Biopharmaceuticals: Approvals and Approval Trends in 2004

Currently, some 160 biopharmaceuticals (defined as recombinant therapeutic proteins, monoclonal antibody-based products used for therapeutic or in vivo diagnostic purposes, as well as nucleic acid-based products) have gained marketing approval in the US and EU. Approx-imately one-fourth of new drugs coming on the market are biopharmaceuticals.¹ These drugs have benefited some 325 million people worldwide² and generate in excess of $30 billion annually, with sales projected to surpass $50 billion by 2010.³ Twelve biopharmaceuticals gained approval within the US and EU in 2004 (Table 1). Six are antibody-based products and two are engineered insulins. The other four are a blood factor, a vaccine containing a recombinant component, a growth factor, and a nucleic acid-based product.

Gary Walsh, Ph.D.

The approvals profile mirrors several trends noted within the sector over the past few years. Antibody-based products continue to represent the single largest category of biopharmaceutical, with a total of 31 now having gained marketing approval. The global market for therapeutic antibodies was estimated at $5.4 billion in 2002 and is projected to reach about $17 billion by the end of the decade.⁴ The trend towards approval of engineered products also continues. Four of the six approved antibodies were engineered (one chimeric and three humanized) as were both insulin-based approvals. The insulin analog Levemir is particularly noteworthy. Its major engineered feature is the attachment of a fatty acid moiety to its amino acid backbone. Several older products have undergone such posttranslational engineering (e.g. pegylated interferons and products such as Cerezyme, which display altered glycocomponents), but Levemir is the first biopharmaceutical engineered by the attachment of a lipid group.

Cancer remains the primary indication (three products), again mirroring recent approval trends. Zevalin seeks to treat non-Hodgkin's lymphoma and Erbitux and Avastin treat colorectal cancer, which is the third most commonly diagnosed cancer worldwide (some 950,000 new cases annually). It is particularly prevalent in western countries.

Only eight approvals represent genuinely new molecular entities. Raptiva, Dukoral, and Zevalin added approval in a new region to an existing approval elsewhere. The recombinant blood factor-VIII product Advate is a modification of the already approved Recombinate.

In terms of expression systems used to produce the approved biopharmaceuticals, seven of the biologically produced products are expressed in Chinese hamster ovary (CHO) or other mammalian cell lines, again confirming the dominance of these cell types in biopharmaceutical production. Two others are produced using Escherichia coli. Tysabri has gained the most attention of all products approved in 2004, for all the wrong reasons. It was on the market for less than four months when sales (and use in ongoing clinical trials) were halted for safety reasons.

Macugen may start a new trend in that it is the first aptamer approved for therapeutic use. Aptamers (also termed decoys) are relatively short nucleic acid-based sequences that adopt a three-dimensional structure capable of binding to, and thereby inhibiting, a specific target molecule. Reference 5 is an excellent review of the topic.

APPROVED PRODUCTS

The remainder of this article focuses upon the 12 approvals in 2004, with product information detailed in monograph format. Information was drawn from regulatory sources

^6,7

as well as the homepages of sponsoring companies (Table 1).

Table 1. Biopharmaceuticals Approved in the US or EU or both in 2004.

Advate Advate (octocog alfa) is a recombinant form of human blood factor-VIII produced in an engineered CHO-cell line. Its amino acid sequence is identical to that of the native factor-VIII. The product is quite heavily glycosylated, with 25 potential N-linked glycosylation sites and 12 O-linked glycans.

The European Commission approved Advate in March 2004. Advate is indicated for the treatment and prophylaxis of bleeding in patients with hemophilia A. The marketing authorization holder is Baxter AG. It is not a new molecular entity but a modification of the licensed factor-VIII product Recombinate (available in the US since 1992).

Advate production incorporates modifications to the original Recombinate procedure to increase product safety. Alterations include elimination of human- and animal-derived raw materials and excipients. Cell culture media is adapted to be serum-free, and human serum albumin is no longer used as an excipient. The purification process has been modified to include a viral inactivation step with a solvent-detergent (1% Triton-X-100, 0.3% polysorbate 80, and 0.3% tri-N-butylphosphate). The purification process consists of immunoaffinity chromatography, cation exchange and anion exchange steps as well as the solvent-detergent step. Final product is presented in lyophilized form and is available in four different strengths. Trials confirmed its efficacy and comparability to Recombinate.

Dukora (oral cholera vaccine) contains inactivated Vibrio cholerae serotype O1 strains along with recombinant cholera toxin B subunit (CTB). The European Commission approved the vaccine in April 2004. It is indicated for active immunization against disease caused by V.cholerae serogroup O1 in adults and children set to visit indemic and epidemic geographical regions. Dukoral contains both killed bacterial cells and the cholera toxin subunit, thereby incorporating all the most important disease-related antigens. The marketing authorization holder is SBL Vaccin AB. The product has been available in Sweden since 1993.

The relevant serogroup O1 cells are cultured in 550-L fermentors, harvested, concentrated and inactivated, either by heat (560C) or formalin (0.5%). The recombinant cholera toxin B subunit, a 102-amino acid polypeptide, is produced by an engineered V. cholerae strain. Precipitation (using sodium hexametaphosphate) and hydroxyapatite chromatography represent the principle purification steps. The finished product is formulated in phosphate buffered saline solution, and is intended for oral administration. A bicarbonate buffer is co-administered to protect the CTB from stomach acid.

The efficacy of Dukoral has been demonstrated in three randomized placebo-controlled trials. It was found to be immunogenic in both children and adults, and an overall protective efficacy of 85 percent after six months was recorded. Commonly reported adverse events were gastrointestinal related, including abdominal discomfort and diarrhea.

Zevalin (ibritumomab tiuxetan) is a murine monoclonal antibody produced in an engineered CHO cell line and administered in radiolabelled form. The product is indicated for refractory or Rituximab-relapsed CD 20+ follicular B-cell non-Hodgkin's lymphoma. The Zevalin antibody specifically binds to the human CD 20 antigen, expressed on the surface of both normal and malignant B-lymphocytes. Cell death is induced by the Y⁹⁰ derived high-energy beta radiation. Treatment is normally in combination with Rituximab (trade names MabThera in EU and Rituxan in US).

Zevalin gained marketing authorization in the EU in January 2004, although it was first approved in the US in 2002. It is manufactured by IDEC pharmaceuticals and the EU marketing authorization holder is Schering AG.

Manufacture starts with cell culture and product recovery, followed by multiple chromatographic steps, and then vital inactivation and removal steps. The antibody is chemically conjugated to a chelator (tiurexan) and formulated as a solution for injection. Immediately prior to its administration, it is chelated to the radionucleotide yttrium-90 (Y⁹⁰), which is not supplied with the product.

The dossier submitted to the EMEA was supported by six clinical trials (306 patients in total). A post-treatment response rate of 75 to 80 percent was observed in patients no longer responsive to plain Rituximab or to chemotherapy. Adverse effects included a severe reduction in white blood cell and platelet counts, and gastrointestinal and respiratory complications.

Erbitux (cetuximab) is a chimeric monoclonal antibody directed against the human epidermal growth factor receptor (EGFR) and is produced by an engineered murine myeloma cell line. It is usually used in combination with irinotecan. Erbitux binds specifically to the extracellular ligand-binding domain of the EGFR found on a range of normal cells (e.g. epithelial cells) as well as a proportion of colorectal and other tumor cells. The product acts as a competitive antagonist, preventing binding of native EGF to its receptor, thereby blocking EGFR activation and signal transduction. This in turn inhibits cell growth and triggers apoptosis.

It was approved in 2004 both in the EU and US for the treatment of (EGFR-expressing) metastatic colorectal cancer in patients refractory to irinotecan-based chemotherapy. Manufactured by ImClone Systems and Boehringer Ingelheim, it is marketed by Bristol-Myers Squibb in the US and by Merck in the EU.

Erbitux manufacture is initiated by batch culturing the producing cell line in serum-free media in 10,000-L and 12,000-L bioreactors. Multistep chromatographic purification is undertaken following initial recovery and concentration. Downstream processing also incorporates two specific viral inactivation and removal steps, and the final product is formulated as a sterile, 2-mg/mL-cetuximab solution, intended for administration via infusion.

Both in vitro and animal studies confirmed that Erbitux inhibits the growth and survival of tumor cells over-expressing EGFR. Clinical trials also showed that a combination of Erbitux and irinoectan outperformed Erbitux monotherpay in terms of objective response rate, disease control rate, and progression-free survival. Reported adverse events include severe infusion-related reactions, pulmonary complications, dermatological toxicity, and gastrointestinal disturbances.

Avastin (bevacizumab) is a humanized monoclonal antibody produced in an engineered CHO cell line. It binds specifically to human vascular endothelial growth factor (VEGF). Avastin brings about its therapeutic effect by inhibiting angiogenesis (the formation of new blood vessels), a process required to support tumor growth. Binding to VEGF prevents VEGF interaction with its cell surface receptor, a process central to the triggering of new blood vessel growth in both normal and diseased tissue.

FDA approved Avastin in 2004. It is indicated for first line of treatment for patients with metastatic carcinoma of the colon or rectum, in conjunction with specified small molecule chemotherapeutic agents (5 fluorouricil, folinic acid, and irinotecan). The product has subsequently also gained marketing approval in the EU (in January, 2005). It is manufactured by Genentech and marketed in the EU by Roche.

Production-scale manufacture entails culture of the CHO producer-cell line in 12,000-L fermentors using a serum-free growth media. Product purification is achieved via protein A affinity chromatography followed by sequential anion and cation exchange steps. Two viral inactivation steps are also included in the downstream processing protocol. The final product is concentrated via ultrafiltration and is formulated as a sterile solution for intravenous infusion.

Clinical trials confirmed the product's efficacy, with Avastin administration (in conjunction with the specified small molecule chemotherapeutics) resulting in a statistically significant increase in overall survival, higher objective response rates, and longer progression-free survival. Adverse effects that were noted included gastrointestinal perforations, hemorrhage and arterial thromboembolism.

Raptiva (efalizumab) is a humanized monoclonal antibody produced in an engineered CHO cell line. Approval by the European Commission was in September 2004 (and by FDA in 2003) for the treatment of adult patients with moderate to severe chronic plaque psoriasis who failed to respond to or were intolerant of other specified systemic therapies. Genentech manufactures the product but Serono markets it.

Psoriasis is a chronic inflammatory skin disorder caused by T-lymphocyte mediated stimulation of keratinocyte hyperproliferation. By preventing LFA-1/ICAM binding, the product inhibits several stages of this immunological disorder. Raptiva specifically binds lymphocyte function-associated antigen 1 (LFA-1), a leukocyte cell surface protein. Binding inhibits interaction of LFA-1 with intercellular adhesion molecules 1, 2 and 3 (ICAM-1, -2 and -3). LFA-1 is present on activated T lymphocytes, and ICAM-1 is up-regulated on endothelial cells and keratinocytes in psoriasis plaques.

Manufacture entails initial culture of producer CHO cells in suspension, followed by multiple chromatographic purification and viral inactivation/removal steps. The final product is presented in lyophilized form, intended for subcutaneous (SC) administration subsequent to reconstitution in water for injections (WFI).

Clinical trials confirmed that 12 weeks of treatment with Raptiva significantly improved the severity and area of psoriatic lesions. As an immunosuppressive agent however, the product has the potential to increase the risk of serious infections and malignancies.

Levemir (insulin detemir) is a long-acting insulin analogue produced in an engineered Saccharomyces cerevisiae cell line. The product differs from native human insulin in that the theronine residue at position 30 of the insulin B chain (B30) has been omitted and a myristic fatty acid (a C14 saturated fatty acid) has been attached (acylated) to the amino group of the lysine residue found at position B29. Albumin harbors three high-affinity, fatty-acid, binding sites and, as such, Levemir binds tightly but reversibly to albumin, both at the site of injection and in the blood. This in turn ensures a constant and prolonged release of free insulin, bestowing upon it an extended duration of action of up to 24 hours.

The European Commission approved the product for the treatment of diabetes mellitus in June, 2004. It is used as a basal insulin preparation in combination with meal-related, rapid-acting insulin products. The marketing authorization holder is Novo Nordisk.

Manufacture entails an initial fermentation step, followed by product recovery, an ion exchange-based concentration step and two crystallization steps. The insulin is then extensively purified via several chromatographic steps, followed by product acylation. Final polishing purification and precipitation follow, and the product is formulated as a solution for subcutaneous administration. Clinical trials illustrated the product's efficacy in maintaining glycemic control and a low within-patient variability. Common side effects were episodes of hypoglycaemia and injection site reactions.

Apidra (insulin glulisine) is a rapid- acting insulin analog produced in an engineered E. coli strain. It was approved in 2004 for the treatment of diabetes mellitus in both the US and EU and is manufactured and marketed by Aventis.

Apidra differs from native human insulin in that lysine replaces asparagine at position 3 of the insulin B chain (B3) and glutamic acid replaces the B29 lysine residue. The amino acid substitutions characteristic of Apidra reduce the interchain interaction between individual insulin molecules within the insulin hexamer. As a result, the hexameric species dissociates rapidly at the site of injection, facilitating more rapid product entry into the bloodstream.

Manufacture is initiated by culture of the producer E. coli cell line. Product accumulates intracellularly in the form of inclusion bodies (IB). The product is chromatographically purified after cell harvest, IB recovery, and solubilization. Downstream processing also incorporates enzyme-based excision of the final insulin from a fusion protein precursor. Product is formulated as a solution intended for SC administration. Clinical data clearly illustrated product safety as well as efficacy in terms of glycemic control. Negative effects included the potential to trigger hypoglycemia as well as local or systemic allergic reactions.

Kepivance (palifermin) is a recombinant, truncated form of human keratinocyte growth factor (KGF), produced in engineered E. coli cells. It was approved by FDA in December 2004, and is indicated to decrease the incidence and duration of severe oral mucositis in patients with hematologic malignancies receiving chemotherapy in preparation for bone marrow transplants. It is manufactured and marketed by Amgen.

Mucositis (ulcers in the lining of the mouth) represent a prominent, common, and sometimes very severe side effect of high-dose chemotherapy or radiotherapy. The KGF receptor is present on epithelial cells in many tissues including the tongue and buccal mucosa, and binding of KGF to its receptor triggers the proliferation and differentiation of these epithelial cells. It is by this means the product is believed to bring about its effect.

Manufacture entails initial E. coli fermentation, product recovery, chromatographic purification, and formulation. Final product is supplied in lyophilized form, intended for IV injection after reconstitution with WFI. Kepivance is a 16.3 kDa, 140-amino acid polypeptide, differing from native KGF only in that it is devoid of the first 23 N-terminal amino acid residues.

The truncated form retains the biological activity of the parent molecule while displaying improved stability. Clinical trials involving patients with leukemia and lymphoma established the product's safety and efficacy. Ninety-eight percent of patients receiving a placebo developed serious mucositis, whereas only 63 percent of KGF-treated patients did. Median duration of mucositis was also reduced, from nine (placebo group) to three days (treated group). The most frequently noted adverse effect was a skin rash.

NeutroSpec (fanolesomab) is a murine IgM monoclonal antibody produced by suspension culture of hybridoma cells. It is indicated for the imaging of patients with equivocal signs and symptoms of appendicitis. Fanolesomab specifically binds the CD15 antigen expressed on the surface of polymorphonuclear neutrophils (PMNs), eosinophils, and monocytes (all white blood cells). Of these, PMNs represent the greatest proportion found in circulation. These cells migrate to the site of active infections in the body such as those underpinning appendicitis.

Approved in July, 2004 by FDA, the product was developed by Palatin Technologies. It is contract manufactured for Palatin by Ben Venue Laboratories and is distributed by Mallinckrodt Inc.

Product manufacture includes initial cell culture, product recovery, and multi-step chromatographic purification. It is presented in lyophilized form, intended for reconstitution immediately prior to use in a Tc^99m-containing solution. The Tc^99m radionucleotide complexes with the antibody. Localization of (gamma radiation emitting) radiolabelled NeutroSpec in the appendix is easily detectable using a gamma camera and is indicative of an active infection characteristic of true appendicitis.

Clinical trials clearly illustrate the effectiveness of NeutroSpec in confirming appendicitis in suspected cases. Hypersensitivity reactions represent a serious potential product side effect. Side effects noted during trials included hypotension, worsening of sepsis, flushing, and dyspena.

Macugen (pegaptanib sodium injection) is a synthetic pegylated oligonucleotide aptamer that specifically binds vascular endothelial growth factor (VEGF). It is indicated for the treatment of age-related neovascular ("wet") macular degeneration, which results from the proliferation of abnormal blood vessels in the eye, leading to retinal damage and vision loss. The process of vascularization (angiogenesis) is fueled by VEGF. Macugen adopts a three-dimensional shape that allows it to interact specifically with VEGF, thereby inhibiting its activity (note that the monoclonal antibody-based product Avastin achieves a similar effect in the context of cancer).

FDA approved it in December, 2004. It is manufactured by Gilead Sciences for Eyetech Pharmaceuticals and Pfizer.

The RNA-based oligonucleotide consists of 28 nucleotides of pre-defined sequence, chemically modified to render the product resistant to nuclease degradation. Two 20-kDa polyethylene glycol (PEG) molecules are covalently attached at one end of the nucleotide (to increase in vivo half life), yielding an overall product molecular mass of some 50 kDa. Final product is presented as a sterile, WFI-based solution containing sodium chloride as well as sodium phosphate as excipients. The product is administered by direct intravitreous injection.

Clinical assessment centered upon two studies involving almost 1,200 wet AMD patients. While both control and product groups continued to experience vision loss, the rate of vision decline experienced by Macugen-treated patients was significantly slower than in the case of control patients. The most frequent potentially serious side effects noted included endophthalmitis, retinal detachment, eye inflammation, irritation, and blurred vision.

Tysabri (natalizumab) is a recombinant, humanized IgG-based monoclonal antibody produced in engineered murine myeloma cells. It is indicated for the treatment of patients with relapsing forms of multiple sclerosis to reduce the frequency of clinical exacerbations. Subsequent to its biosynthesis and purification, Tysabri is formulated as a concentrate (20 mg/mL), indicated for once monthly IV infusion.

Tysabri binds specifically to selected integrins found on the surface of most white blood cells (with the exception of neutrophils). These integrins serve as recognition docking markers, allowing binding to vascular endothelial cells and thereby facilitating leukocyte migration across the endothelium. Tysabri administration blocks this interaction and increases the numbers of circulating leukocytes in the blood by inhibiting transmigration out of the vascular space.

While the exact underlining causes of multiple sclerosis remain to be elucidated, the condition is believed to be an autoimmune one in which selected immune system cells (leukocytes) attack the central nervous system. Tysabri therefore may work by blocking migration of such leukocytes across the blood-brain barrier (comprised of endothelial cells lining brain capillaries), and hence preventing them from damaging the neuronal tissue.

The product is manufactured by Biogen Inc. and distributed by Elan. Initially approved by FDA in November, 2004, marketing and its use in ongoing clinical trials were halted in February 2005. The product received accelerated approval as it appeared to provide a substantial benefit to MS sufferers. One clinical trial recorded a reduction in the frequency of relapses by two-thirds (relative to placebo). A second trial (in which patients also received the interferon-based product Avonex, but for which Avonex did not prevent relapses) was also undertaken. Tysabri reduced the frequency of relapse by 50 percent in these cases.

FDA evaluation was based upon results from trials ongoing for one year. At that stage the most serious adverse events noted were infections and temporary hypersensitivity reactions. Approval was contingent upon continuing the trials for a second year. This data initially revealed one fatal and one additional case of multifocal leukoencephalopathy (PML). Both patients had received Tysabri in conjunction with Avonex for two years. The product remains withdrawn for all clinical use until ongoing investigations are completed.

Gary Walsh, Ph.D., is senior lecturer in the Industrial Biochemistry Program of University of Limerick, Limerick City, Ireland, 353.61.202.664, fax 353.61.202.568, Gary.walsh@ul.ie

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