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Associate Professor in the Industrial Biochemistry Program at the University of Limerick. He is also a member of BioPharm International's Editorial Advisory Board.
The year 2007 witnessed the approval of fifteen biopharmaceuticals in the United States and European Union.
The year 2007 witnessed the approval of 15 biopharmaceuticals in the United States and/or European Union (EU). The 15 approvals included six erythropoietin (EPO)-based products, three antibodies, and two hormones, as well as a one therapeutic enzyme, one recombinant vaccine, one growth factor, and a fusion product (Table 1).
Gary Walsh, PhD
The target indications included anemia (six products) hereditary genetic conditions (four products), cancer prevention or treatment (two products), and neovascular macular degeneration, rheumatoid arthritis, and infertility (one product each).
Only nine of the 15 products were genuinely new to the market in 2007. The other six, although approved in one region last year, had gained approval before 2007 in another world region (Table 1).
Table 1. Biopharmaceuticals (defined as recombinant protein, monoclonal antibody, and nucleic acid-based products) approved in the United States or European Union in 2007. Biosimilars are in shaded rows.
In terms of expression systems used, 11 of the 15 approved biopharmaceuticals are produced using mammalian cell lines; two in E. coli, one in Saccharomyces cerevisiae; and notably, one in a baculovirus/insect cell-based system. Overall, this trend confirms the prominence of mammalian-based expression systems in the biopharmaceutical sector. On the other hand, Cervarix's approval is particularly noteworthy as it is the first biopharmaceutical approved for human use that is produced using an insect-cell–based expression system.
A wide range of therapeutic proteins (e.g., tPA, interferon γ, and hepatitis B surface antigen) have been produced at laboratory scale using such systems. A small number of approved veterinary medicines (e.g., Intervet's porcilis pesti subunit vaccine) are commercially manufactured using such systems. The advantages of recombinant insect-cell–based production include high levels of intracellular expression and rapid cell growth on relatively inexpensive media. However, issues including low extracellular expression levels and the exact nature of post-translational modifications achieved have been cited as disadvantages of this system.
Perhaps the single most notable feature of the 2007 approvals is the number of biosimilar products that entered the market in the EU. Of the 14 products approved for the first time last year in Europe, five are biosimilars (Abseamed, Binocrit, Epoetin alfa Hexal, Retacrit, and Silapo; Table 1). This clearly illustrates that the biosimilar legislative framework developed by the EU works in practice. Another notable fact is that all five products are EPO-based. This is not surprising given that EPO is the single most lucrative biopharmaceutical on the market. EPO-based products represent three of the top 10 blockbuster biopharmaceuticals (Aranesp, Procrit/Eprex, and Epogen)1 , and relevant company annual reports show a cumulative annual market value in excess of $10 billion for these three products. The approval of biosimilar EPOs also confirms the practical feasibility of illustrating comparability of a glycosylated biosimilar to its reference medicine.
Further analysis of the nine products entering the market for the first time last year provides additional insights. The five biosimilar products contain only two different active ingredients: The active ingredient in Abseamed, Binocrit, and Epoetin alfa Hexal (recombinant human erythropoietin alfa) is identical in each case, while Retacrit and Silapo also share an identical active substance (epoetin zeta). Furthermore, Mircera is a PEGylated version of a product already on the market (Neorecormon) and the twin active substances in Pergoveris are already marketed as individual products (Gonal F and Luveris).
The remainder of this article focuses on individual products approved for the first time in 2007, with product information detailed in a monograph format. The information was drawn from regulatory sources and the web sites of sponsoring companies.2,3 Monographs detailing the products listed in Table 1 that had gained prior approval in some world region were included in previous articles.4–6 Therefore, they are not considered below.
Abseamed (recombinant human erythropoietin alfa) is a recombinant form of human erythropoietin (EPO) produced in a Chinese hamster ovary (CHO) cell line. Native human EPO is a 165 amino acid, 36 kDa glycoprotein housing three N-linked and one O-linked glycosylation sites. It is synthesized almost exclusively in the kidney and is primarily responsible for stimulating and regulating erythropoiesis (red blood cell production). Abseamed is a biosimilar product, with Janssen-Cilag's erythropoietin Eprex/Erypo having served as reference medicine. The product is identical to both reference and native EPO in amino-acid sequence, but does differ in exact glycosylation detail. Compared to Eprex it displays somewhat higher sialylation levels, particularly associated with its O-linked glycan chain. The biosimilar product also displays higher levels of mannose-6-phosphate containing glycans associated with the Asn 24 N-linked glycosylation site. However, these specific glycans display only weak binding affinity for the mannose-6-phosphate receptor, and therefore are unlikely to trigger rapid product uptake from circulation.
Abseamed gained approval in the EU in August 2007. It is indicated for the treatment of anemia associated with chronic renal failure and for anemia accompanying chemotherapy of certain cancer types. It also can be used to reduce exposure of patients undergoing major orthopedic surgery to blood transfusions if transfusion related complications are anticipated.
The product is manufactured by batch cell culture in media supplemented with recombinant human insulin. After recovery from the cell media, the product is purified to homogeneity by a combination of standard chromatographic techniques. Downstream processing also incorporates a nanofiltration-based viral-removal step.
Glycine, sodium chloride, polysorbate 80, and phosphate buffer components are added as excipients, and the final product is filter sterilized, followed by aseptic filling into presterile syringes. The product is commercially available at two active ingredient concentrations, 16.8 μg/mL and 84 μg/mL, equating to 2,000 and 10,000 IU/mL, respectively.
The exact administration regime of Abseamed will depend on the indication being treated and will also be tailored to the individual patient. Product comparability to the reference medicine was established primarily by a double- blind, randomized, parallel group multicenter study involving 479 patients displaying anemia associated with chronic kidney disease. The primary endpoint measured was the mean absolute change in hemoglobin levels. A second study comparing the effects of Abseamed to Eprex in 114 cancer patients undergoing chemotherapy also was reported, with both studies confirming product comparability to Eprex. The most common adverse effect associated with product administration was an increase in blood pressure.
Abseamed's active ingredient is manufactured by Rentschler Biotechnologie GmbH (Laupheim, Germany). Germany-based Medice Arzneimittel Putter GmbH markets the product.
Binocrit. This product, along with its indications and all other characteristics, is identical to Abseamed. It differs only in the tradename used and in that Sandoz GmbH (Kundl, Austria), holds marketing authorization throughout the EU.
Cervarix (Papillomavirus, human, types 16,18) is a divalent vaccine containing the purified recombinant (C-terminally truncated) major caspid L1 proteins of human papillomavirus (HPV) types 16 and 18. The L1 proteins are produced separately using a recombinant baculovirus expression system coupled with an insect cell line derived from Trichoplusia ni. It is the first recombinant biopharmaceutical approved anywhere for human use that is produced using a baculovirus-based expression system. The product gained approval in the EU in September 2007 and is indicated for the prevention of cervical cancer and high-grade cervical intraepithelial neoplasia causally related to HPV types 16 and 18. High-grade cervical intraepithelial neoplasia refers to precancerous abnormal cervical cellular growth with a high probability of progression to cancer if left untreated. Over 100 genotypes of these double-stranded DNA-based enveloped viruses have been identified. Of these 100, 16 strains are regarded as highly oncogenic, with strains 16 and 18 believed responsible for over 70% of all cervical cancers.
Cervarix manufacture requires separate recombinant production of the major caspid (L1) proteins of HPV strains 16 and 18. Subsequent to upstream processing the caspid proteins are released from the cultured insect cells by osmotic shock. This is followed by multiple chromatographic purification steps, and nanofiltration. The purified L1 protein antigens are then adsorbed separately onto aluminium hydroxide. An adjuvant material, monophosphoryl lipid A (MPL), is also separately adsorbed onto an additional aluminium hydroxide. MPL is a detoxified derivative of the lipopolysaccharide moiety of the gram negative bacterium Salmonella minnesota. The final vaccine product is then produced by mixing defined amounts of all three adsorbed species, along with sodium chloride and phosphate buffer components as excipients. The final vaccine suspension is filled into either vials or syringes. Each dose contains 20 μg of both HPV type 16 and type 18 L1 caspid proteins.
Product administration (to girls and women 10 years and older) is by intramuscular injection and a typical administration schedule entails follow-on doses one and six months after administrating the initial dose. Clinical trials assessing product safety and efficacy involved more than 21,000 females between the ages of 10 and 25, and clearly illustrated a protective effect. The most common side effects noted were headache, muscle pain, and injection site reactions. The product is manufactured and marketed by GlaxoSmithKline Biologicals (London, UK).
Epoetin alfa Hexal. This product, along with its indications and all other characteristics, is identical to Abseamed and Binocrit. It differs only in the tradename used and in that Hexal biotech Forschungs GmbH, Germany, holds marketing authorization throughout the EU.
Mircera (methoxy polyethylene glycol epoetin beta) is a PEGylated recombinant form of human EPO. The erythropoietin used to generate Mircera is the active substance of Neorecormon (epoetin beta; Roche's recombinant EPO first approved for general medical use in the EU in 1996). The PEG moiety used is methoxypolyethylene glycol–succinimidyl butanoic acid (PEG–SBA), a 30 kDa linear chemically activated PEG. When co-incubated under appropriate conditions, the PEG–SBA spontaneously forms amide linkages with either EPO's N-terminal amino group or with the ε-amino group of an accessible surface lysine residue (Lys 45 or Lys 52). The final product generated is a 60 kDa monopegylated product.
Mircera was approved for general medical use in both the EU and the USA in 2007. The approved indication in both regions is restricted to the treatment of chronic kidney disease, and the label explicitly states that the product's safety and efficacy in the context of other indications has not been established. In fact, one dose-ranging clinical study aimed at assessing the product for treating chemotherapy-induced anemia was terminated prematurely because of a significantly greater death rate amongst patients receiving the product.
The recombinant EPO is manufactured by initial cell culture of the producer CHO cell line in serum-free media. Purification entails five sequential chromatography steps (dye affinity, hydrophobic interaction, and hydroxyapatite chromatographies, followed by a reverse phase HPLC step and finally an ion-exchange step). The purified EPO is then co-incubated with the chemically activated PEG, followed by chromatographic separation of the PEG–EPO conjugate from unreacted monomers or reaction by products. Phosphate buffer constituents, mannitol, poloxamer 188, sodium sulphate, and methionine are added as excipients and the filter-sterilized product is filled into glass vials or prefilled syringes.
In vitro studies indicated that the PEGylated EPO has a lower affinity for the EPO receptor compared to unmodified EPO. However, in vivo studies (using both mouse and dog models, and echoed in clinical studies) showed the PEGylated product to be a more potent stimulator of erythropoiesis in terms of magnitude and, in particular, duration of response. These effects are likely indirect, being linked to reduced product-tissue penetration and retardation of product elimination. Although exact dosage regimes are tailored to individual patients, typical regimes entail product administration once every 2–4 weeks. Neorecormon, in contrast, is administered at least once weekly. The most common adverse reaction associated with Mircera administration is hypertension. The product is manufactured and marketed by Roche (Basel, Switzerland).
Pergoveris (follitropin alfa/lutropin alfa) contains twin active substances—recombinant human follicle stimulating hormone (FSH; follitropin alfa) and recombinant human luteinizing hormone (LH; lutropin alfa). Both are separately produced using an engineered CHO cell line. Both active ingredients are already approved individually for general medical use as stand-alone products (tradenames Gonal F and Luveris, respectively).
Both of these gonadotropic hormones are dimeric glycoproteins, consisting of identical 92 amino acid α subunits and a distinct β subunit (111 amino acids in the case of FSH; 121 amino acids in the case of LH). The distinct β subunit confers biological specificity on these hormones. Both subunits are glycosylated.
Pergoveris was approved for general medical use in the EU in June 2007. It is indicated for stimulating follicular development in women with severe LH and FSH deficiency (and who are therefore usually infertile). FSH naturally stimulates follicular development with subsequent rupture, and hence, egg release being largely under the influence of LH.
Manufacturing the active substances entails separate cell culture of the anchorage-dependent producer CHO cells in microcarrier -containing bioreactors. The products are subsequently purified by multiple chromatographic steps and are then combined in a fixed concentration. The final product is filled into vials and lyophilized. Each vial contains 11 μg (150 IU) FSH and 3 μg (75 IU) LH.
Pergoveris is typically administered once daily until the recipient has developed a suitably matured follicle. This may take up to five weeks.
Some clinical data generated during previous studies of the individual active ingredients were presented in support of safety and efficacy. Additional studies of the combination product, including bioequivalence studies, were also conducted. The most common side effects noted were headaches, injection site reactions, and ovarian cysts. The product is manufactured and marketed by Serono (Geneva, Switzerland).
Retacrit (epoetin zeta) is a recombinant form of human erythropoietin produced in a CHO cell line. It is a biosimilar product with Janssen-Cilag's EPO Eprex/Erypo having served as reference medicine.
The product is identical to both reference and native EPO in amino-acid sequence. A combination of HPLC, NMR, and Mass spectrometry-based analysis indicated the molecule's overall glycocomponent to be substantially similar to the reference EPO. The number of glycoforms devoid of the O-linked glycan chain was slightly higher although the level of sialylation was similar. Overall, the product's carbohydrate content was judged to be free of any unusual or potentially immunogenic structures or epitopes.
Retacrit gained approval in the EU in December 2007. Its approved indications are similar to those of Abseamed, Binocrit, and Epoetin alfa Hexal. Specifically, it is indicated for the treatment of anemia associated with chronic renal failure and for anemia accompanying the chemotherapy of certain cancer types. It also can be used to increase the yield of autologous blood from patients in a predonation program.
The upstream element of product manufacture is based on a feed-batch cell-culture process. The cell-culture media is proteins, insulin, and free of antibiotics, and is also devoid of ingredients derived from human or animal origin. After initial recovery, the product is purified by a combination of chromatographic techniques, and downstream processing also incorporates a nanofiltration-based virus-removal step. The excipients added include phosphate buffer components, calcium, and sodium chloride, polysorbate 20, and several amino acids (glutamic acid, glycine, isoleucine, leucine, phenylalanine, and threonine). The final product is filter sterilized and aseptically filled into presterile syringes. The product is commercially available at active ingredient potency levels ranging from 3,333 IU/mL to 40,000 IU/mL.
Retacrit's exact dosage regime will be tailored to the individual patient. The product can be administered subcutaneously to patients receiving chemotherapy and intravenously for other indications.
Product comparability to the reference medicine was established primarily by two studies involving a total of 922 patients displaying anemia associated with chronic kidney disease, as well as a third study in which the product was administered to 261 cancer patients receiving chemotherapy. Primary endpoints measured were invariably generation and maintenance of blood hemoglobin levels. The most common adverse effect associated with product administration was an increase in blood pressure.
Retacrit's active ingredient is manufactured by Norbitec GmbH (Uetersen, Germany). Netherlands-based Hospira enterprises B.V., markets the product.
Silapo. This product, along with its indications and all other characteristics, is identical to Retacrit above. It differs only in the tradename used and in that Germany-based STADA Arzneimittel AG, holds marketing authorization throughout the EU.
Soliris (eculizumab) is a 148 kDa humanized IgG2/4 kappa antibody produced by recombinant means in an NS0 murine myeloma cell line. The antibody is composed of two 448 amino acid heavy chains and two 214 amino acid light chains, and displays a characteristic IgG oligosaccharide attached to Asn 298 of the heavy antibody chains. The product, which binds to the human C5 complement protein, was approved in 2007, both in the EU and the US, and is indicated for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH), to reduce hemolysis. It has orphan status.
PNH is a rare genetic condition in which patients harbour a mutant gene coding for CD 59, a complement inhibitory protein normally found on the surface of red blood cells. As its name suggests, this protein serves to inhibit the formation of a complement complex (i.e., the membrane attack complex) on the red blood cell (RBC) surface, thereby preventing RBC destruction (hemolysis). This RBC protective mechanism is absent in PNH sufferers and the predominant clinical manifestation of the condition is intravascular hemolysis. Patients typically have a 15-year median survival period from diagnosis. The treatment approaches are largely palliative, with transfusion therapy often used to maintain RBC levels. By binding to the C5 complement protein, the antibody effectively inhibits final activation of the complement system, hence preventing complement-mediated hemolysis.
Antibody manufacture entails an initial cell-culture step using a 5,000-L bioreactor. The product is subsequently purified by a combination of chromatographic steps, and downstream processing also incorporates viral inactivation (low pH) and virus-removal (nanofiltration) steps. The excipients added include phosphate buffer components, sodium chloride, and polysorbate 80. The final product is filter sterilized and presented in vials as a concentrate (30 mL of a 10 mg/mL solution) destined for IV infusion after dilution to 5 mg/mL. A typical dosage regime entails initial infusion of 600 mg of product weekly for four weeks, a 900-mg infusion at week five, followed by 900-mg infusions every second week thereafter.
Product safety and efficacy were primarily investigated by one main study involving 87 PNH patients. All had received at least four blood transfusions for anemia in the previous year. By the 26-week endpoint, 49% of treated patients displayed stabilized hemoglobin levels, as opposed to none in the untreated group. Likewise, 51% of the treated group had avoided the necessity for a blood transfusion while all of the nontreated group required transfusions. The most commonly reported side effects included headaches, urinary tract infections, and nasopharyngitis. The most significant adverse reaction experienced was meningococcal infections. As a result, patients should be vaccinated against meningitis caused by Neisseria meningitides before product administration.
The active ingredient in Soliris is manufactured by Lonza Biologics (Newington, New Hampshire). The product is marketed in the United States and EU by Alexion (Cheshire, CT).
Gary Walsh, PhD, is an associate professor in the Industrial Biochemistry Program at the University of Limerick, Limerick City, Ireland, +353.61.202664, Gary.email@example.com He is also a member of BioPharm International's Editorial Advisory Board.
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2. Available from: www.fda.gov
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