Chemistry: analytical and immunological testing – Clotting or clotting factor level tests
Reexamination Certificate
2001-03-22
2003-07-22
Low, Christopher S. F. (Department: 1653)
Chemistry: analytical and immunological testing
Clotting or clotting factor level tests
C424S001210, C424S009100, C424S009321, C424S009322, C424S450000, C424S460000, C530S381000
Reexamination Certificate
active
06596543
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of prothrombin time reagents for determining dysfunction in the coagulation system and more specifically to reagents made from native thromboplastin or purified or recombinant tissue factor and phospholipids from a natural or synthetic source.
BACKGROUND OF THE INVENTION
Tissue factor is a membrane-associated glycoprotein which functions by forming a complex with blood coagulation factors VII and VIIa. The complexing of these factors greatly enhances the proteolytic activity of factors VII and VIIa. Functional activity of tissue factor has an absolute dependence on the presence of phospholipids. Bach, Ronald R.
Initiation of Coagulation by Tissue Factor
. CRC Critical Reviews in Biochemistry 1988:23(4): pp. 339-368. The factor VII/VIIa/tissue factor complex activates a series of specific enzymes that comprise the extrinsic and common pathways of the coagulation cascade ultimately leading to the formation of thrombin, fibrin, platelet activation and finally clot formation.
Diagnostic tests such as the prothrombin time (PT) test, utilize this series of enzymatic events in vitro under controlled conditions to diagnose dysfunction in the blood coagulation system of patients. In the PT test, the time it takes for clot formation to occur is the prothrombin time or PT value.
There are many commercially available PT reagents. They contain membrane-bound proteins, for example tissue factor, incorporated into liposomes. Some are crude tissue factor preparations (thromboplastins) extracted from rabbit brain, rabbit brain/lung mixtures, human placenta or ox brain. Others are based on purified or recombinant tissue factor (human, rabbit etc) combined with phospholipids. The protein/phospholipid mixture or crude thromboplastin extract is generally also combined with calcium ion and buffers. The final reagents may include stabilizers such as glycine, dextrans, detergents and the like and salts such as sodium chloride.
Methods for incorporating, i.e., reconstituting, proteins into liposomes are known in the art. See Rigaud, J-L., et al., “Liposomes as Tools for the Reconstitution of Biological Systems,” p. 71-88, in Liposomes as Tools in Basic Research and Industry, ed. Philippot, J. R. and Schuber, F., CRC Press, Boca Raton, Fla. (1995). One method is disclosed in U.S. Ser. No. 09/459,137 filed Dec. 10, 1999, incorporated herein by reference in its entirety, which describes a method to incorporate membrane proteins into liposomes without the use of detergents. The method comprises providing the membrane protein in solution, then providing a solution of preformed liposomes; and incubating the mixture. Prior to the step of providing a solution of preformed liposomes, the liposomes are formed by combining a mixture of phospholipids with a solution of at least one type of unsaturated fatty acid. U.S. Ser. No. 09/459,137 discloses that it is preferred that the liposomes have a generally uniform size. In general, the smaller and larger liposomes, e.g., 50 nm and 400 nm, show a longer clotting time. In U.S. Ser. No. 09/459,137 it is disclosed that the liposomes may have a preferred size ranging from 75 to 150 nm, most preferably about 100 nm. U.S. Ser. No. 09/459,137 also discloses that addition of phospholipids and fatty acids into the tissue factor reagent modulates the clotting activity.
SUMMARY OF THE INVENTION
The present invention relates to methods to make a diagnostic reagent that includes a membrane-bound protein incorporated into a liposome and having additional empty liposomes (liposomes without membrane-bound protein incorporated therein) added to the solution.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of preparing diagnostic reagents that include liposomes as a constituent. The liposomes used in the present invention are selected and prepared to have a particular and/or known mean size. The present invention also includes diagnostic reagent compositions comprising liposomes selected as to have a particular mean size. The present method enables the preparation of such reagents, such as prothrombin time (PT) reagents and Activated partial thromboplastin times (APTT), having improved performance and improved manufacturability compared with currently available PT or APTT reagents. After a reagent is formulated and prepared it may lack certain characteristics such as the time it takes to clot certain types of samples (e.g. normal and abnormal samples or controls). In this method liposomes of a uniform size are added to current formulated reagents as an additional step in the manufacturing process after the membrane-bound protein has been incorporated into liposomes. If necessary, additional buffer and stabilizers may also be added after this step. The added liposomes are sometimes referred to herein as “empty liposomes” to distinguish them from liposomes that have the membrane-bound protein incorporated therein.
The liposomes used in the present invention may be synthetic or natural and include without limitation saturated or unsaturated mono or disubstituted fatty acids and combinations thereof. These phospholipids may include dioleoylphosphatidylcholine, dioleoylphosphatidylserine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid, palmitelaidoyloleoylphosphatidylcholine, palmitelaidoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylethanolamine, palmitelaidoyloleoylphosphatidylglycerol, palmitelaidoyloleoylphosphatidic acid, myristoleoyloleoylphosphatidylcholine, myristoleoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylethanoamine, myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidic acid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine, dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol, dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine, palmiticlinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylethanolamine, palmiticlinoleoylphosphatidylglycerol, palmiticlinoleoylphosphatidic acid These phospholipids may also be the monoacylated derivatives of phosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine (lysophosphatidylserine), phosphatidylethanolamine (lysophosphatidylethanolamine), phophatidylglycerol (lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidic acid). The monoacyl chain in these lysophosphatidyl derivatives may be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or myristoleoyl. Preferably, the mixture of phospholipids comprises dioleoylphosphatidylcholine (1,2 Dioleoyl-sn-glycero-3-phosphocholine) (“DOPC”) and dioleoylphosphatidylserine (“DOPS”) or 1-Palmitoyl-2-Oleoyl-sn-glucero-3-phosphoserine (“POPS”) in a ratio of from about 5 to about 1. In preferred methods, the DOPC and DOPS or POPS are in a ratio of from about 7 to about 3. In preferred methods the phospholipids are synthetic. Synthetic phospholipids are readily available commercially from various sources, such as AVANTI Polar Lipids (Albaster, Ala.); Sigma Chemical Company (St. Louis, Mo.).
In one embodiment of the present invention, the mean sized liposomes are added to current PT or APTT reagent compositions to adjust the clotting activity and sensitivity during the manufacturing process. The final reagent is active as demonstrated by showing clotting activity which is comparable to that of optimized current reagents.
Naturally occurring phospholipids used to prepare liposomes for use in a PT reagent containing recombinant or native TF include natural phosphatidyl serine (PS) suitably in the range 2.5-50 mole % (“%”), generally in the range from about 25 to 35% of total phospholipid with the most common at about 30% and natural phosphatidyl choline (PC) suitably in the range from 20-95%, generally in the range from about 65 to 75% of total phospholipid with the m
Doobay Hema
Johnson Kevin Bruce
Tejidor Liliana Maria
Wang Jianfang
Dade Behring Inc.
Low Christopher S. F.
Schnizer Holly
Tymeson Cynthia G.
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