Status: FDA approved in Feb. 2009, previously approved in EU

Organizations involved:

rEVO Biologics Inc. – Manuf.; R&D; Tech. Genzyme Transgenics Corp. – Former

Genzyme Corp. – USA mark.; Parent

Leo Pharma A/S – Europe mark.; Canada mark.; Mid. East mark.

Cambrex Bio Science – Manuf. other

Lonza Biologics plc – Parent

MedImmune, Inc. – Manuf. other

AstraZeneca plc –Parent

Ovation Pharmaceuticals, Inc. – Former

Lundbeck A/S - Parent; Former

Cambrex Bio Science, Inc. – Parent; Former

Cross ref.: See also the Antithrombin III Products entry for information about antithrombin III (AT-III), and the other AT-III product entries in the blood products section.

Description: ATryn is a lyophlized (freeze-dried) formulation of recombinant human antithrombin III (AT-III; rhATIII) glycoprotein expressed in the milk of transgenic goats. rhATIII is expressed in goats transformed to have the human antithrombin gene linked to a milk-protein expresion-promoting gene. The expressed glycoprotein is further purified, nanofiltered and terminally heat-treated (pasteurized). Purification includes affinity chromatography using a heparin immobilized resin. The goats in which the AT-III is produced are USDA certified scrapie-free, and controlled for specific pathogens.

The antithrombin III blood protein has anticoagulant and anti-inflammatory properties. This was the first transgenic protein to successfully receive approvals in the U.S. and European Union, a significant precedent for transgenic animal-derived therapeutic proteins.

The amino acid sequence of Antithrombin (Recombinant) in Atryn is identical to that of human plasma-derived antithrombin. Like human AT-III, goat-expressed AT-III is a 432 amino acid single-chain glycoprotein with a molecular weight of ~57,215 Daltons and a molecular formula of C2191H3457N583O656S18. AT-III has a carbohydrate content of about 15%. AT-III, both Antithrombin (Recombinant) and plasma-derived antithrombin, has six cysteine residues forming three disulfide bridges and 4 N-linked glycosylation sites (Asn 96, 135, 155 and 192), considered essential for efficacy (which is why the product in not manufactured using a microbial expression system). Antithrombin (Recombinant) and plasma-derived antithrombin both contain six cysteine residues forming three disulphide bridges and 3-4 N-linked carbohydrate moieties.

However, the glycosylation profile of Antithrombin (Recombinant) is different from plasma derived antithrombin, resulting in an increased heparin affinity. When assayed in the presence of excess of heparin, the potency of the recombinant product is not different from that of plasma-derived product. N-terminal variability (N-2 and N-3) is observed (≤10%); larger truncation has not been clearly detected, although SDS-PAGE analysis (Coomassie reduced conditions) reveals faint presence of low molecular weight species. Four methionines have been identified as potential oxidation sites (Met314, 315, 17 and 20).

The human AT purified from transgenic goat’s milk is structurally indistinguishable from human plasma-derived AT, with the exception of the carbohydrates (glycosylation). The main glycosylation differences are the presence of fucose and GalNAc in the Antithrombin (Recombinant) in Atryn, a higher level of mannose, and a lower level of galactose and sialic acid. There is substitution of 40-50% of the N-acetyl neuraminic acid with N-glycolyl-neuraminic acid. The glycosylation profile of goat-expressed Antithrombin (Recombinant) in Atryn differs from plasma-derived antithrombin in terms of monosaccharide composition (fucosylated, less sialylated, high proportion of NGNA), oligosaccharide population, as well as glycoform species (different pI distribution). Differences are observed on heparin binding affinity analysis. Plasma derived AT-III is usually composed of ~90% alpha isoform (low binding affinity to heparin) and ~10% beta isoform (high binding affinity species). The goat-expressed antithrombin alfa demonstrates a plasma-derived antithrombin “beta-like” affinity without any detectable impact on the in-vitro bioassay (assay performed in excess of heparin).

As approved by FDA, each vial of ATryn is tested for potency stated on the product label using a reference standard calibrated against the World Health Organization international standard for antithrombin concentrate. Potency is approximately 1,750 IU AT-III. In addition to Antithrombin (Recombinant), each vial of the product contains 100 mg glycine, 79 mg sodium chloride, and 26 mg sodium citrate. When reconstituted with 10 mL Sterile Water for Injection, the pH is approximately 7.0. Following reconstitution, the solution may be further diluted into 0.9 % sodium chloride for injection. Atryn contains no detectable heparin (<0.0002 IU heparin/IU AT-III) in the final product. The product is stored at 2-8˚C (refrigerated).

As approved in the U.S. Atryn is packed in 1750 IU/vials as a sterile white to off-white lyophilized powder for reconstitution.Each carton contains one single dose vial of ATryn.

As approved in the European Union, ATryn is packaged in 20 mL type I glass vials containing AT-III at a concentration of 175 IU/mL, with 250 mg (1750 IU) per vial; glycine, 133 mM (104.8 mg/vial); sodium chloride, 10 mM (82.8 mg/vial); and sodium citrate, 135 mM (27.1 mg/vial). Vials are closed with a siliconized bromo-butyl rubber stopper, sealed with an aluminium seal and a flip-off cap. ATryn is at a concentration of 175 IU/mL (25 mg/mL) after reconstitution with 10 mL water for injection.

Milk contaminants are allergens that may be present in trace amounts in Atryn. These are monitored with a series of assays. The European Union SPC has a special warning for patients with hypersensitivity to goat proteins or goat milk components. The product has an EU expiration date of 18 months. ATryn does not contain any preservatives, and is not formulated with Albumin or other human plasma proteins.

Nomenclature: Antithrombin III [BAN]; Antithrombin III, rDNA [BIO]; ATryn [TR]; Antithrombin alfa [INN]; human antithrombin-III from the milk of transgenic goats (glycoform alfa) [CAS]; 9000-94-6 [CAS RN]; AT-III, recombinant [SY]; rhATIII [SY]

Biological.: The anticoagulant activity of antithrombin alfa and human plasma-derived antithrombin have been compared in several in vitro studies: in vitro thrombin and factor Xa inhibition assay, effect on fibrinogen levels in blood obtained from Sprague-Dawley rats and humans, and in vitro coagulation in blood from cardiac surgical patients. Antithrombin alfa and plasma-derived antithrombin had equivalent activity. Heparin binding studies indicated that heparin affinity is 4 fold higher for antithrombin alfa than for plasma-derived antithrombin. In vivo studies have been carried out with several acquired AT deficiency models, including sepsis, disseminated intravascular coagulation (DIC) and organ transplantation. Goat-expressed antithrombin alfa prevented sepsis and effects caused by septic shock similar to plasma-derived antithrombin. A 100% bioavailability in humans is assumed. Atryn’s altered glycosylation results in 7-10 fold decrease in serum half-life vs. natural, and requires reinfusions.

Companies.: Recombinant transgenic goat expressed AT-III or ATryn was developed by Genzyme Transgenics Corp. (GTC), now rEVO Biologics Inc., along with Genzyme General, both subsidiaries of Genzyme Corp. Cambrex Bio Science is GTC’s contractor for protein purification and finishing. Lonza Biologics plc acquired the contract manufacturing subsidiary of Cambrex in mid-2006.

rhATIII is GTC’s lead therapeutic. GTC has a 300+ acre facility in Massachusetts which houses about 2,000 goats certified free of scrapie (prions that cause a transmissible spongiform encephalitis) by the USDA. This herd was derived from goats imported from New Zealand. GTC has facilities for purification and other downstream processing with an annual processing capacity of multiple kilograms.

In Nov. 2005, GTC granted Leo Pharma A/S exclusive rights for clinical/regulatory development and marketing of ATryn in Europe, the Middle East and Canada. GTC is eligible for $73 million in clinical, regulatory and sales milestone payment, plus royalties. These payments included $2 million upon signing, and $5 million for European Union approval of ATryn. GTC will be responsible for manufacture of ATryn and receives a transfer price for all product used by LEO. GTC will also receive a royalty on net sales. Leo handles marketing in its territories, and clinical development for acquired AT-III deficiency indications:, while GTC retains responsibility for clinical development of hereditary antithrombin deficiency indications:. Leo has long been involved with therapeutics for thromboembolic disorders. This includes, Innohep, a low molecular weight heparin product involving blood-derived AT-III bound with heparin to produce a greater anticoagulant effect. Leo has over 250 sales reps in Europe, the Middle East, and Canada focused on critical care and coagulation management, out of a total marketing work force of 1,000.

In Aug. 2008, Ovation Pharmaceuticals, Inc. entered into a collaborative agreement with Genzyme, and received exclusive U.S. marketing rights for Atryn. The agreement includes $257 million in potential payments to GTC for meeting clinical, regulatory, and sales milestones; and development and commercialization of ATryn in the hereditary antithrombin deficiency, or HD, indication. The milestone payments include a total of $9 million through approval of ATryn for HD in the U.S., including $3 million paid upon closing and an additional $2 million expected in 2008. The collaboration also plans for further development in larger market acquired antithrombin deficiencies, such as the treatment of heparin resistance in patients undergoing surgery requiring cardiopulmonary bypass and the treatment of disseminated intravascular coagulation associated with severe sepsis. GTC receives a transfer price on commercial product it manufactures, a royalty on net sales, and payment for product used in clinical trials. As the collaboration proceeds with the acquired deficiencies, Ovation will fund GTC’s costs of clinical development; and will be responsible for sales and marketing of ATryn in the U.S., including all launch activities.

In March 2009, Lundbeck A/S acquired Ovation Pharmaceuticals.     

In July 2010, GTC regained U.S. commercialization rights to ATryn from Ovation, then Lundbeck, Inc., H. Lundbeck A/S. Presumably, the parent Genzyme Corp. handles U.S marketing. The two companies agreed to a defined transition period of up to six months in which Lundbeck performs certain services on behalf of GTC to ensure that ATryn will continue to be available as commercialization responsibilities are transitioned to GTC. ,Lundbeck will earn an undisclosed royalty on net sales beginning in two years, with a predefined cumulative maximum.

GTC Biotherapeutics Inc. was acquired by LFB in Dec. 2010 and was renamed rEVO Biologics.

Manufacture: Simply stated, DNA for human AT-III linked with DNA for a goat milk protein promoter gene (beta-casein promoter), which directs transgene expression to the goat mammary gland during lactation, was spliced into a transgene expression vector, which was microinjected into isolated fertilized goat embryos, which were microinjected into the uterus of host female goats, which carried the embryo to term (normal birth). The resulting first generation transgenic (“founder”) animals carry the human AT-III gene and expression promoter, and females (doe) express human rhAT-III in their milk. The founder animals serve as the production herd, and are further propagated. Several modifications have been introduced in the active substance and finished product manufacturing process between 1991 and filing for approval. The timeline from initiation of transgene transfer to natural lactation of resulting transgenic does is 16-18 months for goats.

The founder transgenic goat was obtained after microinjection of the transgene in the pro-nucleus of a goat embryo. This transgene is composed of the gene of interest (human AT-III cDNA) and the regulatory regions of goat beta casein gene (CSN2) to direct tissue specific expression in goat mammary gland. The selection of the first transgenic goat F0 was based on its transgene status (mosaicism and germline transmission) and its capacity for producing large quantities of antithrombin alfa in its milk. Goat breeding was then accomplished through a combination of natural breeding and artificial insemination. The herd is predominantly constituted of “Swiss breeds” dairy goats (Saanen, Alpine, Toggenburg breeds and mixtures thereof). Transgenic goats may be obtained from any combination of these breeds. The GTC farm in Massachusetts houses about 1,500 goats, most derived from New Zealand breeds that are notably free of Scrapie disease.

A 2-tiered banking system was established: the Master Transgenic Bank (MTB) is comprised of semen from qualified F0 and F1 males, and the qualified P0 and P1 females, and the Working Transgenic Bank (WTB) is comprised of qualified male and female animals from qualified animals, and the semen from these qualified males. Considerable efforts were made to identify the genotype of the goats, and to improve the “genetic consistency” of the herd. The qualification of a production doe, and production group was mainly based on the transgenic animal capacity to produce antithrombin alfa in sufficient quantity, and on microbiological screening.

Source material (milk) is then collected from qualified doe at GTC Biotherapeutics Inc. Source material is then shipped to Cambrex for further processing.

Purification and the manufacture of the bulk active agent are performed by Cambrex BioScience. Frozen milk is received, thawed, AT-III concentrated using tangential flow filtration (TFF; primarily for removal of casein micelles, fat globules, bacteria and somatic cells); heparin affinity chromatography (for removal of DNA, lactose, mineral salts, hormones and milk proteins); and nanofiltration for virus removal. This is followed by anion exchange chromatography (for removal of lactoferrn and milk proteins); hydrophobic-interaction column chromatography to remove goat (not the desired recombinant human) AT-III, which is also carried over in milk and has about 80% homology with rhAT-III; and a buffer exchange step to bring the final rhAT-III concentration to 25 mg/mL. Overall, yield is 300 grams at about 55% yield, with purity >99.99%. This bulk final product is then filtered to remove any residual microorganisms. The active substance has a shelf life of 18 months at 2-8 °C. The validation of removal of goat milk impurities by the purification process has been demonstrated by validation studies. More than 4 logs of removal are observed for all measured impurities, except for goat antithrombin (2.9 logs). A 300 Liter batch of milk provides about 300 grams of purified rhAT-III in 10-15 L of bulk product. This is roughly comparable to the yield from purifying AT-III from 4,000 units of Plasma. The human AT purified from transgenic goat’s milk is structurally indistinguishable from human plasma-derived AT (hpAT) with the exception of the carbohydrates.

The formulated bulk drug active is shipped to MedImmune BV, Nijmegen, The Netherlands, and stored at 2 to 8°C. MedImmune was acquired by AstraZeneca plc in April 2007. The formulated antithrombin alfa is filtered (0.22 µm) and aseptically filled. The product is then lyophilized, subjected to heat inactivation and sealed in vials.

Biological activity is assayed using an antithrombin inhibition test using an excess of heparin. For the European Union EEU), this involves the the Thrombin Inhibitory Activity assay consistent with the European Pharmacopeia monograph on Assay of Human antithrombin III. The calculated potency (IU) must not be less that 80 % nor more than 120 % of the potency stated and the confidence interval (p=0.95) not greater than 90 % to 110 % of the estimated potency. ATryn and plasma-derived antithrombin behave differently in this thrombin inhibitory analysis (without heparin and with progressive amounts of heparin), even though the potency of both is expressed in international units (IU). Therefore, a statement is included in the EU SPC indicating that both products are not interchangeable.

A viral inactivation heat treatment step was added during development. The formulations used in preclinical studies, with the exception of prenatal and postnatal toxicity studies, were non-heat treated. Additional data submitted to the EU indicate there appear to be only minor biochemical differences between the non-heat treated, heat treated and the nanofiltered plus heat treated ATryn, which may not impact on the pharmacokinetics, distribution or activity of the antithrombin alfa in animals or humans. Bioequivalence was shown in a clinical pharmacokinetics study (GTC AT PK 011-04). Viral validation studies showed a significant virus reduction of >8.5 to >25.3 log10 (roughly 300 million fold to septillion fold) was accomplished across the rhAT process.

The viral safety of this product mainly relies on the quality of starting materials (health status of animals, the quality of milk) and the capacity of the production process to remove or inactivate viruses. The viral safety strategy of starting materials relies on a closed herd of selected and limited origins, together with a health monitoring program, and some viral screenings. During the manufacturing process, specific viral removal and inactivation steps include nanofiltration and dry heat treatment (pasteurization). Measures are taken at the GTC farm to ensure health status and monitoring of transgenic animals (closed herd, controlled farm, good manufacturing practices, virological controls on animals). In case a confirmed infections occurs, as part of its EU approval, GTC has committed to communicate the description of the case and the subsequent decisions to the competent authorities. Adventitious agents are searched on bulk milk. The only biological material used during the purification process is heparin of porcine origin.

To assure transmissible spongiform encephalitis (TSE) and other prion safety aspects, the goats were maintained in a closed herd in the U.S. by GTC. All GTC goats have been certified scrapie-free by the USDA. Milk is considered as a product with no detectable TSE infectivity. Moreover, some steps of the production process were subjected to scrapie removal/inactivation studies. As part of its EU approval, GTC committed to further investigate the use of a systematic scrapie search by ELISA or Western Blot on the CNS of each slaughtered animal as part of a health monitoring program. The rhAT purification process has been validated for its ability to reduce scrapie contamination over a 100 billion fold (>11.3 log10 scrapie removal).

A typical goat expresses about 3 liters of milk daily containing about 8.3 grams of AT-III (also reported as 2-8 grams/Liter), and about 3 kilograms annually. The concentration of AT-III in milk is usually about 2 g/L. Transgenic dairy goats have an average milk output per doe on the order of 600 to 800 L per natural lactation. This is unlike human plasma, the source for current AT-III products, which can only be taken from donors about once every six weeks. Goat-expressed antithrombin is properly folded and fully functional. In early 2006, GTC had 57 transgenic goats for manufacture of Atryn. In order to increase the consistency of the source material, which shows variability in amounts of specific proteins, lactose, total protein and fat levels, a milk pooling strategy was revised .

GTC’s transgenic goat production technology is based on use of the goat beta-casein promoter sequence which directs the expression of recombinant DNA sequences in mammary epithelial cells upon lactation of the transgenic animal, with the protein secreted in the animal’s milk. The expression cassette used comprises 7.2 kb of 5’ and 71 kb of 3’ goat beta-casein sequences surrounding a Xho I restriction site. A 2.4 kb insulator sequence, the direct repeat of the 1.2 kb of the chicken beta-globin insulator sequence (5’HS4 element), is use to flank the mammary gland regulatory sequences. The 5’CHS4 element significantly reduces transgene silencing due to chromosomal position effects and promoter interference.

The purification and drug product manufacturing processes have been validated to demonstrate its capacity for removal and/or inactivation of viruses. As reported for U.S. product, total/global tested virus reduction (log10 reduction) is ≥18.7 for pseudorabies virus; ≥20.0 for xenotropic murine retrovirus; ≥13.2 for human adenovirus; and NA for porcine parvovirus. [Note, these are very high virus reduction values]. Viral log10 reductions (removal and/or inactivation) for each of the relevant steps are:

a) Tangential Flow Filtration - ≥5.1 for pseudorabies virus, NA for the others tested

b) Affinity Chromatography - 1.6 for pseudorabies virus; 1.2 for xenotropic murine retrovirus; NA for human adenovirus; and 1.4 for porcine parvovirus.

c) Nanofiltration - ≥3.8 for xenotropic murine retrovirus; ≥6.3 for human Adenovirus; and ≥3.7 for porcine parvovirus.

d) Ion Exchange Chromatography - 3.6 for pseudorabies virus; 1.0 for xenotropic murine retrovirus; ≥7.1 for human adenovirus; and NA for porcine parvovirus

e) Hydrophobic Interaction Chromatography (HIC) - ≥5.6 for pseudorabies virus; ≥4.4 for xenotropic murine retrovirus; ≥4.8 for human adenovirus; and ≥5.7 for porcine parvovirus

f) Heat Treatment - 2.8 for pseudorabies virus; ≥5.0 for xenotropic murine retrovirus; ≥1.8 for human adenovirus; and 2.4 for porcine parvovirus

Particuarly with its non-human glycosylation, immunogenicity was an issue that had to be resolved during development. GTC developed assays to detect antibodies directed against antithrombin (Recombinant), goat AT, and goat-milk proteins. No confirmed specific immunological reaction was seen in any of the patients tested, nor were there any clinical adverse events that might indicate such a response.

FDA class: Biologic BLA

Approvals: Date = 20090204; BLA (STN 125284/0)

indications: [full text of the "Indications and USAGE" section of the product insert]:

ATryn is a recombinant antithrombin indicated for the prevention of peri-operative and peri-partum thromboembolic events in hereditary antithrombin deficient patients.

It is not indicated for treatment of thromboembolic events in hereditary antithrombin deficient patients.

Status: Expectations for initial U.S. approval of AT-III for treatment of heparin resistance in patients about to undergo cardiopulmonary bypass surgery were abandoned in Feb. 2001. GTC had reported efficacy and safety in two completed pivotal Phase III trials (a major precedent for therapeutic transgenic proteins). However, FDA requested further clinical trial data that the company considered were not economically justified by the limited market size of the heparin resistance indication. This is also an indication for which the product would compete against current plasma-derived AT-III products.

GTC then concentrated on first obtaining European Union (EU) approval for the treatment of high risk patients with a genetic deficiency of antithrombin protein, specifically for prophylaxis of deep vein thrombosis (DVT) and thromboembolism in patients with hereditary AT-III deficiency (HD) in “clinical risk situations” such as surgical procedures, including during labor and delivery. A MAA for EU approval was filed on Jan. 26, 2004 and was accepted for filing on Feb. 27, 2004. In Dec. 2004, GTC submitted responses to a consolidated list of questions from EMEA/EU; and reported that it expected an EMEA/EU response by the end of April 2005, with plans to launch ATryn in mid-2005. However, in April 2005, GTC reported it would complete its response to a List of Outstanding Issues raised by EMEA/EU by July 8, and that the expected approval decision date had been extended to late Oct. 2005. Subsequently, due to ongoing inspections and reporting of the results, which affect completion of the overall review process, GTC anticipated the EMEA’s opinion in Feb. 2006.

The submission of this EU MAA was the first completed application submitted by any company to any U.S. or European regulatory authority requesting approval of a recombinant therapeutic protein produced using transgenic technology. It also was GTC’s first filing for a therapeutic product. GTC press releases do not mention EU orphan designation for ATry. If not already granted, this will presumably be granted upon approval (unless rhATIII is considered insufficiently novel by the EU, e.g., taking into account the plasma-derived products, but this is highly unlikely).

In April 2005, GTC completed enrollment for a pivotal international open-label Phase III trial in 17 patients undergoing high-risk procedures. For its BLA, data from this trial were planned to be combined with the prior similar 14-patient study (used for the EU filing), and compared to historical data for patients treated with human plasma-derived antithrombin.

In Feb. 2006, EMEA/European Union issued a negative opinion (rejected) the application for marketing of Atryn. The Committee for Medicinal Products for Human Use (CHMP), EU, adopted a negative opinion, effectively rejecting approval of Atryn, for use in surgical patients with congenital antithrombin deficiency for the prophylaxis of deep vein thrombosis and thromboembolism in clinical risk situations, i.e., during the peri-surgical period. The CHMP, after excluding data from nine pregnant patients, determined that an insufficient number of surgical patients had been enrolled in the pivotal trials to support approval. CHMP excluded the childbirth patients and concluded that data from five other patients was not enough for approval. The problem was that the administration schedule of ATryn to childbirth patients ended up being different from the one that was originally proposed in the Phase III protocol. Unlike other patients, the childbirth patients did not consume ATryn at a constant rate, primarily needing it during contractions. The CHMP also had concerns about sufficient immunologic/antigenicity data, i.e., regarding induction of antibodies and their effects, and the lack of sufficient clinical data concerning ATryn produced with an additional filtration step (added for product used in the pivotal Phase III trials). GTC attributed apparent safety-related problems cited by CHMP as having been caused by the dosing protocol, with some physicians with less experience with Atryn administering it too quickly. Genzyme promptly filed an appeal and the application was reexamined.

On June 2, 2006, the EU CHMP recommended that ATryn be granted market authorization for the prophylaxis of venous thromboembolism in surgery of patients with congenital antithrombin deficiency, optionally, in association with heparin or low molecular weight heparin. GTS provided some additional data and analyses, and the CHMP accepted GTC’s data from childbirth patients as relevant to deciding the product’s approval (although this indication was not approved). At the time, eight EU countries (with a total populations of 23 million) did not have any approved plasma-derived products.

In Aug. 2006, Atryn receive European Union approval. The approved EU indications: are: “ATryn is indicated for the prophylaxis of venous thromboembolism in surgery of patients with congenital antithrombin deficiency. ATryn is normally given in association with heparin or low molecular weight heparin.”

On Aug. 8, 2008, GTC/Genzyme completed its fast-track BLA filing for priority review of Atryn approval for prophylactic treatment of deep vein thrombosis and other thromboembolisms in patients with hereditary antithrombin deficiency who are undergoing high-risk surgical procedures or are in childbirth. The FDA had granted both Fast Track review and Orphan Drug status to ATryn.

On Sept. 5, 2008, FDA granted the BLA priority review, and set a PDUFA (target approval date) of Feb. 7, 2008. ATryn has been designated as an orphan drug.

In Jan. 2009, the Blood Products Advisory Committee, FDA, voted that ATryn is safe and efficacious for the prevention and treatment of venous thromboembolism in hereditary antithrombin deficient patients undergoing surgery or childbirth procedures.

On Feb. 4, 2009, FDA granted full approval to Atryn. Atryn was launched in the U.S. on May 6, 2009.

A post-marketing patient registry has been established to collect additional data on the immunogenic potential of ATryn in patients treated with ATryn on more than one occasion.

Tech. transfer: Referring to patented transgenic, including goat, technology, including specialized promoters and genetic constructs, GTC claims its "intellectual property includes a patent in the United States through 2021 for the production of any therapeutic protein in the milk of any transgenic mammal."

U.S. 6,441,145, Transgenically produced Antithrombin III," assigned to Genzyme Transgenics," expires Feb. 21, 2015. U.S. 5,843,705, "Transgenically produced antithrombin III," expires Dec 1, 2015.

Trials: In Feb. 2004, GTC reported results from its pivotal open-label clinical trial in hereditary antithrombin deficiency patients undergoing high risk procedures, such as surgery and childbirth. This trial was expected to support EU approval. This safety and efficacy study enrolled 14 patients from Europe and the U.S. The high risk situations treated included five surgeries, including four hip replacements, and nine childbirths, including natural, induced and Cesarean section procedures. The study used serial Duplex ultrasound scans of the lower extremities to monitor for the development of deep vein thromboses (DVTs), regardless of the presence or absence of symptoms of DVT. ATryn was well tolerated and any adverse events that occurred were consistent with the surgical or childbirth procedures performed. There were no clinical signs or symptoms of DVTs in the patients treated with ATryn.

The EU MAA included data from two pivotal trials in a total of 19 hereditary antithrombin-deficient patients (14 patients from study GTC AT III 01002 and 5 patients in the compassionate-use treatment GTC AT III 011003) treated during and after 20 high risk situations. The Compassionate Use Study (GTC AT III 011-003) involved retrospective collection data performed on 5 patients (6 treatments) with non-heat treated ATryn administered by multiple daily infusions (due to a shortage of human-derived product in the U.S.), The Efficacy Study (GTC AT III 01002) was a prospective study performed on 14 hereditary AT deficient surgery (n=5) and delivery (n=9) patients with heat treated antithrombin alfa administered by continuous infusion with central independent evaluation of standardized ultrasound examinations for detection of deep vein thrombosis (DVT), a potential adverse event. The incidence of DVT was assessed both locally and centrally by an independent group of reviewers. One patient in the Efficacy Study was excluded since an acute DVT was detected by central evaluation at baseline ultrasound. Patients received ATryn treatment for periods of time ranging from 3.0 to 18.6 days and total doses from 37,884 IU up to 227,817 IU. Infusion rates ranged from 3360 to 64800 IU per 24 hours for varying periods of time. The primary efficacy endpoint was defined as “incidence of acute DVT at any time point after start of study drug treatment as assessed by a blinded, independent central review of duplex ultrasonography and/or venography testing.” There has been no pediatric development program for ATryn. FDA generally requires larger studies than the pivotal 14-patient open-label efficacy trial submitted with the EU application, and also favors placebo-controlled trials. However, larger trials with ATryn are difficult, since it took a year to recruit the patients into the Phase III trial, and placebo-controlled trials would be unethical due to the risks involved for placebo recipients.

In early 2008, ATryn met the primary endpoint of a pivotal comparative study, required by FDA approval, demonstrating non-inferiority to plasma-derived antithrombin in prophylactically treating hereditary deficient (HD) patients undergoing surgical or childbirth procedures with a high risk of developing deep vein thrombosis or thromboembolism. This was submitted in the BLA filing. A minimum of 31 evaluable HD patients were required for this trial. Results for 14 of these patients were already used to support European Union approval for HD patients undergoing surgical procedures. Seventeen evaluable patients have since been treated to complete the pivotal study. There were no clinically relevant deep vein thromboses or other thromboembolisms during the evaluation period in any of the patients treated in the pivotal study. The full clinical data package will include 90-day follow-up tests for antibody generation to AT-III.

Disease: Individuals with a genetic deficiency of antithrombin are at risk to develop thromboses (excessive blood clotting), particularly during surgery, pregnancy and childbirth. About 1 in 2,000 to 1 in 5,000 people have hereditary deficiency of antithrombin. AT-III deficiency patients are prone to hypercoagulation and thrombosis, and are often treated with an oral anticoagulation drug, e.g., warfarin, prior to surgery. However, due to the excessive risk of bleeding from use of blood thinners and anticoagulants in AT-III deficient patients, e.g., mothers in labor, AT-III deficient patients have often received plasma-derived antithrombin products to control coagulation and inflammation during surgery.

Medical: Atryn is administered intravenously after reconstitution. The dosage of ATryn is individualized for each patient. The treatment goal is to restore and maintain functional antithrombin (AT) activity levels between 80% - 120% (0.8 - 1.2 IU/mL) of normal. Typically, a loading dose is administered as a 15-minute intravenous infusion immediately followed by continuous infusion of the maintenance dose. The U.S. product insert includes formulas for dosage calculations.

Patients with hereditary antithrombin deficiency (HD) have an increased risk for the development of deep vein thromboses (DVTs) or thromboemboli, especially during high-risk medical procedures. These patients are normally treated with an orally available anticoagulant, e.g., Warfarin. DVT occurs in about 30% of hip replacement surgeries in patients with normal coagulation (not AT-III deficient). However, anticoagulant maintenance therapy often must be discontinued during events such as childbirth or surgery to reduce the risks of excessive bleeding associated with oral anticoagulants. Currently, human plasma derived antithrombin products are often given to cover the period of high risk for the development of DVT. Recombinant AT-III may also help reduce the supply constraints that can occur with antithrombin derived from human blood plasma, and should avoid potential viral and other infection risks associated with plasma-derived products. However, expression in goats may, theoretically, involve other risks, e.g., currently undetectable prions.

Supplementation of low antithrombin levels with ATryn may be utilized during high risk surgical procedures to allow for appropriate functioning of the natural coagulation management system. ATryn is administered by infusion in a hospital setting during the high risk procedure. The therapeutic goal is to increase to and maintain antithrombin activity between 80-120% (0.8-1.2 IU/ml) for the duration of treatment. Initial treatment starts with a loading dose of ATryn targeting an antithrombin activity level of 100%. This initial loading dose is based on body weight and on the pretreatment antithrombin activity level. The usual loading dose in surgical patients (baseline AT activity 50%, bodyweight 75 kg) with congenital antithrombin deficiency in clinical risk situations is 20-25 IU/kg bodyweight. The loading dose should be given as a 15 minute infusion immediately followed by initiation of the maintenance infusion. The required maintenance dose for surgical patients is given as a continuous infusion and is determined using the following formula: Maintenance Dose (IU/hour) = [(100 – patient’s pre-treatment AT activity level in %) /10.22] x Body Weight in kg. The usual maintenance dose in surgical patients with congenital antithrombin deficiency in clinical risk situations is 4-5 IU/kg/h. During consumptive states (e.g. major surgery, concomitant use of heparin) the actual dose may be higher

Upon approval, FDA reported that Atryn had not been tested or proven safe and effective in pediatric patients..

Market: ATryn compete against human plasma-derived AT-III, e.g., Thrombate (see related entry).

The market for AT-III products is very limited (see the Antithrombin Products entry in the Blood Products, Human section). Analysts project that first year sales of ATryn may reach $50 million,

The market for Atryn could expand significantly, e.g, to a multi-$billion annually, if it is found safe and effective and approved for broader indications:, e.g., for treatment of disseminated intravascular coagulation (DIC) associated with severe sepsis; and a smaller market (potentially $150-$200 million/year) for restoration of heparin responsiveness in heparin-resistant patients

Ongoing: LEO Pharma, in partnership with GTC Biotherapeutics, is developing ATryn in Europe as a potential treatment for disseminated intravascular coagulation (DIC) associated with severe sepsis. In fall 2008, LEO was conducting a Phase II dose ranging study of ATryn as a potential treatment for DIC associated with severe sepsis. GTC will have access to LEO’s European Phase II study.An international Phase III program is anticipated, in which LEO Pharma and Ovation will share clinical development costs required for approval for this indication in the U.S. and Europe.

Ovation and GTC also expect to develop ATryn as a supplement to restore heparin responsiveness in heparin-resistant patients. Coronary artery bypass graft and related surgeries where a cardiopulmonary bypass machine, or CPB, is used requires that patients are anticoagulated prior to going on bypass, in order to avoid clot formation. Heparin is used to prevent the formation of blood clots in the CPB. Heparin’s ability to prevent clotting depends on the presence of sufficient antithrombin in the bloodstream to achieve the desired anticoagulant effect. Over 20% of patients in CPB related surgeries exhibit heparin resistance.