Status - approved; marketed; being replaced in 2008 by next-generation product, Xyntha

Organizations involved:

Wyeth - Manuf. other; R&D; Tech.; World mark.

Genetics Institute, Inc. - R&D; Tech.; Former

Biovitrum AB - Manuf.; R&D; Tech.; Europe mark.

Pharmacia AB - R&D; Tech.; Former

Bayer Schering Pharma AG - Tech.

New York Blood Center, Inc. - Tech.

Behringwerke AG - Tech.; Former

CSL Behring GmbH - Tech.

CSL Ltd. - Parent

Genentech, Inc. - Tech.; Patent dispute

Columbia University - Tech.; Patent dispute

Cross ref: See the entry below for Xyntha, which contains the same active agent (recombinant Factor VIII SQ), was approved by FDA in 2008 and is replacing ReFacto. See also the Factor VIII Products entry in the Blood Products, Human section, and the other Factor VIII product entries.

Description: Antihemophilic Factor (Recombinant) or ReFacto is a lyophilized (freeze-dried) formulation of a mutein (modified form) of recombinant Factor VIII glycoprotein (rVIII SQ) expressed by a transformed Chinese hamster ovary (CHO) cell line, with purification including monoclonal antibody-based immunoaffinity chromatography, viral inactivation by a solvent-detergent process, and formulation without addition of Albumin (Human) as a stabilizer. rVIII SQ is essentially Factor VIII with most of the nonessential B-domain portion of the Factor VIII glycoprotein deleted. rVIII SQ, a 170 kDa glycoprotein, is comparable to the smallest active form of naturally occurring, active, human Factor VIII, with a sequence comparable to the 90+80 kDa form of Factor VIII. Removal of the B--domain of the protein facilitates manufacture of a product with a higher specific activity (higher activity per weight) and provides a stable molecule not requiring addition of Albumin (Human) as a stabilizer in the final product. ReFacto is the only hemophilia A therapy indicated for short-term routine prophylaxis and was the first human "albumin-free" formulation of recombinant Factor VIII.

Native human Factor VIII protein exists in a series of active forms, varying in molecular mass from 170 to 280 kDa. Human Factor VIII is normally expressed in vivo as a single chain molecule consisting of the structural domains A1-A2-B-A3-C1-C2, with the molecule retaining this structure at high dilution (e.g., physiological concentration). At high concentrations, Factor VIII is primarily in the form of a heterodimer (dimer composed of two different polypeptide chains), consisting of a light chain (domains A3-C1-C2; m.w. = 80 kDa) and various heavy chain derivatives (domains A1-A2-B; m.w. = 90 to 200 kDa). The two-chain structure results from intracellular proteolytic processing of the precursor molecule. Heterogeneity of the heavy chain is due to limited, variable proteolysis within the carboxy (C)-terminus of the B-domain. The heavily glycosylated B-domain portion, which is deleted from the recombinant Factor VIII in rVIII SQ (ReFacto), is not required for hemostatic activity. Deletion of the B-domain yields a smaller yet comparably effective protein less prone to proteolytic degradation (and not requiring added albumin to stabilize the protein). CHO-expressed rVIII SQ glycoprotein has post-translational modifications (e.g., glycosylation, folding) and activity similar to those of the plasma-derived Factor VIII.

rVIII SQ has a molecular weight of 170 kDa; 1,438 amino acids total; and is composed of two non-covalently bound Factor VIII polypeptide chains - a 90 kDa heavy chain and a 80 kDa light chain. The N-and C-terminal regions of the B-domain are retained and fused at Ser 743-Glu 1638. rVIII SQ has structural, functional and pharmacodynamic properties very similar to that of plasma-derived Factor VIII. The ratio of Factor VIII activity to Factor VIII antigen is close to one, and the rVIII SQ interacts in a normal fashion (like plasma-derived product) with thrombin, von Willebrand's factor, Factor IIa, Factor Xa and activated Protein C. The specific activity of rVIII SQ in Refacto is 15,000 IU/mg protein - higher than any other recombinant Factor VIII product. rVIII SQ is antigenic like plasma-derived and other recombinant Factor VIII products and, like these, its use may result in inhibitors (neutralizing antibodies) which may adversely affect treatment.

Refacto contains no Albumin (Human) added to the final bulk as a stabilizer, no preservatives, and no added human or animal products. However, Albumin (Human) and recombinant human insulin are used during the early stages of culture of the CHO cells. No albumin is added during purification or in final formulation. Following purification, the albumin concentration is below the limit of detection. All other recombinant Factor VIII products in the U.S. market, except for Kogenate FS (and equivalent Helixate FS) have Albumin (Human) added both during host cell culture and to the final bulk. Formulation without Albumin (Human) obtained from pooled human blood plasma as a protein/formulation stabilizer and/or cell culture nutrient, theoretically, reduces risk for transmission of difficult-to-inactivate viruses, such as parvo-virus B19, and possibly prions, including transmissible spongi-form encephalitis (TSE) agents. Manufacture of ReFacto does not involve use of fetal calf serum or bovine insulin in the culture medium of the mammalian host cells, as do some other recombinant Factor VIII products. This theoretically reduces the risk for bovine pathogen transmission.

ReFacto is packaged as a lyophilized powder in vials nominally containing 250, 500, 1000 or 2000 IU of rVIII SQ for reconstitution (with 4 ml of 0.9% w/v sodium chloride solution) for intravenous injection, along with a double-ended needle, alcohol swabs, filter needle, infusion set, and disposable syringe. The specific activity is of ReFacto is ~13,000 IU/mg protein (11,200-l 5,500 IU per mg of protein). Excipients in ReFacto are sucrose, calcium chloride, L-histidine, polysorbate 80 (Tween 80), and sodium chloride. Reconstitution (dissolution) provides Factor VIII at a concentration of 62.5 IU/mL. Refacto should be stored at 2-8ûC (refrigerated), and may be stored at room temperature not to exceed 25 ¡C (77ûF) for up to 3 months.

The R2 ( Rapid Reconstitution) Kit, the first needle-less reconstitution device with a prefilled diluent syringe for hemophilia, was launched in the U.S. in early 2005. . The R2 kit contains a syringe prefilled with diluent, a vial adapter, and a single-use vial of ReFacto containing either 250, 500, 1,000, or 2,000 IUs. The adapter is placed on the vial of ReFacto, and the adapter and prefilled syringe allow ReFacto to be reconstituted without the risk of needle exposure. The R2 kit is provided in a smaller package and has fewer components, reducing required storage space.

The potency of rVIII SQ is assayed using a Chromogenic Substrate method. The APTT-based one-stage assays commonly used for plasma-derived Factor VIII products are not used. One-stage assays, which can be used with plasma-derived products, often underestimate the potency of rVIII SQ and other highly purified Factor VIII preparations. The activity of the final product is assigned and calibrated relative to the Fourth International Standard (WHO 88/804) for Factor VIII concentrates. For Refacto marketed in Europe, the potency (IU) is determined using the European Pharmacopoeia chromogenic assay against the World Health Organization (WHO) international standard for Factor VIII:C.

Nomenclature: Factor VIII, rDNA/Wyeth [BIO]; ReFacto [TR]; Antihemophilic Factor (Recombinant) [FDA EMEA]; moroc-tocog alfa [USAN INN]; Recombinant coagulation Factor VIII SQ [SY]; rVIII-SQ [SY]; rVIII SQ [SY]; r-VIIISQ [SY]; B-domain deleted recombinant Factor VIII [SY]; BDDrFVIII [SY]; NDC 58394-007-01; NDC 58394-006-01; NDC 58394-005-01; NDC 58394-011-01 [NDC]

Companies: This product was originally developed by Pharmacia AB, which later became Pharmacia & Upjohn AB, which was acquired by Pfizer, which spun-off its plasma products and other business lines to Biovitrum AB (formerly Kabivitrum AB) in mid-2001. In August 1997, Genetics Institute (GI), a subsidiary of Wyeth (now fully integrated into Wyeth), purchased exclusive development and commercialization rights for rVIII SQ from Phar-ma--cia AB. At the time, ReFacto was in Pharmacia-sponsored Phase III trials in Europe and U.S. GI/Wyeth continued its clinical development.

ReFacto is marketed worldwide by Wyeth and affiliates. In 2008, ReFacto is being replaced by Xyntha (see entry below)

Biovitrum AB continues to manufacture ReFacto for GI/Wyeth under contract at facilities in Stockholm, Sweden, originally Pharmacia AB. Biovitrum also co-promotes ReFacto in the Nordic area and the Middle East.

GI/Wyeth had obtained manufacturing rights from Pharmacia/Biovitrum and BLA approval for its own manufacture of rVIII SQ at its St. Louis, MO, facility. European Union approval was granted in May 2002 to GI/Wyeth for ReFacto manufacture at facilities in St. Louis, MO, and Algete, Spain. Wyeth invested $200 million in this expansion of manufacturing capacity. These facilities supply product for marketing in Europe, allowing a larger proportion of Refacto manufactured at Biovitrum's Stockholm facility to be available to the U.S. and the rest of the world.

In late 2003-early 2004, Wyeth closed its St. Louis facility, and consolidated all manufacture of Refacto at Biovitrum's Stockholm, Sweden, facility. In Jan. 2004, Wyeth signed an agreement with Biovitrum for it to be the sole supplier of ReFacto.

Note, Wyeth manufactures and markets other recombinant Factor VIII products, e.g., Recombi-nate and Bioclate; and Xyntha is replacing ReFacto (see entry below).

Manufacture: The rVIII SQ gene, encoding a single chain 170 kDa polypeptide, was derived from full-length human Factor VIII cDNA by removing the major part of the region encoding the B-domain. The rVIII SQ vector system is inserted into Chinese hamster ovary (CHO) cells, and the cells are cultured in a medium containing Albumin (Human). Production begins with thawing of an am-poule of production CHO cells maintained in the Working Cell Bank (WCB), expanding the cells by culture in T-flasks, then spinner flasks and 50 L seed reactors, and finally in a [redacted/censored by FDA, but reported by Biovitrum as 500 L] bioreactor. rVIII SQ is expressed into the culture medium. Cell culture is performed in a continuous perfusion mode, involving continuous harvesting of culture medium for Factor VIII purification, and reintroduction of fresh medium. Approximately 500 L of culture media is exchanged daily. The original medium (for manufacture of ReFacto) was well-defined with 60 components and free of antibiotics and serum, but containing Albumin (Human) meeting European Pharmacopeia specifications. The current medium (for manufacture of Xyntha) is presumably much the same, but is antibiotic-, serum- and Albumin (Human)-free.

The production cell line has been cryopreserved as a MCB, from which the WCB has been derived. The production cell line for ReFacto has been adapted to use defined growth medium containing Albumin (Human) and recombinant human insulin. The Master Cell Bank (MCB) and WCB are maintained in the absence of human or animal serum. The MCB, the WCB, and end-of-production cells have been characterized and found to be stable in genotype and free of any detectable bacterial, mycoplasma, or fungal contamination. No adventitious virus was detected from a conventional testing program. The manufacturing process has been validated for consistency, robustness and for removal of impurities.

rVIII SQ (ReFacto) is purified by primary capture using ion exchange chromatography, followed by viral inactivation, immunoaffinity chromatography (murine monoclonal antibody affixed to 8A4 Sepharose), ion exchange chromatography (Q Sepharose), hydrophobic interaction chromatography (HIC), gel permeation chromatography [with the last step also reported in the same source by Biovitrum as hydrophobic interaction chromatography (HIC)], and buffer exchange, yielding highly purified active bulk drug substance. This is followed by formulation, lyophilization, packaging and labeling. The new process, for Xynta, is much the same. Somewhere in this process (at least for Xyntha), most likely towards the end, a virus-retaining nanofiltration step is performed.

The immunoaffinity purification step for ReFacto uses chromatography medium-bound murine monoclonal antibodies with binding specificity for rVIII SQ, while this step for Xyntha uses a custom synthetic affinity peptide.

 Purification involves five different chromatography steps. This includes affinity chromatography using an unspecified medium (8A4 Sepharose)-bound murine monoclonal antibody ligand with binding specificity for the 90 kDa heavy chain of Factor VIII, with VIII SQ binding to the column and subsequently eluted. Bulk rVIII SQ is subjected to a solvent-detergent virus inactivation step using the solvent tri-n-butyl phosphate (TNBP) and the detergent Triton X-100 (octoxynol; polyethylene glycol p-isooctylphenyl ether). rVIII SQ is stabilized by addition of polysorbate 80 (Tween 80), L-histidine, sucrose, sodium chloride and calcium chloride. The bulk is then lyophilized and formulated without Albumin (Human) for stabilization.

Purity testing includes Specific activity; SDS-PAGE; SEC-HPLC; RP-HPLC; DNA; CHO cell protein; Mouse IgG; tri-butyl n-propyl phosphate (TNBP; from solvent-detergent viral inactivation); Triton X-100 (from solvent-detergent viral inactivation) and ethylene glycol. Structure is affirmed by Amino acid composition; Amino acid sequence; Carbohydrate composition; Carbohydrate sequence; SDS-PAGE/ Western Blot; Tryptic map; Gel filtration; MALDI-MS; RP-HPLC; and Circular dichroism. As disclosed by Biovitrum, substances used in manufacture and tested for as impurities include polysorbate 80 (Tween 80); polyethylene glycol (PEG) with m.w. of about 4,000 (PEG 4000), human serum albumin [Albumin (Human)]; Haemaccel (gelatin, presumably porcine or less likely bovine-derived, polypeptides cross-linked via urea bridges; a human therapeutic product); and hydroxyethyl starch (HAES).

The major steps in manufacture providing virus reduction/inactivation are the solvent-detergent (S/D) inactivation stepl immunoafffinity purification; and gel filtration. The S/D treatment reduces murine xenotropic retrovirus (model for Retroviridae) by ³4.6 log; mink cell focus virus (Retroviridae) by ³4.1 log; infectious bovine rhinotracheitis virus (Herpesviridae) by ³4.9 log; parainfluenza 3 virus (Paramyxoviridae) by ³6.2 log; with no reduction in polio virus (Picornoviridae). The immunoafffinity step reduces murine xenotropic retrovirus (model for Retroviridae) by 5.1 log; mink cell focus virus (Retroviridae) by 5.5 log; infectious bovine rhinotracheitis virus (Herpesviridae) by ³7.9 log; parainfluenza 3 virus (Paramyxoviridae) by 6.3 log; and polio virus (Picornoviridae) by 3.8 log. The gel permeation chromatography step reduces polio virus (Picornoviridae) by 1.8 log. Global/overall reduction is murine xenotropic retrovirus (model for Retroviridae) by ³9.7 log; mink cell focus virus (Retroviridae) by ³9.6 log; infectious bovine rhinotracheitis virus (Herpesviridae) by ³12.8 log; parainfluenza 3 virus (Paramyxoviridae) by ³12.5 log; and polio virus (Picornoviridae) by 5.6 log.

In a study using filtered brain homogenate from TSE-infected hamsters, the immunoaffinity and anion-exchanges steps demonstrated 3.8 log reduction and >5.2 log reduction, respectively, for TSE.

rVIII SQ and Refacto for the U.S. are manufactured at the Pharmacia AB, later Pharmacia & Upjohn AB, now Biovitrum facility in Stockholm, Sweden. Pharmacia/Biovitrum also tests the final containers for sterility and particulates, labels and packages the product, and ships the released product to distribution centers. Product testing includes potency, protein concentration, specific activity, SDS-PAGE (identity), SEC-HPLC (aggregates and fragments), sterility, endotoxin, appearance before reconstitution, dissolution time and appearance (coloration and clarity) after reconstitution, residual moisture, pH, and concentrations of the major excipients. Assays of the active substance and formulated product have been validated for accuracy, precision and reproducibility.

Various steps in the purification process have been validated for their ability to remove viruses that may not have been detected in the production cells. Two of the chromatography steps and the solvent-detergent viral inactivation step were evaluated using appropriate model viruses. Overall, the purification processes has been shown to reduce these viruses by a factor of at least 107 (7 log). At time of approval, no testing or purification validation had been carried out for Bovine Spongiform Encephalopathy (BSE). However, neither bovine components nor bovine serum are used in the cell culture manufacturing process. Components used in the development of the cell line up to cell banking, including fetal bovine serum, were sourced from countries known to be free from BSE. The possibility of introduction of TSE/BSE into the ReFacto manufacturing process is considered to be negligible.

FDA class: Biologic BLA

CBER class: Blood And Blood Derivatives

Approvals: Date = 20000306; BLA (STN 103779); first approval; orphan designation (expired 3/6/2007)

Date = 20021001; launch (not approval), of 2,000 IU vial

Date = 20040913; supplemental BLA; Indication = approval of the R2 Kit, the first needle-less reconstitution device with a prefilled diluent syringe for hemophilia

Indications: [full text of the "INDICATIONS AND USAGE' section of product insert/labeling, 12/2002]:

ReFacto Antihemophilic Factor (Recombinant) is indicated for the control and prevention of hemorrhagic episodes and for surgical prophylaxis in patients with hemophilia A (congenital factor VIII deficiency or classic hemophilia). ReFacto is indicated for short-term routine prophylaxis to reduce the frequency of spontaneous bleeding episodes. The effect of regular routine prophylaxis on long-term morbidity and mortality is unknown. ReFacto can be of a significant therapeutic value for treatment of hemophilia A in certain patients with inhibitors to factor VIII. In clinical studies of ReFacto, patients who developed inhibitors on study continued to manifest a clinical response when inhibitor titers were < 10 BU. When an inhibitor is present, the dosage requirement of factor VIII is variable. The dosage can be determined only by a clinical response and by monitoring of circulating factor VIII levels after treatment (see DOSAGE AND ADMINISTRATION). ReFacto does not contain von Willebrand factor and is not indicated in von Willebrand's disease.

Status: The BLA was submitted to FDA on Feb. 2, 1998, received standard review, and was approved on March 6, 2000. The International Nonproprietary Name (INN) and USAN of the approved active pharmaceutical ingredient (API) is moroctocog alfa.

European Union was granted on April 13, 1999 to the Genetics Institute (Europe) subsidiary of Wyeth..

U.S. product launch was planned to begin in the 3rd quarter of 2000. However, this was delayed into 2001, due to insufficient supplies for U.S. launch (partially due to increased demand in Europe). In Nov. 2000, Bayer (now Bayer Schering) was awaiting approval (presumably now granted) for use of a 200 L fermentor to replace a 100 L fermentor at its Berkeley, CA, facilities to expand manufacturing capacity.

In June 2003, the European Union approved a new assay standard for use in the manufacture of ReFacto. As a result, the labeled amount of clotting protein in each vial in Europe increased by approximately 20%.

Tech. transfer: See the Factor VIII Products entry in the Blood Products, Human section (and other Factor VIII entries) for discussion of Factor VIII patents and technology transfer.

Relevant patents include U.S. 4,868,112, " Novel procoagulant proteins," assigned to Genetics Institute, Inc., now Wyeth. This claims Factor VIII molecules with a substantial portion deleted, including the amino acid sequence for Factor VIII SQ.

U.S. 5,733,873 and 5,919,766, "Composition comprising coagulation factor VIII formulation, process for its preparation and use of a surfactant as stabilizer, are assigned to Pharmacia & Upjohn AB, now Biovitrum AB. This concerns Factor VIII formulation containing a non-ionic surfactant e.g., block copolymers, e.g., polyoxamers or polyoxyethylene, e.g., polysorbate 80 as stabilizer. The compositions can also included sodium chloride, calcium chloride, L-histidine and/or sugars or sugar alcohols.

U.S. 6,005,082, "Process for purification of factor VIII," assigned to Genetics Institute, Inc., now Wyeth, includes claims for Factor VIII purification using hydrophobic interaction chromatography (HIC) gels (gel permeation chromatography) in the presense of a surfactant.

U.S. 5,831,026, "Process for purifying factor VIII," assigned to Pharmacia & Upjohn AB, now Biovitrum AB, concerns methods for reducing degradation of recombinant coagulation Factor VIII caused by metal-dependent proteases requiring Zn2+ for activity or containing Zn2+ as an integral part of their structure by adding an inhibitor of Zn2+ dependent proteases to a Factor VIII in solution, including use of L-histidine as a complexing agent.

Solvent detergent viral inactivation technology was developed by and nonexclusively licensed from the New York Blood Center. For example, see U.S. 4,820,805. See the entry for Pooled Plasma, Solvent Detergent Treated (SD Plasma) for further information about solvent detergent viral inactivation, used primarily for inactivation of enveloped viruses (e.g., HIV, hepatitis B and C viruses).

Genetics Inst./Wyeth in 2000 licensed from Aventis Behring GmbH, now CSL Behring AG, technology for the manufacture of high purity Factor VIII without stabilization using Albumin (Human). This includes U.S. 5,565,427 and other "Freudenberg" patents, origin-ally assigned to Behringwerke AG (later Aventis Pharma, now Sanofi Aventis S.A.) for which CSL also has certain rights through licensing or co-assignment. These patents includes claims use of amino acids, non-ionic detergents and a carbohydrate (e.g., sucrose), to stabilize Factor VIII. For example, U.S. 5,565,427 (EP 508 194) describes Factor VIII preparations containing combinations of detergent and amino acids, specifically arginine and glycine, in addition to excipients such as sodium chloride and sucrose.

Wyeth (originally Genetics Inst.) was a licensee of Columbia University's patents concerning cotransformation, a broadly-useful genetic engineering method allowing selection and isolation of transformed cells. Presumably, Wyeth paid (or reimbursed) royalties for Factor VIII manufacture by Pharmacia/Biovitrum. The original patents and license expired in 2000, but Columbia received another patent in 2002 and was again seeking royalties, which Wyeth and other companies challenged in court. Recently, the University decided not to continue to press infringement suits and seek royalties, but the patent office is reexaming the relevant patent, and the university could against pursue infringement and royalties at a later date. See the Tech. transfer section of the Recombinant DNA Products entry (#100) for further information.

Market.: Total 2007 worldwide sales were $335 million, ~$306 million in 2006; and $224 million in 2003. The author's rough guess for 2005 total sales is $250-$275 million, $240 million in 2004.

The 2007 Average Wholesale Price (AWP) is $1.36/IU for all vial sizes, and the Direct Price (manufacturer's discount price) is $1.09/IU (Red Book, 2007). This is unchanged since at least 2004.

Medicare reimbursement is set at $1.29/IU for inpatient and home care, and $1.01/IU for outpatient care. Estimated Acquisition Costs (for hospitals, treatment centers) is $.84-$1.09/IU [from NHF].

In its April 18, 2005 price list, FFF Enterprises, a major biologics distributor, reported its price as $1.16/IU ($1.15/IU in March 2004).

On June 1, 2005 Wyeth launched the Wyeth Factor Resource Program to provide free hemophilia factor to customers who experience a temporary lapse in insurance coverage. Qualified individuals receive product for up to one year or until a 200,000 IU maximum is reached. Participants must have insurance and have been using Wyeth products ReFacto (Factor VIII) or BeneFIX (Factor IX) for three consecutive months when they apply. Other key components of the Program include the Patient Assistance Program and the Reimbursement Information Line.