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Read Taylor's Story
An HD patient discusses learning about,
and living
with hereditary antithrombin deficiency.
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What are the HD Treatment Options?
Treatment of hereditary antithrombin deficiency depends upon a patient's risk of thromboembolic disease.
When a patient has had a venous clot, that person will receive anticoagulation. This is accomplished by
several different medications:
1) heparin, 2) warfarin and 3) low-molecular-weight heparins.
Patients that have had multiple thromboembolic episodes, or patients who are at risk of further episodes
(for example, they have multiple coagulation deficiencies) may be considered for long-term oral anticoagulation.
Because studies have demonstrated an increased risk of recurrent venous thromboembolic disease in patients with
hereditary antithrombin deficiency, long-term oral anticoagulation may be recommended.
However, the use of long-term anticoagulation has risks associated with it (approximately a 3% chance per
year of a major hemorrhage, of which approximately 1/5 are fatal) [1]. Therefore, the decision to begin
long-term anticoagulation is influenced by the patient's overall risk of recurrent thrombosis balanced with
the risks of long-term anticoagulation.
In addition to the anticoagulants mentioned above, antithrombin concentrates are also used for the treatment of
hereditary antithrombin deficiency. Typically, antithrombin concentrates are used during periods of risk
such as major surgery, where oral anticoagulation must be discontinued, or pregnancy, where oral anticoagulation
is contraindicated.
Plasma Antithrombin
Antithrombin products available today are derived from human plasma. Plasma, taken from pooled whole
blood, is separated or fractionated into many therapeutic products, including the coagulation protein antithrombin.
Pooled whole blood, plasma and its component protein fractions are derived from the blood of many
thousands of donors.
After fractionation, plasma antithrombin is purified including at least one viral inactivation step. Some
commercial suppliers include an additional viral removal step called nanofiltration.
Plasma derived antithrombin is available in Europe, Canada and the United States.
Recombinant Human Antithrombin, rhAT
GTC Biotherapeutics has developed recombinant human antithrombin (rhAT). Because the complex structure
of antithrombin precluded its efficient production in traditional bioreactors, the company utilized an innovative
production system it had developed for expressing recombinant proteins in the milk of transgenic dairy goats.
Key objectives for recombinant production are purity, safety, consistency and unlimited supply. These
were the same objectives GTC sought with the development of their novel expression system, and with the production
of recombinant human antithrombin.
Using GTC technology, recombinant antithrombin is isolated from the milk of transgenic female goats and purified
using both conventional and proprietary methods. The rhAT production process also incorporates one viral
treatment step and one viral removal step (nanofiltration). Purified recombinant antithrombin is structurally
indistinguishable from plasma derived AT in amino acid sequence.
In August, 2006, the European Commission granted market authorization to ATryn®, GTC's recombinant form of human antithrombin,
for the prophylaxis of venous thromboembolism in surgery of patients with congenital antithrombin deficiency. ATryn® is the
first recombinant antithrombin product approved anywhere in the world and the first antithrombin product, whether recombinant or
derived from the human blood supply, approved through the centralized EMEA procedure for use in all 25 countries of the European Union.
Click here to read the press release describing ATryn® approval.
Click here to read a recent press release describing ATryn's US development status.
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Plasma & Recombinant Human Antihrombin -- Detailed Discussion
Manufacturing Process of rhAT and hpAT
Commercially available human plasma-derived AT (hpAT) concentrates are isolated and purified from the plasma of
thousands of human blood donors. The manufacturing processes for all hpAT concentrates differ in their
details depending on the manufacturer. Each of the processes also includes at least one viral inactivation
step (usually heat treatment) and in some cases a nanofiltration step has been incorporated to provide an additional
level of viral safety. Viral validation studies for the hpAT manufacturing processes have demonstrated a
³ 2 to ³ 14 log10 reduction of a variety of viruses [2-6].
RhAT is isolated from the milk of the transgenic goats and conventionally purified using tangential flow filtration,
heparin affinity chromatography, nanofiltration, anion exchange chromatography and hydrophobic interaction chromatography [7].
The production goat herd for recombinant AT is highly controlled. A high level of donor control and
testing is a key parameter in the viral safety strategy for rhAT. In addition to the high level of donor control
and goat testing, the rhAT-containing milk must test negative for viruses when assessed in vitro on four cell
cultures (human MRC-5, monkey Vero, BHK-21, and goat turbinate) to screen for adventitious viruses. The manufacturing
process for rhAT has also been validated for its viral removal capacity. The rhAT viral validation studies demonstrated
that a significant virus reduction of greater than or equal to 8.5 to greater than or equal to 25.3 log10 was accomplished across
the distinctly different modes of the rhAT process [8].
Transmissible spongiform encephalopathies (TSE), such as new-variant Creutzfeld-Jacob disease (nvCJD) in humans, bovine
spongiform encephalopathy (BSE) in cattle and scrapie in sheep and goats, also must be considered in assuring the safety
of products made from human or ruminant sources. Human donors are monitored for CJD and nvCJD and potentially
contaminated blood, plasma pools, and products made from them have been recalled or traced when a contributing donor has
been diagnosed with CJD. All GTC goats are certified free of scrapie in the United States Department of Agriculture
(USDA) Scrapie Flock Certification Program and various risk minimization measures have been instituted to reduce any potential
risk from this TSE in this highly controlled closed donor goat population. In addition, the rhAT purification process
has been validated for its ability to remove greater than or equal to 11.3 log10 scrapie [8].
Structural Characteristics of rhAT and hpAT
The amino acid sequence of rhAT purified from transgenic goat's milk is structurally indistinguishable from hpAT. Both
rhAT and hpAT contain the same 4 N-linked glycosylation sites (Asn 96, 135, 155, 192). The monosaccharide composition
of rhAT differs from that of hpAT [7]. The main glycosylation differences are the presence of fucose and GalNAc, a higher
level of mannose, and a lower level of galactose and sialic acid in rhAT. There is also substitution of 40-50% of the
N-acetyl neuraminic acid with N-glycolyl-neuraminic acid.
There are two forms of AT in human plasma having different heparin affinities, but the same molar inhibitor activity toward thrombin
[9, 10]. Approximately 85-95% of circulating hpAT has glycosylation on 4 Asn residues. This fully glycosylated
form is referred to as the alpha-form. The remainder (5-15%) of circulating hpAT, referred to as the beta-form, lacks
glycosylation at Asn 135 and has a 3-10 fold higher heparin affinity than the alpha-form [10]. RhAT has a four-fold higher
affinity for heparin as compared to hpAT, similar to that reported for the beta-form of hpAT [7].
Several laboratories have determined that differences in glycosylation of AT do not affect the intrinsic rate constant of the uncatalyzed or
heparin catalyzed inhibition of thrombin, indicating that the carbohydrate chains solely affect heparin binding and not heparin activation or
proteinase binding functions [11, 13]. Thus, glycosylation differences do not impact the major biological activity of AT which is
thrombin inhibition, but do explain the differences in affinity for heparin and in pharmacokinetics. The specific activity of rhAT
is identical to various hpAT preparations in in vitro thrombin and factor Xa inhibition assays in the presence of excess heparin
(~6 IU/mg) [7, 14, 15]. A lower concentration of heparin was required for rhAT than for hpAT for inhibition of both enzymes, but
similar to the beta form of hpAT. Thus, rhAT closely resembles hpAT with respect to its activity for both thrombin and factor Xa
in the presence of saturating levels of heparin [7].
It is important to note that differences in glycosylation between hpAT and rhAT do not appear to elicit immune reactions, since none of the
patients treated with rhAT during various clinical studies have developed an antibody response, suggesting that rhAT is immunologically
tolerated [16-18].
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Learn more about HD in the Disease Profile 
References
1 Van der Meer FJM, Rosendaal FR, Vandenbroucke JP, Briet E. 1993.
Bleeding complications in oral anticoagulant therapy. An analysis of risk factors. Arch Intern Med 153:1557-1562.
2. Pharmacia ATenativ Brochure.
3. Antithrombin III Immuno Brochure.
4. Kybernin P Brochure.
5. Groner, A., Schmitt, K., and Schuler, E. 1999. Elimination of
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6. Thrombate – Summary of Basis for Approval, 1991.
7. Edmunds T, Van Patten SM, Pollock J, et al. 1998.
Transgenically produced human antithrombin: structural and functional comparison to human
plasma-derived antithrombin. Blood 91:4561.
8. Ziomek C, Kutzko J, Sherman L, et al. 2003. Viral and prion
safety of recombinant human antithrombin.
Ann Hematol 82(Suppl 1):S95.
9. Turk B, Brieditis I, Bock SC, Olson ST, Bjork I. 1997.
The oligosaccharide side chain of Asn-135 of a-antithrombin, absent in b-antithrombin, decreases
the heparin affinity of the inhibitor by affecting the heparin-induced conformational change.
Biochemistry 36:6682.
10. Swedenborg J. 1998. The mechanism of action of a- and b-isoforms
of antithrombin. Blood Coagul Fibrinolysis 9(Suppl3):S7.
11. Ersdal-Badju E, Lu A, Peng X, et al. 1995. Elimination of
glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by
expression of a beta-like variant in baculovirus-infected insect cells.
Biochem J 310:323.
12. Olson ST, Frances-Chmura AM, Swanson R, Bjork I, Zettlmeissl G. 1997.
Effect of individual carbohydrate chains of recombinant antithrombin on heparin affinity and on the
generation of glycoforms differing in heparin affinity.
Arch Biochem Biophys 341:212.
13. H. Biescas, M. Gensana, J. Fernandez, et al. 1998. Characterization
and viral safety validation study of a pasteurized therapeutic concentrate of antithrombin III obtained
through affinity chromatography. Haematologica 83:305.
14. Hellstern P, Mober U, Ekblad M, et al. 1995. In vitro characterization
of antithrombin III concentrates--a single-blind study. Haemostasis 25:193.
15. Barrowcliffe TW, Eggleton CA, Mahmoud M. 1983. Studies of the
heterogeneity of antithrombin III concentrates.
Br J Haematol 55:37.
16. Konkle BA, Bauer KA, Weinstein R, et al. 2003. Use of recombinant
human antithrombin in patients with congenital antithrombin deficiency undergoing surgical procedures. Transfusion 43:390.
17. Tait RC, Morfini M, Walker ID, et al. 2002. A Pharmacokinetic Model for
Dosing of rh AT by Continuous Infusion in patients with Hereditary AT Deficiency. Blood 100:Abst3970.
18. Tait RC, Konkle BA, Bauer KA, et al. 2003. Clinical application of recombinant human
antithrombin (AT) in patients with hereditary deficiency. Ann Hematol 82(Suppl 1):S84.
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