Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-03-22
2004-05-25
Wax, Robert A. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S380000, C530S416000, C530S417000
Reexamination Certificate
active
06740736
ABSTRACT:
This invention is concerned generally with protein purification and specifically with the purification of fibrinogen from the milk of transgenic animals using cation exchange chromatography.
Fibrinogen, the main structural protein in the blood responsible for the formation of clots exists as a dimer of three polypeptide chains; the A&agr; (66.5 kD), B&bgr; (52 kD) and &ggr; (46.5 kD) are linked through 29 disulphide bonds. The addition of asparagine-linked carbohydrates to the B&bgr; and &ggr; chains results in a molecule with a molecular weight of 340 kD. Fibrinogen has a trinodal structure, a central nodule, termed the E domain, contains the amino-termini of all 6 chains including the fibrinopeptides (Fp) while the two distal nodules termed D domains contain the Carboxy-termini of the A&agr;, B&bgr; and &ggr; chains. Fibrinogen is proteolytically cleaved at the amino terminus of the A&agr; and B&bgr; chains releasing fibrinopeptides A and B (FpA & FpB) and converted to fibrin monomer by thrombin, a serine protease that is converted from its inactive form by Factor Xa. The resultant fibrin monomers non-covalently assemble into protofibrils by DE contacts on neighboring fibrin molecules. This imposes a half staggered overlap mode of building the fibrin polymer chain. Contacts are also established lengthwise between adjacent D domains (DD contacts) leading to lateral aggregation. Another serine protease, Factor XIII is proteolytically cleaved by thrombin in the presence of Ca
2+
into an activated form. This activated Factor XIII (Factor XIIIa) catalyses crosslinking of the polymerised fibrin by creating isopeptide bonds between lysine and glutamine side chains. The first glutamyl-lysyl bonds to form are on the C-terminal of the &ggr; chains producing D—D crosslinks. Subsequently, multiple crosslinks form between adjacent A&agr; chains, the process of crosslinking imparts on the clot both biological stability (resistance to fibrinolysis) and mechanical stability [Sienbenlist and Mosesson, Progressive Cross-Linking of Fibrin y chains Increases Resistance to Fibrinolysis,
Journal of Biological Chemistry,
269: 28414-2841, 1994].
The coagulation process can readily be engineered into a self sustained adhesive system by having the fibrinogen and Factor XIII as one component and thrombin and Ca
2+
as the second component which catalysis the polymerization process. These adhesion systems, know in the art as “Fibrin Sealents” or “Fibrin Tissue Adhesives” have found numerous application in surgical procedures and as delivery devices for a range of pharmaceutically active compounds [Sierra, Fibrin Sealent Adhesive Systems: A Review of Their Chemistry, Material Properties and Clinical Applications,
Journal of Biomaterials Applications,
7:309-352, 1993; Martinowitz and Spotnitz, Fibrin Tissue Adhesives,
Thrombosis and Haemostasis,
78:661-666, 1997; Radosevich et al., Fibrin Sealent:Scientific Rationale, Production Methods, Properties and Current Clinical Use,
Vox Sanguinis,
72:133-143, 1997].
It has been estimated that the annual US clinical need for fibrin sealents is greatly in excess of the 300 kg/year that can be harvested using the current cryoprecipitation methods used by plasma fractionaters. Alternative sources of fibrinogen, by far the major component in fibrin sealant, have therefore been explored with recombinant sources being favored [Butler et al., Current Progress in the Production of Recombinant Human Fibrinogen in the Milk of Transgenic animals,
Thrombosis and Haemostasis,
78: 537-542, 1997]. While cell culture systems have demonstrated the ability to produce small amounts (1-4 ug/ml) of fibrinogen, it has been shown that mammals are capable of producing transgenic human fibrinogen at levels of up to 5.0 g/L in their milk making this a commercially viable method for the production of human fibrinogen [Prunkard et al., High-level expression of recombinant human fibrinogen in the milk of transgenic mice,
Nature Biotechnology,
14:867-871, 1996; Cottingham et al., Human fibrinogen from the milk of transgenic sheep. In:
Tissue Sealants: Current Practice, Future Uses
. Cambridge Institute, Newton Upper Falls, Mass., Mar. 30-Apr. 2, 1996 (abstract)].
Differences have been identified between recombinant human fibrinogen and fibrinogen which has been purified from human plasma. Fibrinogen which has been purified from human plasma has two alternately spliced gamma chains (&ggr; and &ggr;′). In contrast, recombinant human fibrinogen only has the major form &ggr;. Further, the glycosylation of the beta and gamma chains (there is no N-linked glycosylation of the alpha chain) of recombinant human fibrinogen differs slightly from that on plasma derived fibrinogen, but is similar to the glycosylation found on other proteins expressed in the milk of transgenic animals. In addition, the Ser3 of the alpha chain of recombinant human fibrinogen is more highly phosphorylated than Ser3 of the alpha chain of plasma derived fibrinogen, although the difference in phosphorylation does not result in functional differences. Also, there are detectable differences in heterogeneity caused by C-terminal proteolysis of a number of highly protease-sensitive sites on the alpha chain. Differences of a similar magnitude are also observed between plasma-derived fibrinogen from different sources.
Another driving force for the development of totally recombinant fibrin-based sealents stems from the fact that commercially available adhesives originate from pooled plasma. As blood-derived products have been associated with the transmission of human immunodefieciency virus (HIV), hepatitis virus and other etiological agents, the acceptance and availability of such adhesives is limited. While the incorporation of viral removal and inactivation procedures has increased the safety of these products (for example, U.S. Pat. Nos. 4,960,757 and 5,116,950), plasma derived fibrinogen is still not without risk. The use of autologous plasma reduces the risk of disease transmission; however, autologous adhesives can only be used in elective surgery when the patient is able to donate the blood in advance.
While the main use of fibrinogen is thought to be for the preparation of adhesive or sealing agents, fibrinogen also has other applications in the field of medicine, for example as a coating for polymeric articles as disclosed in U.S. Pat. No. 5,272,074, and the concern for safety apply to any of these other uses in medicine.
Various methods for the purification of fibrinogen have been described where, in most cases, the starting material has been either plasma or more usually cryoprecipitate or Cohn Fraction 1, both of which are rich in fibrinogen. Schwartz et al., [U.S. Pat. Nos. 4,362,567; 4,377,572; 4,414,976] have disclosed a process for the manufacture of fibrinogen tissue adhesive using cryoprecipitation of plasma as the major purification step. Burnouf et al., (1990) [Biochemical and Physical Properties of a Solvent-Detergent-Treated Fibrin Glue,
Vox Sang
58:77-84] describe a process for the preparation of fibrinogen concentrate from 150 L of human plasma. After thawing anticoagulated plasma at 37° C., the plasma was subjected to a 10% ethanol precipitation. Following dissolution of the precipitated fibrinogen a Solvent-Detergent viral inactivation technique was used followed by a further two ethanol precipitation procedures to remove the Solvent-Detergent chemicals. The final material is quoted as being 93.5% pure with respect to protein content and is sold under the trade name Biocoll©.
Vila et al., (1985) [A Rapid Method for Isolation of Fibrinogen From Human Plasma by Precipitation with PEG 6000,
Thrombosis Research
39:651-656] describe a method whereby fibrinogen is precipitated from citrated plasma using an 8% solution of PEG 6000. Following dissolution the fibrinogen is then further precipitated using 2 mol/l acetic acid-acetate buffer pH 4.6. A final precipitation with ammonium sulphate is used to give a produc
Drinker Biddle & Reath LLP
PPL Therapeutics (Scotland) Limited
Wax Robert A.
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