stainless steel vessels. After recovery of the product from the cells, a number of chromatographic purification steps are undertaken, essentially within

Table 8.9 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 AIDS-related Kaposi's sarcoma Multiple myeloma Chronic myelogenous leukaemia Non-Hodgkin's lymphoma aBenign growths (papillomas) in the larynx. bA fungal disease. cGenital warts.

Laryngeal papillomatosisa Mycosis fungoidesb Condyloma acuminatac Chronic hepatitis B Hepatitis C Chronic hepatitis D

Chronic hepatitis, non-A, non-B/C hepatitis a large cold-room adapted to function under cleanroom conditions. Crystallization of the IFN-a2b 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).

More recently, a number of modified recombinant interferon products have also gained marketing approval. These include PEGylated interferons (PEG IntronA and Viraferon Peg (Table 8.8 and Box 8.2) and the synthetic interferon product Infergen. PEGylated interferons are generated by reacting purified IFN-as with a chemically activated form of PEG. Activated methoxypoly-ethylene glycol is often used, which forms covalent linkages with free amino groups on the interferon molecule. Molecular mass analysis of PEGylated interferons (e.g. by mass spectroscopy, gel

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