CA2448188A1 - Method for manufacturing a tissue factor-based prothrombin time reagent - Google Patents
Method for manufacturing a tissue factor-based prothrombin time reagent Download PDFInfo
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- CA2448188A1 CA2448188A1 CA002448188A CA2448188A CA2448188A1 CA 2448188 A1 CA2448188 A1 CA 2448188A1 CA 002448188 A CA002448188 A CA 002448188A CA 2448188 A CA2448188 A CA 2448188A CA 2448188 A1 CA2448188 A1 CA 2448188A1
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- phospholipid
- mixture
- rtf
- detergent
- tissue factor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/7454—Tissue factor (tissue thromboplastin, Factor III)
Abstract
A method for manufacturing a tissue factor-based prothrombin-time reagent includes combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture. In this combining step, the quantity of TF is predetermined based upon its measured TF activity. Next, the detergent is removed from the TF/phospholipid mixture to produce an essentially detergent-free TF/phospholipid mixture that is useful as a tissue factor-based PT reagent. The method can include the use of hydrophobic resin that has been prescreened based on phospholipide recovery to accomplish the removal of the detergent.
Description
METHOn FOR i~YANUF'ACTURIi~TG A
BACKGROUND OF IhTVENTION
[8001] 1. Field ofthe lnvention~ ' ' ~ . The present ifivetition relates, in general, to the field of prathrombin time .. . ~ ~ reagents and, in particular, to a method for manufacturing PT
reagents. .. .
BACKGROUND OF IhTVENTION
[8001] 1. Field ofthe lnvention~ ' ' ~ . The present ifivetition relates, in general, to the field of prathrombin time .. . ~ ~ reagents and, in particular, to a method for manufacturing PT
reagents. .. .
[0002] 2. De~,scription of the Related Axt ' . ~ . . . . ' . ~ Blood coagulation tests may be performed for a variety of purposes;
including determination of the bleading susceptibility of patients undergoittg_surgery and ' ~ .
. monitoring of patients undergoing anticoagulant therapy foi prevention of blood clots, A number of coagulation tests are currently in use, one ofwhich.is the '.'prothmnibin time" (PTA test. The PT test relies upon activation of the~extrinsiG
coagulation . ' ~ . pathway by thrombopIsstin that has been added ~to a: blood sample undergoing the PT
test. Activation of the extrinsic coagulation pathway Leads to the production of .
thrombin, a proteolytic crizyme that catalyzes the conversion of fibrinogen to ~fibrin,.a..
catalysis that~is essential to the clotting process. ., ' y ~ ..
. . . [0003] ~ Thromboplastin, also knrnvn as tissue factor ('fF), is a membrane associated .~ ~ .
. - ~ ~ glycoprotein that forms a complex with fsctor'Va3a. The.factor V~
campleic initiates the.blood coagulation process. Once formed, the factor VIIafI'F
complex ' ~ ~ activates a series of specific enzymes that are involved in the extrinsic and~inhi~asie, . ' . . ~ pathways of the coagulation cascade, ultimately leading to the~formation ofthminbin,. ' fibrin, platelet activation, and finally clot formation. For a related, discussion, s~ " . .
Nemersoh, Yale, Tissue F~etor araat Herr~ostasis, Blood, 71, pp.l-S (198$). ' .
. . '. ~ [0004] Conventional PT tests utilize the above-described series of enzymatic events~in an irt vitro environment and, under controlled conditions to diagaosc'dysfuncti~ons of deficiencies in the blood coagulation system of patients. The time period it.takes for ~i clot formation to occur is referred to as the Prothrombin Time or PT value.
including determination of the bleading susceptibility of patients undergoittg_surgery and ' ~ .
. monitoring of patients undergoing anticoagulant therapy foi prevention of blood clots, A number of coagulation tests are currently in use, one ofwhich.is the '.'prothmnibin time" (PTA test. The PT test relies upon activation of the~extrinsiG
coagulation . ' ~ . pathway by thrombopIsstin that has been added ~to a: blood sample undergoing the PT
test. Activation of the extrinsic coagulation pathway Leads to the production of .
thrombin, a proteolytic crizyme that catalyzes the conversion of fibrinogen to ~fibrin,.a..
catalysis that~is essential to the clotting process. ., ' y ~ ..
. . . [0003] ~ Thromboplastin, also knrnvn as tissue factor ('fF), is a membrane associated .~ ~ .
. - ~ ~ glycoprotein that forms a complex with fsctor'Va3a. The.factor V~
campleic initiates the.blood coagulation process. Once formed, the factor VIIafI'F
complex ' ~ ~ activates a series of specific enzymes that are involved in the extrinsic and~inhi~asie, . ' . . ~ pathways of the coagulation cascade, ultimately leading to the~formation ofthminbin,. ' fibrin, platelet activation, and finally clot formation. For a related, discussion, s~ " . .
Nemersoh, Yale, Tissue F~etor araat Herr~ostasis, Blood, 71, pp.l-S (198$). ' .
. . '. ~ [0004] Conventional PT tests utilize the above-described series of enzymatic events~in an irt vitro environment and, under controlled conditions to diagaosc'dysfuncti~ons of deficiencies in the blood coagulation system of patients. The time period it.takes for ~i clot formation to occur is referred to as the Prothrombin Time or PT value.
[0003) Different types of thromboplastins can either enhance or dimipish the ability of a PT test to discriminate between blood samples having different protbrombin×
Thromboplastins with greater discrimination arc termed'~more sensitive". The liquid phase sensitivity of s thromboplastin preparation is graded by use,of the International Sensitivity Index (ISn. An ISI value can be obW fined by plotting, oxr s icigarithmic . ~ t'~~
. ' scale, the prothrombin time value obtained with a given thmmboplastirz lot versus the ' . a ,' ' . ' prothrombin time values obtained with a standardized reference,lot ofthromboplastin.
The 18I value of the given tlammboplastin lot is the slope of the resulting line multiplied by the 1SI of then standardized reference lot of thrvmboplastiri.
More sensitive thrombopl~stins have. lower ISI e~urribers,.around 1.0 and less sensitive ' thromboplastins have higher ISI numbers, typically around 2.0 to 3Ø . ' [0006] For use, in PT testing, a highly sensitive 1PT reagent with ari ISI of ~ ' ~ ' approximately'I .0 is considered ti~ost beneficial and suitable since, with ~
ISI:of 1.0, .
. : the calculation of an Intemational~Normaliaed Ratio (INR) is simplified and piecise. .. .
O~ae skilled in the art will recognize that use. of the. INR can compensate fog '. thromboplastin variation due to differences in sensitivity. INR is calculated usii~g~the ~ ~ v ~ . v following equation (see Hirsh et al., Oral Anticor~ulants:~ Mechanism ofActio~
' Cltnfcal E~''eclivenear, anti Optamal Therapeutic Range, Chest 2001 i 119:88 =~2I S):
. . ~g = (patient PT/mean normal P3~~ ~ ~ . ' where: . ' patient PT = the prothmmbin time of a patient's blood sample, and . ' .~
mean normal PT (or MNP'I~ = the mean prathrotztbin time of blood samples from at least twenty normal (reference) sample donors. . ' Conventional methods for manufacturing PT reagents generally use TF' from , .
natural or recombinant sources, natural or synthetic phospholipids, calcium and a l..
l.
.l .... .., l:
l .
. buyer composition. U.S. Patent No. 5,314,695, which is fully incorporated'herein by , ' reference; discloses a method for manufacturing PT reagents: The reagents.include , ~ . .. .
liposome compositions, in which natural or recombinant tissue ~etvr~is associated ,. . y~,i~ ~d inserted into, the liposomes' phospholipid bilayer. In the PT
reagent ' ' manufavturing methods described therein, a determination of the guantity of TF
~ . _ ' employed in manufacturing the PT reagent is determined entirely on a mass to volume .
basis: . . . y . y ' : .
. (0009) .U.S. Patent No. 5,625,036, which is fully incorporated by reference, discloses ' a PT reagent for use in PT testing and a method fos creating lipid vesicles containing, .. _ . . tissue factor. This PT reagent'utilizes recombinant human tissue factor, phaspholiplds . ~~ . . .
of a natural or synthetic.origin, i~ buffer composition and calcium ions.
St'sbilizers and salts rn,ay also be utilized in the PT reagent. ' ~ ' ._ X0008] ~ Use of dried thromboplastin in PT test devices is described in U.S.
Patent 5,418,141 and U. S: Patent Application 2002/0110922; both of which are fttlly incorporated herein by reference., U.S. Patent 5,415,141 descrrlbe~ the use .of . . , .. , . ~ ..
recombinatrt thromboplastins in dry reagent prothrombin time assays. The 1 . .
. . recomt~inant thmmboplastins are employed for 2he~pvirpose'of improving test . , precision. In addition, this patent compares recombinant thromboplastin be natural. ' ' v thromboplastins, which, when used in dry reagent PT assays, ara Iess sensitive arid .. ~ , , ' ~ . : ~ result in less precise ISI values. In U.S. Patent Application 20D2/0110922, d fluidic . ~ . ' . test device .is described that incorporates throniboplastiw into three measurement , , ' . ' areas, one of which is used for measuring the PT time of a blood sample.
The ether ' , . , two measurement areas are used as control areas, 'thereby increasing the reliability of PT times measured using the fluidle test device. . The contml ar8as contain , . .
. , components in addition to thrortiboplastin in order ~to partially or completaely overcome ~.
. ~ ~ the effect of an anticoagulant present in the blood sainpie. . : . . ; .
(0009 . With experience, it has become apparent that conventional methods for .
manufacturing PT reagents do not reproducibly yield a suitably sensitive PT
reagent. , Still needed in the field, therefore, is a method for rnanufacturing~a PT
re~get~t that y reproducibly yields a suitably sensitive PT reagent., In addition,'the.methad~sbould be .
v.. ..::. . . .
' . simple and provide for acceptance testing of the manufactured PT reagent.
SUIViMARY OF INYEhTTION
[00010] , The present invention provides a method for manufacturing a tissue factori based prothrombin time (PT) reagent that reproducibly yields a suitably sensitive ' tissue factor-based PT reagent. In addition, the method is simple and provides. for acceptance testing of the rnanufaetured tissue 'factor-based PT reagent.
. [OOOI 1l] ' In striving at the present invention, it was recognized that the reproducibility ~. . .
(e.g., lot-to-lot variation) and suitability (e.g., sensitivity) oftissue factor based PT
reagents is determined by a variety of previously unrecognized critical ~faetorS during . ; ~ ~ . manufacturing. These, critical factors include (~ the specific activity ofthe TF
erriployed during manufacturing; and, (i~ the phospholipid concentration both during manufacturing and in the final tissue-factor~based pT reagent. Since the activity of TP .
arid phosphoIipid concentration are both critical, factors, the ratio of active TF
'. (measured as units of activity) to phospholipid concentration employed during ..
' ~' rtxanufacturing is also of interest. Embodiments of methods according to~the present:.
invention, therefore, include steps such that~one or more ofthe critical facfors is v - , controlled. Such control provides a manufacturing method (process) that reproducibly . ~ y yields a suitable sensitive tissue factor-based P'1' reagent. , (00012] . A method for manufacturing a tissue~fa.ctor-based PT reagent according~to an .
exemplary embodiment of the present invention includes cornbiningvprvdetermined .~ . , quantities of tissue factor ('fly, phospholipid and detergent-containing buffer to create a TF/phospholipid mixture. . In this combining step, the quantity of TF is predetermined based upon its measured TF activity, Next, the detergent is removed from the TF/ptiospholipid mixture to produce an essentisllydetergent-free TF/phospholipid vesicle mixture. Such a detwgent-free TF/phospholipid vesicle v . .
mixture is'uscful as a tissue factor-basod PT reagent. . ' .. . . d BRIEF DESCRLI~IOI~ OF lIRAWINGS
. , . . .
A better understanding of the features and advantages of the present invemion .
will be obtained by reference to the following detailed deseription'thttt_sets_forth . . .' iilusu'ative eit~bodiments, in which the principles of the invention stet utilized, and the . ~ ' ~ accornPanY~b swings of which: , ', . . ' F1C3. 1 is a flow chart illustrating.a sequence of steps is s process aocortling to~ ' ' ., '. ~ one exemplary embodiment ~of the present invention;
FIG. 2 is a graph showinE the efI"eet of phospholipid eonoentration on Interriationsl-Sensitivity Index; and ~ . ' ~ : ' ;
FIG. 3 is a graph showing the effect of phospholipid concentration on .Mea» .
Normal Prothrombin ?ime: ~ ' .
~ DETAILED DESCRIPTION OF T1EIE 1NVF,.NTI~N .
I, . . . . . . ..
a [000131 FIG.1 is a flowi chart of a process 100 for manufacturing a tissue factor-based , ~ : ~ PT reagent in accordance with_an exemplary embodiment of the pint invention:
. Froeess .100 includes combining predetermined quantities of tissue factor (TIC,' ~ ' phospholipid and a detergent-containing buffer to date a TF/phosphnlipid rxitxture, $s set forth ~in step I 10. The Quantity of TP is pr~de2ermined in this step based upon .. its measured~TF activity. . ~ . , ' ~00014~ The tissue factor employed in processes according to the present invention, ' . ~ , , ~
including process 100, can be any suitable tissue factor known to one skilled izi the art. .
' ' .Suitable tissue factors~can include, for example, tissue factors from natural or~ ' ~' . ~ ~ . synthetic sources. Use of recombinant tissue factor (aTF) is particularly beneficial in ~ ~' ~ providing a tissue factor-based PT reagent that is highly sensitive~wheri employed~in, ' ~.~ ~ ' .
~ for example, a dry state. .
. (0p015] ~. Ttie TF activity of the predetermined guaatity of tissue factor esn be measured ' using any suitable TF activity assay known to'those skilled in the art and/or ~ ' ~ ' ' ..i specifically developed for use in conjunction with methods according to the.
present .
invention. Those skilled iri the art will recognize that such TF. ~tivity assays ~neastue TF' functional activity and not simply the quantity of TF. . ~ ~ ' ~ . . '.
. ~ [pp0~ 6] A particularly beneficial and simple TF aceivity assay that can be employed to . .
measure TF activity is described in co-pending U.S. Patent Application No: ~ ' (tentatively identified by Attorney's Docket No. MBl-iH~02~1049 and itaeorporated, ~ ~ .
. herein by reference as if fully set forth) entitled "Assay for Tissue Factor. and Factor v VIia" and filed on an even date. This TF activity assay utilizes a fluorogenic substrato .. ' .' ., . to measure the ability of TF to function as a cofactor to factor VIIa, in doing so, the .
. TF activity assay determines the capacity of factor VDa to cleave a fluorogenic .
y ' ~ substrate (e.g.; the fluorogenic substrate G-peptidylamino-1-naphthalenesulfonomides described iri U.S. Patent No. 5,399,d8f, which'is fully incorporated herein by . , ' reference) in the presence of tissue factor. .1n this assay, the ~bindlng of factor Vlla to 1~ enhances this capacity by approximately 100 fold. Comparison of a.subject TF
, . preparation,to a standard preparation of tissue factor (available frora, for example,. the , ~ . .~
World Health Organization Standatt~ enables an activity determination of the subject TF preparations. . Further details of the TF activity assay are disclosed ixi.'the ' , .. aforementioned U.S. Patent Application No. ~. '(tentatively identified by.
. , ... ~ ' Attorney's Docket.No. MBHB-02~1049).. ' . . ' [0001'7] ' In the circumstance that the.TF activity assay of U.S. Paterrt Application No. , . ' . (tentatively identified by Attorney's Docket No. MBHB-02-1049) is . employed to measure the activity of the TF, ~it has bin aseertaine~ that adding °TF
.. ~ such that the.,TF/phospholipid mixture has a'fF activity inane range of 9000 to 18000 .
Units per liter is~paitieularly benef cial in terms ofproviding a.reproducilile and ~ .
sensitive tissue factor-based PT reagent. In this circumstance, the TF
activityunits are . . ~ .defined.by assi,gnin$ a TF activity of 1 Unitliz~l to .WNO standard thromboplastin . reconstituted according to instructions supplied by the WHO. .One skilled in the art v, , ~ ' will recognize that the range of 9.000 to 18000 Units per liter has been'speeified.in relationship to the TF/phospholipid mixture and not the detergent-free . .
TF/phospholipid vesicle mixture.
6. . . . : .:.
z .. ' (80018] The detergent-containing buffer employed, in step 1.10 can tie any suitable detergentrcuntaining buffers known to those skilled,in the art. For example;
the detergent containing buffer can be a 100mM 3-((3-cholamidogropyl)- ~ ' ' dimethylammonioJ-1-propanesulfonate (CHAPS, buffer andlor CHAPS/>3ovine Gamma Globulin.(BGG) buffer. Tl~e phospholipids utilized in step 110 can be any suitable phospholipids or.mixture of phospholipids known io ane skilled in the art. .
For example, a phospholipid mixture containing phosphatidylcholine, ~pliosphaiidyl- . ..
glycerol, phosphatidylethanolarnine, and phosphatidylserine can b8 used. . ' (000X9] Combining step 11'0 can include, for example, adding the predetertilined ' . quantity of TF to a miarture of phospholipid and detergent-containing buffer. Such a ' , ;.
combining step provides for the simple and successful incorporation ofTF into the phospholipid and detergent-containing buffer mixture). In PT reagent manufacturing .
methods known in the art, the addition of TF~ to such a phospholipid and detergent.
. containing m~iatture is done entirely on a mass to volume basis (see, for example, U.S.. '. ..
Patent 5,314,695). Using this 'conventional approach, the mass quantity of TF~
added during manufacturing of a~PT reagent is known, but notfing is known about the , .
functional quality (i.e., activity) of'the added TF.
[00020] Next, the detergent is removed from the TF/phospholipid mixture to provide an essentially detergentrfiee TF/phospholipid vesicle mixture, as set forth~in step 120' ofFIG. 1. One skilled in. the art wil3'recognize that such a~detergent-free TF/phospholipid vesicle mixture can serve as a tissue factor-based'IrI°
reagent:
[0002I] The removal of the detergent can be accomplished using any suitable .technique known to those skilled in the art. Suitable techniques include, ~or example, adding a hydrophobic resin (e.g., XAD-16 hydrophobic resin) to the TF/phospholipid mixture, incubating .the 1'F/phospholipid mixture and the added resin .until the .
. 1T'F/phospholipid mixture is essentially free of detergent; and then removing said ~ .
hydrophobic resin from the ~'F/phospholipid mixture. ~ Other suitable techniques include gradual rernQVal of the detergent by dialysis or by tangential flow filtration, :. . j0002~] The essentially deterge»t-free TF phospholipid vesicle mixtut~e calf-include a ~ ' .
functionally insignificant amount pf detergent while still being considered i'esseritially .
detergent-free." The concentration of detergent that is ~consider~ed~ fux~~vy , . . , ~ .
insignificant can be determined by one of skill in the art throw rbutine experimentation. By way of example, 0.2 mM or less of CIiAPs dete=rgent was .
determined to be functionally insignificant in txrtain embodiments of.~e pn. =
invention. ~ ' ~ .
j000231 As explained above, embodiments of the present invention cap itjvolve the . ~ . _ ' v.
addition of a predetermined quantity of TF to s phospholipid arid dete~gent~a~ining. .
buffer mixture. In such embodir»ents, the phosphoiipid earn be mixed With a non-'. . denaturing zwitberionic detergent to create s phospholipid Gild datetgeat ~cott~ining ~ .
mixture that includes muted micelles ofphospholipid and detergent. ,An epy~y ~~ ~.
. ~ ' non-denaturing zwittcrionic detergent is 3-[(3-cholamidopropyl)dimetllyhammo~nio]-.1-. . _ .. , ~ propanesulfonate (CHAPS). . , . . . .
COp0~4) , Any suitable hydrophobic resin can be employed to remove detergent in ' .
methods according to the present invention; including but not limits .tp, p~b~l~. .
XAD-16, which is commercially available from Sigma Chemicals, St. Louis, ~ ~ ' , Missouri. Those= skilled in the art will recognize that the removal of detergent'in processes aecarding to the present invention can, for example, muse the phospholipids to form unilamellar vesicles; into which °TF becomes incorporated by . ' ~ ~ virtue of the TF's membrane binding domai;r. The resultant,essentially dete~~gent=free ~.~ : '.
TF/phospholipid vesicle mixture is a unifoim mixture of unilamellar vesicles containing TF, some of which is oriented with an active portion exposed.on the , .
surface of the unilamellar vesicles: The effeptivenass of such a tissue factor-bases P'f ~ .
reagent to serve as a cofactor for factor ~IIa is believed to be dictated bar the number , .
of functional TF molecules avaiisble at the surface of the vesicles, 'The addition ~of TF.: . ~ .
. based on a conventional mass-to-volume basis merely assures 'the quantity of such TF, but not the functional quality thereof. Hy basing the predeterrniried~
quantity of TF on activity instead, the amount of functional TF available at the surface of the vesicles ~. ~ . ' .
can be more precisely coxitrolled and, in effect, "standardized" from lot-to-Iot'of the' tissue factor-based PT reagent.
. , ~ [00025) In addition to.the amount of functional TF, it has been recognized that the : . ' ~ ' ' qu.atttity of phospholipid employed during the manufacturing of ~a tissue factotbased , _ ~ .
' ~ ' . PT reagent also influences its suitability and reproducibility. I:
has~been~further ,~ .~ . ., . " ~ ~ ~ recognized that the amount of phospholipid present is the.essentialiy detergent-free.
TFlphospliolipid vesicle mixture is linked to phospholipid loss during removal~ofthe . ,~ . ~ '- detergent ' , [00026] To control t'he amount of phospholipid present irr the tissue factor-based PT . .
reagent, an acceptance.test for phospholipid,coneentration can be ertipIoyed in. . . .
processes~according to the present invention.. 1~or example, the processes can;eW plod steps to (t) measure the total phospholipid concentration of the,essentially.
detergent- ' :
free TFlphospholipid vesicle mixture; and (ii) determine acceptability of the.
essentially detergent-free TF/phospholipid vesicle mixture for use ass tissue factor-based PT reagent based on the measurad total phospholipid concentration. ~ .
.. ' ]0002'7] The total phospholipid. concentration can be measured using, for example, assays for inorganic phosphate that are well, know in the.art and for which procedt~
' ai~e .readily available (see Chen et al., .Mfcrodetetmtnar3on of Pho~phorur,. Anal ytical '.~
. . Chemistry; 28(~i 1), 1756-178 (1950. Since, phospholipid is the.onty potential ' .
significant source of inorganic phosphate in processes according to the present: y _ . ~ ~ . , . invention, the molar concentration of phosphate is.directly proportional. to the ~molat concentration of phospholipid in a sample. A particularly beneficial pho'spholipid . ' .
concentrafian (measured as inorganic phosphate) in the essentially detergent-free TF/phosphoiipid vesicle mixture is in the range.of from 2.5 to 5.0 ttiIVI. An even more ~.
beneficial phospholipid concentration is in,the range of y3.0 to ~.S:QmM; with a, 3.8 mM phospholipid concent'ratian being excepiiahally lienefcial. , , ~ ., .. = ~ .. ~ ~ 9 , .
[0002$ 3 In another exemplary embodiment of processes according~to the presec~t invention, resin employed in the removal of detergent is prescreened ~prior~to use'in , , the process. Such prescreening is based on a percent recovery ofphc~spholipid (see .
EJ~AMP1..E. 7 below) and can, for example, employ the inorganic assays described above. Prescreening according to embodiments ofthe present invention calf .
beneficially control loss ofphospholipid during a detergent removal step and serve po insure that the essentially detergent-free TF/phosphnlipid vesicle. mixture colltaitts a ~ ~' ' controlled concentration of phospholipid. - . . . .' . . ;.
..
EXAI~IPLfi 1: Prs screer~irrR ofI-iydrophobic ChronoatoEranhi_e~tesin foa' Use ~.
Manufacturinsz a Tissue Factor-Based_PT_Reeszezrt ., ' : , [pp029~ - The following study demonstrates an exemplary technique by which . hydrophobic resin, lots were successfully prescre~.-ned, based on perecs~nt recovery of . ' phospholipid, for use in a tissue factorbased PT' reagent nlarstttheturing process ~~qpg to ~e present invention. In order to pre-screemthe hydrophobic resin lots, 4.95 mM,of a solubilized phospholipid mixtureycontaining phosphatidylciioiine, ~ ' ~ :.
phosphatidyl-glycerol, phosphatidylethanolamine.and phosphatidylset~,e~
available:: ' .~ . .
. tom Avanti Polai Lipids, ADabaster, Alabama) and 30 grams ofAmberlite~XAD-i6 ~ ~~ '.
' - hydrophobic resin (commercially available from Sigma Chemical Compairy, St, Louis, MO) were combined in 104mL of 20 mM Tris and 150 mM NaCI (TBS) btiifer containing 0.8% glycine, 150 mM trehalose, 100 mM CHAPS and 0.05'Yo sodiutti ' azide. The pH of the buffer,was 9.4. , ~ . ~ . .
[00030) . The phospholipid and hydrophobic resin werevmixed for apprQ~imately:W
hour at room temperature. The mixture'.s stipentataat was co~leeted gna assayed foi .
phospholipid (measured as inorganic phosphate using .a oonventiotxai inorganic 1. .
phosphate assay)... The percent (~o) recovery of phospholipid was~then caJetUated~fro~zi - the inorganic_phosphate assay results. Data from the testing of six.lots ~of hydrophobic resin are reported in Table 1. Tissue factor-based PT reagent lots prepared with resin loi number 2 consistentDy failed fatal release testing. Based on the data ofTabDe 1 and .. . , an understanding of the measurement accuracy associated with percent (°.''o) reco~rer,~, '. 10 only those lots ofhydropho6ic resin yielding a 75% to 95°/a recovery ofphospholipid' : .
were deemed accegt~ble for use in manufacturinf the tissue faetor~based P.T
reagent, , .
. I,ot of Final inorganic . .
Resin ~osphate _ % Recovery~
' .
, J1 . .
Concentration m .: , . 1 4.6 93 ' ~ . : . . .
. . . . :, ~ .. ' . 2 3.3 ~ 67 . ~ . , . ~ . .
3 4.4 ~ ~ 88 . ' . ' a 4.4 ss ~ . . .
. . . . , S 4.1 ~ 82 . .
.
.
.
.
. .
y w . . ' . 6 3.9 79 .
' EXEMPLE 2: Exem 1 Process fo Manufacturin a'i'"'ssu Factor- seed R ~ nt ~. ' . ' . , '. . ~~ ~j00031] Lyophilized phospholipids (1.5 grams) obtained $om Avanti,Polaz Lip~idS, .. . ~ .~
. ' ~ . . (Alabaster, AL)~
at a mole ratio of 67;1d:10:7.(pho'sphatidyl, eholine: phosp3iatidyl ., . . .~
' glycerol: phosphatidyl ethanolamine:
phosphatidyl serine) were solubiHzed in 100 W L : ;
' . : ~ of'I'BS buffer containing 0.$% glycine, l50 mM trehaloise, 100 tM CHAPS slid 0.05% sodium azide. At alt steps in this exensplary process, the reagent solutioa was mixed while being maintained at 30 - 37 C. Based upon the assayed inor~anie~
. " phosphate concentration ofthis soluljilized'phospholipiil, 2.25 mmol,of solubilized .
phospholipid was removed for further processing. , . ~
~ . , '. [0003aj The volume~eontaining 2.25 mmol of phospholipid that was removed for . ' further processing was adjusted to 100 tnl by the addition of TBS buffer. containing.
. '.
0.8% glycine, I50 mM trehalose, 100 mM CHAPS and 0'.05% sodium azide. Fifty .
milliliters~(SOmI) of a bovine gamma globulin-solution (O.laY
in TBS containing '20mM CHAPS, 0.8% giycine, 150 mM trehalose and 0:05Y
sodium ~azide) was then .
'. . . ~ added to the solubilized phospholipid mixture. Next, rfiF tii~ TBS containing l~OmIVi , w , .
CLAPS) and additional buffer ('fBS buffer containing 0.89 glyrine; '150 ruM
.-..~,, .', ' , .. Z~
Table, l : Summary of Phospholipid Recovery.Data for Resin Lots trehalose ant':0,059'o sodium azide) was added to the phospholipid mixture to yield _ , .
_. . ' S00 ml of?FJphospholipid rnixture~ wish a r'TF.concentratian of 13,500 unitslL.
(00033] ARer the rTF/phospholipid mixture was mixed for one hour, 150 gains of pre-,' . ~ screened (per the exemplary technique described above) and pre-washed XAD-16 .
. , hydrophobic. resin was added, Aifiez mixing 3.5 hours, ~tlae r'TFlphospholipid. mixture' ~ . ~ .
was filtered to remove the hydrophobic resin and stored at 2-8°C.
The;addition of ~ ~ ~ ~ ' . ~ . resin, iilcubation and removal ofthe resin produced an essentially detergent-free .~~ ~ . , . : ' ' ~ rTF/phospholipid vesicle mixture (i.e., tissue factor-based PT reagent). ~
~ ' ' .
' j00034) The~tissue factor-based PT reagent was then assayed to determine its final , ' . ' phospholipid concentration (measured as inorganic phosphate).. The tissue.
factor.
based PT reagent was also coated and dried into a test strip format, The ISI
acid ' ' ' ' ~ . ~ MNPT of each multiple tissue~factor-based ~PT reagent' lots were then deteimiried by testing test strips with at last 100 whole blood capillary samples; at least 20 of which . . , ' : . were from noamal patients and at lcas~t 80 of which were ~frorn,patients on oral . anticoagulant therapy. The ISI value was obtained by plotting, on a logarithmic scale, '.
the prothrombin time obtained with a lot irt question versus the prothrombin, time .
values~obtained with a standardized lot of thromboplastiri feared on a reference instrument. 'The ISI value is the slope ofthe resulting line multiplied by the ISI oftlie, .
reference thmmboplastin. The M,NPT is the mean prothrornbin tune of at~ least twenty .. , ' normal donors. ' ~~ . . ~ . .
[00035) Table 2 shows the ISI and MNPT for tissue factor-based PT reagent Iota ~ ~ .
., ; r . ~ . manufactured using methods that encompassed a relatively widz range of added~itn'F ' .
and that utilized a resin that was nvt prescreened. An .acceptable range for 1SI and . ' . . . ~ .
y ' MIfPZ' is 1.0 -1.3 and 7.0 to 9.0 seconds, respectively. ?'he data in 'Table .2 indicate ' ' ~t certain combinations of TF and phospholipid cause the lots to fail ~tbe ISI
at~d : ;
MNPT release criteria. For. example, when both a low TF. and a low phospholipid 7 _ ' .,~ w concentration are used to make 1?T reagent, the Tesulting ISI is above the acceptable , .~ . range. ' ~ .:
= Table 2: Summary of ISI and MNPT for nI'F-based PT Rea,Bent Lots ;
Manufactured Using Resin that lhad not been Prescreened.
RTF- , .. . ' based ~ ConcentrationInorganic . ' ~
. 'T (I,1/Lj in Phosphate . . .
p the ReagentTF/phospholipidConcentrationISI (seconds)pass/Fail, Lot mixture . (mlVJ7 , Number , ; , I 9 250 1.93 1.37' '8.88F
.
2 9 250 2.56 ' . 9.72 F ' ~ . ' .
l .f~
3 9 250 2.78 I.2'.i8.52 p . . . . ' 4 9 218 3.02 1,113'8.17 P, ' . 5 ~ 9,248 3.80 1.238.36 P ' . .
6 9 000 5 .20 Z 8..03 ' P : . ' .10 7 11 500 2.42 1.258.62 ' P~ ~ .
8 ~ ~ 13 250 3.00 1.23.7.95 p. ~. .
9 13,856 3.40 1.088.12 P
10 13 500 3.60 1.117.66 P . .
I 1 I 3 s o0 3:90 1.1 . 7.s4 'p . ~
. , . 12, 7 000 I .92 I.32.7.92 ~ F
I3 1 ~ 244 3.60 1.097.39 P ~ ~~
.
14 ' 18,000 3.80 1.12 7.42 . . p ' . , 100036y Table 3 summarises the ISI and MNPT
results fox tissue factor-based PT ' ~ ~ .
reagent lots manufactured according to an exert~plary pYOCess (described above) and . . ' ,. adjusted to provide various t'I~, and phospholipid concentrations:
The results in. Table v.
3 indicate that tissue'factor-based PT reagent lots manufactured.accordit~~
to the. , .~ , present invention include consistept (1.e., reproducible) amounts of rTF and phospholipid and lave more reproducible and suitable ISI
and MNpT.vaJues than lots ~ . ' manufactured using conventional methods.
None ofthe lots ofTabl~
3 failed the'ISI
' . ' .
arid 31~VPT criteria:
, . ' .. .
...
' ' :..
~- . . . . - , -.:..
.
based'rZ'F ConceniratiotiInorganic ' . , ' .: - '. ~ pr ' NIL) in ' Phosphate MNPT ' . .. ' ' the ISI pa r ' . ~gen TF/PhospholipidConcentration (seconds)~ _ , .
y .
. , L,ot mixture (~ . ' .: . .
. . _.
' . . . . .
b 15 9 056 2.5 1.18 ?.93 ;p . .
. 16 . . 9 056 2.5 ' 1.15 ' .7.80 p . , _ , .
.
. ' . ;
'. . ~ 17 ~ 9 056 2.S 1.15 ' 8.07 ~ ~ .
' ~ p I S '13 500 . 3.9 i.11 ?.39 p ' . ; "
~ ~
19 . 13 500 3.2 1.16 7:42 ' p ~ ~ , . ~ .
' 20 13 500 3.9 1.09 7.44 p ' ' , :' ~
~ ' ' .
, ' . . 2a is sa0 3.9 1.11. , 7.ss ' ~ . ~ ' .
.
p .
' ~ 22 i3 500 3.6 1.14 7.59 .p ' ~ v13 500 3.6 '1.11 . ~ ?.7l' ~ ~ . ' ..
, ' P .
..' ' ' 24 I3 500 '3.2 1.19 7.72 . ~ ~ ' ~' p .~
. . . . a5 18 000 3.s 1.10 ' 7.48: P
~
. . 26 13 s0o . 3.2 1.15 ' 7:64 . ~. . . . ' : ~ p . "
27 13 500 3.7 '' 1.14 ?.93 p _ y ~ ;
' ~
28 ' 13 500 . 3.9 1.11 7.58. ~ ' .
' . p . 29 I3 300 3.4 1-.13 . 7.65 p ~ ~ : ~ ~ .' ' ' [0p03?3 Based.
on the data of Table 3, suitable r'I~ activity concentrations are in the range of9;000 to 13,500, while suitable phospholipid concentrations are in the range .~~ . ' ' .
of 2.5 to 3.9 mM. Various ratios of added fIF
activity and phosphate conce~ation . : ~ ' .
that racy be of interest to those skilled in the art can be routinely derived ~6rcim the. data .
of.Tables 2 and 3. Horwover, is believed that employing predeterminod concentration ~
~ . ' . . . ranges for added TF activity and phospholipid is sufficient in terms.of reproducibly ; ~ ' . .
.
.
.
: .
.. .
yielding a suitably sensitive TF-based PT reagent. ~
[00038 She effect of the phospholipid concentration (measured ~as inorgat:ic phosphate) on ISI and MNPT'at s constant conce~tiat'ton of iTF are ~~
yFIC3s:.2. .
. .
and 3, respectively.
FIG. 2 dernonstrntes that when the inorganic phosghate _ . .
concentration is in the range of approximately 2.5 and 3.9 mM, the ISI is within the ~ ~ .
' ' ' desired range of .1.0 to 1.3. As shown -in F1Q. 3, the same inorganic pho~phatc ~ .
~ ~ v .' .
Table 3: Summary of ISI and MN'fT for rT:E-based pr Reagent Lots '.
Manufactured Using Exemplary Methods According to the Present Invention ...... _.._.__ _._.._.~_~...*...~,~ ~~.»3~,,_., m..._......_~_._..~__ ._.....~. ~_...*.,.~.~.~_ ..,._
Thromboplastins with greater discrimination arc termed'~more sensitive". The liquid phase sensitivity of s thromboplastin preparation is graded by use,of the International Sensitivity Index (ISn. An ISI value can be obW fined by plotting, oxr s icigarithmic . ~ t'~~
. ' scale, the prothrombin time value obtained with a given thmmboplastirz lot versus the ' . a ,' ' . ' prothrombin time values obtained with a standardized reference,lot ofthromboplastin.
The 18I value of the given tlammboplastin lot is the slope of the resulting line multiplied by the 1SI of then standardized reference lot of thrvmboplastiri.
More sensitive thrombopl~stins have. lower ISI e~urribers,.around 1.0 and less sensitive ' thromboplastins have higher ISI numbers, typically around 2.0 to 3Ø . ' [0006] For use, in PT testing, a highly sensitive 1PT reagent with ari ISI of ~ ' ~ ' approximately'I .0 is considered ti~ost beneficial and suitable since, with ~
ISI:of 1.0, .
. : the calculation of an Intemational~Normaliaed Ratio (INR) is simplified and piecise. .. .
O~ae skilled in the art will recognize that use. of the. INR can compensate fog '. thromboplastin variation due to differences in sensitivity. INR is calculated usii~g~the ~ ~ v ~ . v following equation (see Hirsh et al., Oral Anticor~ulants:~ Mechanism ofActio~
' Cltnfcal E~''eclivenear, anti Optamal Therapeutic Range, Chest 2001 i 119:88 =~2I S):
. . ~g = (patient PT/mean normal P3~~ ~ ~ . ' where: . ' patient PT = the prothmmbin time of a patient's blood sample, and . ' .~
mean normal PT (or MNP'I~ = the mean prathrotztbin time of blood samples from at least twenty normal (reference) sample donors. . ' Conventional methods for manufacturing PT reagents generally use TF' from , .
natural or recombinant sources, natural or synthetic phospholipids, calcium and a l..
l.
.l .... .., l:
l .
. buyer composition. U.S. Patent No. 5,314,695, which is fully incorporated'herein by , ' reference; discloses a method for manufacturing PT reagents: The reagents.include , ~ . .. .
liposome compositions, in which natural or recombinant tissue ~etvr~is associated ,. . y~,i~ ~d inserted into, the liposomes' phospholipid bilayer. In the PT
reagent ' ' manufavturing methods described therein, a determination of the guantity of TF
~ . _ ' employed in manufacturing the PT reagent is determined entirely on a mass to volume .
basis: . . . y . y ' : .
. (0009) .U.S. Patent No. 5,625,036, which is fully incorporated by reference, discloses ' a PT reagent for use in PT testing and a method fos creating lipid vesicles containing, .. _ . . tissue factor. This PT reagent'utilizes recombinant human tissue factor, phaspholiplds . ~~ . . .
of a natural or synthetic.origin, i~ buffer composition and calcium ions.
St'sbilizers and salts rn,ay also be utilized in the PT reagent. ' ~ ' ._ X0008] ~ Use of dried thromboplastin in PT test devices is described in U.S.
Patent 5,418,141 and U. S: Patent Application 2002/0110922; both of which are fttlly incorporated herein by reference., U.S. Patent 5,415,141 descrrlbe~ the use .of . . , .. , . ~ ..
recombinatrt thromboplastins in dry reagent prothrombin time assays. The 1 . .
. . recomt~inant thmmboplastins are employed for 2he~pvirpose'of improving test . , precision. In addition, this patent compares recombinant thromboplastin be natural. ' ' v thromboplastins, which, when used in dry reagent PT assays, ara Iess sensitive arid .. ~ , , ' ~ . : ~ result in less precise ISI values. In U.S. Patent Application 20D2/0110922, d fluidic . ~ . ' . test device .is described that incorporates throniboplastiw into three measurement , , ' . ' areas, one of which is used for measuring the PT time of a blood sample.
The ether ' , . , two measurement areas are used as control areas, 'thereby increasing the reliability of PT times measured using the fluidle test device. . The contml ar8as contain , . .
. , components in addition to thrortiboplastin in order ~to partially or completaely overcome ~.
. ~ ~ the effect of an anticoagulant present in the blood sainpie. . : . . ; .
(0009 . With experience, it has become apparent that conventional methods for .
manufacturing PT reagents do not reproducibly yield a suitably sensitive PT
reagent. , Still needed in the field, therefore, is a method for rnanufacturing~a PT
re~get~t that y reproducibly yields a suitably sensitive PT reagent., In addition,'the.methad~sbould be .
v.. ..::. . . .
' . simple and provide for acceptance testing of the manufactured PT reagent.
SUIViMARY OF INYEhTTION
[00010] , The present invention provides a method for manufacturing a tissue factori based prothrombin time (PT) reagent that reproducibly yields a suitably sensitive ' tissue factor-based PT reagent. In addition, the method is simple and provides. for acceptance testing of the rnanufaetured tissue 'factor-based PT reagent.
. [OOOI 1l] ' In striving at the present invention, it was recognized that the reproducibility ~. . .
(e.g., lot-to-lot variation) and suitability (e.g., sensitivity) oftissue factor based PT
reagents is determined by a variety of previously unrecognized critical ~faetorS during . ; ~ ~ . manufacturing. These, critical factors include (~ the specific activity ofthe TF
erriployed during manufacturing; and, (i~ the phospholipid concentration both during manufacturing and in the final tissue-factor~based pT reagent. Since the activity of TP .
arid phosphoIipid concentration are both critical, factors, the ratio of active TF
'. (measured as units of activity) to phospholipid concentration employed during ..
' ~' rtxanufacturing is also of interest. Embodiments of methods according to~the present:.
invention, therefore, include steps such that~one or more ofthe critical facfors is v - , controlled. Such control provides a manufacturing method (process) that reproducibly . ~ y yields a suitable sensitive tissue factor-based P'1' reagent. , (00012] . A method for manufacturing a tissue~fa.ctor-based PT reagent according~to an .
exemplary embodiment of the present invention includes cornbiningvprvdetermined .~ . , quantities of tissue factor ('fly, phospholipid and detergent-containing buffer to create a TF/phospholipid mixture. . In this combining step, the quantity of TF is predetermined based upon its measured TF activity, Next, the detergent is removed from the TF/ptiospholipid mixture to produce an essentisllydetergent-free TF/phospholipid vesicle mixture. Such a detwgent-free TF/phospholipid vesicle v . .
mixture is'uscful as a tissue factor-basod PT reagent. . ' .. . . d BRIEF DESCRLI~IOI~ OF lIRAWINGS
. , . . .
A better understanding of the features and advantages of the present invemion .
will be obtained by reference to the following detailed deseription'thttt_sets_forth . . .' iilusu'ative eit~bodiments, in which the principles of the invention stet utilized, and the . ~ ' ~ accornPanY~b swings of which: , ', . . ' F1C3. 1 is a flow chart illustrating.a sequence of steps is s process aocortling to~ ' ' ., '. ~ one exemplary embodiment ~of the present invention;
FIG. 2 is a graph showinE the efI"eet of phospholipid eonoentration on Interriationsl-Sensitivity Index; and ~ . ' ~ : ' ;
FIG. 3 is a graph showing the effect of phospholipid concentration on .Mea» .
Normal Prothrombin ?ime: ~ ' .
~ DETAILED DESCRIPTION OF T1EIE 1NVF,.NTI~N .
I, . . . . . . ..
a [000131 FIG.1 is a flowi chart of a process 100 for manufacturing a tissue factor-based , ~ : ~ PT reagent in accordance with_an exemplary embodiment of the pint invention:
. Froeess .100 includes combining predetermined quantities of tissue factor (TIC,' ~ ' phospholipid and a detergent-containing buffer to date a TF/phosphnlipid rxitxture, $s set forth ~in step I 10. The Quantity of TP is pr~de2ermined in this step based upon .. its measured~TF activity. . ~ . , ' ~00014~ The tissue factor employed in processes according to the present invention, ' . ~ , , ~
including process 100, can be any suitable tissue factor known to one skilled izi the art. .
' ' .Suitable tissue factors~can include, for example, tissue factors from natural or~ ' ~' . ~ ~ . synthetic sources. Use of recombinant tissue factor (aTF) is particularly beneficial in ~ ~' ~ providing a tissue factor-based PT reagent that is highly sensitive~wheri employed~in, ' ~.~ ~ ' .
~ for example, a dry state. .
. (0p015] ~. Ttie TF activity of the predetermined guaatity of tissue factor esn be measured ' using any suitable TF activity assay known to'those skilled in the art and/or ~ ' ~ ' ' ..i specifically developed for use in conjunction with methods according to the.
present .
invention. Those skilled iri the art will recognize that such TF. ~tivity assays ~neastue TF' functional activity and not simply the quantity of TF. . ~ ~ ' ~ . . '.
. ~ [pp0~ 6] A particularly beneficial and simple TF aceivity assay that can be employed to . .
measure TF activity is described in co-pending U.S. Patent Application No: ~ ' (tentatively identified by Attorney's Docket No. MBl-iH~02~1049 and itaeorporated, ~ ~ .
. herein by reference as if fully set forth) entitled "Assay for Tissue Factor. and Factor v VIia" and filed on an even date. This TF activity assay utilizes a fluorogenic substrato .. ' .' ., . to measure the ability of TF to function as a cofactor to factor VIIa, in doing so, the .
. TF activity assay determines the capacity of factor VDa to cleave a fluorogenic .
y ' ~ substrate (e.g.; the fluorogenic substrate G-peptidylamino-1-naphthalenesulfonomides described iri U.S. Patent No. 5,399,d8f, which'is fully incorporated herein by . , ' reference) in the presence of tissue factor. .1n this assay, the ~bindlng of factor Vlla to 1~ enhances this capacity by approximately 100 fold. Comparison of a.subject TF
, . preparation,to a standard preparation of tissue factor (available frora, for example,. the , ~ . .~
World Health Organization Standatt~ enables an activity determination of the subject TF preparations. . Further details of the TF activity assay are disclosed ixi.'the ' , .. aforementioned U.S. Patent Application No. ~. '(tentatively identified by.
. , ... ~ ' Attorney's Docket.No. MBHB-02~1049).. ' . . ' [0001'7] ' In the circumstance that the.TF activity assay of U.S. Paterrt Application No. , . ' . (tentatively identified by Attorney's Docket No. MBHB-02-1049) is . employed to measure the activity of the TF, ~it has bin aseertaine~ that adding °TF
.. ~ such that the.,TF/phospholipid mixture has a'fF activity inane range of 9000 to 18000 .
Units per liter is~paitieularly benef cial in terms ofproviding a.reproducilile and ~ .
sensitive tissue factor-based PT reagent. In this circumstance, the TF
activityunits are . . ~ .defined.by assi,gnin$ a TF activity of 1 Unitliz~l to .WNO standard thromboplastin . reconstituted according to instructions supplied by the WHO. .One skilled in the art v, , ~ ' will recognize that the range of 9.000 to 18000 Units per liter has been'speeified.in relationship to the TF/phospholipid mixture and not the detergent-free . .
TF/phospholipid vesicle mixture.
6. . . . : .:.
z .. ' (80018] The detergent-containing buffer employed, in step 1.10 can tie any suitable detergentrcuntaining buffers known to those skilled,in the art. For example;
the detergent containing buffer can be a 100mM 3-((3-cholamidogropyl)- ~ ' ' dimethylammonioJ-1-propanesulfonate (CHAPS, buffer andlor CHAPS/>3ovine Gamma Globulin.(BGG) buffer. Tl~e phospholipids utilized in step 110 can be any suitable phospholipids or.mixture of phospholipids known io ane skilled in the art. .
For example, a phospholipid mixture containing phosphatidylcholine, ~pliosphaiidyl- . ..
glycerol, phosphatidylethanolarnine, and phosphatidylserine can b8 used. . ' (000X9] Combining step 11'0 can include, for example, adding the predetertilined ' . quantity of TF to a miarture of phospholipid and detergent-containing buffer. Such a ' , ;.
combining step provides for the simple and successful incorporation ofTF into the phospholipid and detergent-containing buffer mixture). In PT reagent manufacturing .
methods known in the art, the addition of TF~ to such a phospholipid and detergent.
. containing m~iatture is done entirely on a mass to volume basis (see, for example, U.S.. '. ..
Patent 5,314,695). Using this 'conventional approach, the mass quantity of TF~
added during manufacturing of a~PT reagent is known, but notfing is known about the , .
functional quality (i.e., activity) of'the added TF.
[00020] Next, the detergent is removed from the TF/phospholipid mixture to provide an essentially detergentrfiee TF/phospholipid vesicle mixture, as set forth~in step 120' ofFIG. 1. One skilled in. the art wil3'recognize that such a~detergent-free TF/phospholipid vesicle mixture can serve as a tissue factor-based'IrI°
reagent:
[0002I] The removal of the detergent can be accomplished using any suitable .technique known to those skilled in the art. Suitable techniques include, ~or example, adding a hydrophobic resin (e.g., XAD-16 hydrophobic resin) to the TF/phospholipid mixture, incubating .the 1'F/phospholipid mixture and the added resin .until the .
. 1T'F/phospholipid mixture is essentially free of detergent; and then removing said ~ .
hydrophobic resin from the ~'F/phospholipid mixture. ~ Other suitable techniques include gradual rernQVal of the detergent by dialysis or by tangential flow filtration, :. . j0002~] The essentially deterge»t-free TF phospholipid vesicle mixtut~e calf-include a ~ ' .
functionally insignificant amount pf detergent while still being considered i'esseritially .
detergent-free." The concentration of detergent that is ~consider~ed~ fux~~vy , . . , ~ .
insignificant can be determined by one of skill in the art throw rbutine experimentation. By way of example, 0.2 mM or less of CIiAPs dete=rgent was .
determined to be functionally insignificant in txrtain embodiments of.~e pn. =
invention. ~ ' ~ .
j000231 As explained above, embodiments of the present invention cap itjvolve the . ~ . _ ' v.
addition of a predetermined quantity of TF to s phospholipid arid dete~gent~a~ining. .
buffer mixture. In such embodir»ents, the phosphoiipid earn be mixed With a non-'. . denaturing zwitberionic detergent to create s phospholipid Gild datetgeat ~cott~ining ~ .
mixture that includes muted micelles ofphospholipid and detergent. ,An epy~y ~~ ~.
. ~ ' non-denaturing zwittcrionic detergent is 3-[(3-cholamidopropyl)dimetllyhammo~nio]-.1-. . _ .. , ~ propanesulfonate (CHAPS). . , . . . .
COp0~4) , Any suitable hydrophobic resin can be employed to remove detergent in ' .
methods according to the present invention; including but not limits .tp, p~b~l~. .
XAD-16, which is commercially available from Sigma Chemicals, St. Louis, ~ ~ ' , Missouri. Those= skilled in the art will recognize that the removal of detergent'in processes aecarding to the present invention can, for example, muse the phospholipids to form unilamellar vesicles; into which °TF becomes incorporated by . ' ~ ~ virtue of the TF's membrane binding domai;r. The resultant,essentially dete~~gent=free ~.~ : '.
TF/phospholipid vesicle mixture is a unifoim mixture of unilamellar vesicles containing TF, some of which is oriented with an active portion exposed.on the , .
surface of the unilamellar vesicles: The effeptivenass of such a tissue factor-bases P'f ~ .
reagent to serve as a cofactor for factor ~IIa is believed to be dictated bar the number , .
of functional TF molecules avaiisble at the surface of the vesicles, 'The addition ~of TF.: . ~ .
. based on a conventional mass-to-volume basis merely assures 'the quantity of such TF, but not the functional quality thereof. Hy basing the predeterrniried~
quantity of TF on activity instead, the amount of functional TF available at the surface of the vesicles ~. ~ . ' .
can be more precisely coxitrolled and, in effect, "standardized" from lot-to-Iot'of the' tissue factor-based PT reagent.
. , ~ [00025) In addition to.the amount of functional TF, it has been recognized that the : . ' ~ ' ' qu.atttity of phospholipid employed during the manufacturing of ~a tissue factotbased , _ ~ .
' ~ ' . PT reagent also influences its suitability and reproducibility. I:
has~been~further ,~ .~ . ., . " ~ ~ ~ recognized that the amount of phospholipid present is the.essentialiy detergent-free.
TFlphospliolipid vesicle mixture is linked to phospholipid loss during removal~ofthe . ,~ . ~ '- detergent ' , [00026] To control t'he amount of phospholipid present irr the tissue factor-based PT . .
reagent, an acceptance.test for phospholipid,coneentration can be ertipIoyed in. . . .
processes~according to the present invention.. 1~or example, the processes can;eW plod steps to (t) measure the total phospholipid concentration of the,essentially.
detergent- ' :
free TFlphospholipid vesicle mixture; and (ii) determine acceptability of the.
essentially detergent-free TF/phospholipid vesicle mixture for use ass tissue factor-based PT reagent based on the measurad total phospholipid concentration. ~ .
.. ' ]0002'7] The total phospholipid. concentration can be measured using, for example, assays for inorganic phosphate that are well, know in the.art and for which procedt~
' ai~e .readily available (see Chen et al., .Mfcrodetetmtnar3on of Pho~phorur,. Anal ytical '.~
. . Chemistry; 28(~i 1), 1756-178 (1950. Since, phospholipid is the.onty potential ' .
significant source of inorganic phosphate in processes according to the present: y _ . ~ ~ . , . invention, the molar concentration of phosphate is.directly proportional. to the ~molat concentration of phospholipid in a sample. A particularly beneficial pho'spholipid . ' .
concentrafian (measured as inorganic phosphate) in the essentially detergent-free TF/phosphoiipid vesicle mixture is in the range.of from 2.5 to 5.0 ttiIVI. An even more ~.
beneficial phospholipid concentration is in,the range of y3.0 to ~.S:QmM; with a, 3.8 mM phospholipid concent'ratian being excepiiahally lienefcial. , , ~ ., .. = ~ .. ~ ~ 9 , .
[0002$ 3 In another exemplary embodiment of processes according~to the presec~t invention, resin employed in the removal of detergent is prescreened ~prior~to use'in , , the process. Such prescreening is based on a percent recovery ofphc~spholipid (see .
EJ~AMP1..E. 7 below) and can, for example, employ the inorganic assays described above. Prescreening according to embodiments ofthe present invention calf .
beneficially control loss ofphospholipid during a detergent removal step and serve po insure that the essentially detergent-free TF/phosphnlipid vesicle. mixture colltaitts a ~ ~' ' controlled concentration of phospholipid. - . . . .' . . ;.
..
EXAI~IPLfi 1: Prs screer~irrR ofI-iydrophobic ChronoatoEranhi_e~tesin foa' Use ~.
Manufacturinsz a Tissue Factor-Based_PT_Reeszezrt ., ' : , [pp029~ - The following study demonstrates an exemplary technique by which . hydrophobic resin, lots were successfully prescre~.-ned, based on perecs~nt recovery of . ' phospholipid, for use in a tissue factorbased PT' reagent nlarstttheturing process ~~qpg to ~e present invention. In order to pre-screemthe hydrophobic resin lots, 4.95 mM,of a solubilized phospholipid mixtureycontaining phosphatidylciioiine, ~ ' ~ :.
phosphatidyl-glycerol, phosphatidylethanolamine.and phosphatidylset~,e~
available:: ' .~ . .
. tom Avanti Polai Lipids, ADabaster, Alabama) and 30 grams ofAmberlite~XAD-i6 ~ ~~ '.
' - hydrophobic resin (commercially available from Sigma Chemical Compairy, St, Louis, MO) were combined in 104mL of 20 mM Tris and 150 mM NaCI (TBS) btiifer containing 0.8% glycine, 150 mM trehalose, 100 mM CHAPS and 0.05'Yo sodiutti ' azide. The pH of the buffer,was 9.4. , ~ . ~ . .
[00030) . The phospholipid and hydrophobic resin werevmixed for apprQ~imately:W
hour at room temperature. The mixture'.s stipentataat was co~leeted gna assayed foi .
phospholipid (measured as inorganic phosphate using .a oonventiotxai inorganic 1. .
phosphate assay)... The percent (~o) recovery of phospholipid was~then caJetUated~fro~zi - the inorganic_phosphate assay results. Data from the testing of six.lots ~of hydrophobic resin are reported in Table 1. Tissue factor-based PT reagent lots prepared with resin loi number 2 consistentDy failed fatal release testing. Based on the data ofTabDe 1 and .. . , an understanding of the measurement accuracy associated with percent (°.''o) reco~rer,~, '. 10 only those lots ofhydropho6ic resin yielding a 75% to 95°/a recovery ofphospholipid' : .
were deemed accegt~ble for use in manufacturinf the tissue faetor~based P.T
reagent, , .
. I,ot of Final inorganic . .
Resin ~osphate _ % Recovery~
' .
, J1 . .
Concentration m .: , . 1 4.6 93 ' ~ . : . . .
. . . . :, ~ .. ' . 2 3.3 ~ 67 . ~ . , . ~ . .
3 4.4 ~ ~ 88 . ' . ' a 4.4 ss ~ . . .
. . . . , S 4.1 ~ 82 . .
.
.
.
.
. .
y w . . ' . 6 3.9 79 .
' EXEMPLE 2: Exem 1 Process fo Manufacturin a'i'"'ssu Factor- seed R ~ nt ~. ' . ' . , '. . ~~ ~j00031] Lyophilized phospholipids (1.5 grams) obtained $om Avanti,Polaz Lip~idS, .. . ~ .~
. ' ~ . . (Alabaster, AL)~
at a mole ratio of 67;1d:10:7.(pho'sphatidyl, eholine: phosp3iatidyl ., . . .~
' glycerol: phosphatidyl ethanolamine:
phosphatidyl serine) were solubiHzed in 100 W L : ;
' . : ~ of'I'BS buffer containing 0.$% glycine, l50 mM trehaloise, 100 tM CHAPS slid 0.05% sodium azide. At alt steps in this exensplary process, the reagent solutioa was mixed while being maintained at 30 - 37 C. Based upon the assayed inor~anie~
. " phosphate concentration ofthis soluljilized'phospholipiil, 2.25 mmol,of solubilized .
phospholipid was removed for further processing. , . ~
~ . , '. [0003aj The volume~eontaining 2.25 mmol of phospholipid that was removed for . ' further processing was adjusted to 100 tnl by the addition of TBS buffer. containing.
. '.
0.8% glycine, I50 mM trehalose, 100 mM CHAPS and 0'.05% sodium azide. Fifty .
milliliters~(SOmI) of a bovine gamma globulin-solution (O.laY
in TBS containing '20mM CHAPS, 0.8% giycine, 150 mM trehalose and 0:05Y
sodium ~azide) was then .
'. . . ~ added to the solubilized phospholipid mixture. Next, rfiF tii~ TBS containing l~OmIVi , w , .
CLAPS) and additional buffer ('fBS buffer containing 0.89 glyrine; '150 ruM
.-..~,, .', ' , .. Z~
Table, l : Summary of Phospholipid Recovery.Data for Resin Lots trehalose ant':0,059'o sodium azide) was added to the phospholipid mixture to yield _ , .
_. . ' S00 ml of?FJphospholipid rnixture~ wish a r'TF.concentratian of 13,500 unitslL.
(00033] ARer the rTF/phospholipid mixture was mixed for one hour, 150 gains of pre-,' . ~ screened (per the exemplary technique described above) and pre-washed XAD-16 .
. , hydrophobic. resin was added, Aifiez mixing 3.5 hours, ~tlae r'TFlphospholipid. mixture' ~ . ~ .
was filtered to remove the hydrophobic resin and stored at 2-8°C.
The;addition of ~ ~ ~ ~ ' . ~ . resin, iilcubation and removal ofthe resin produced an essentially detergent-free .~~ ~ . , . : ' ' ~ rTF/phospholipid vesicle mixture (i.e., tissue factor-based PT reagent). ~
~ ' ' .
' j00034) The~tissue factor-based PT reagent was then assayed to determine its final , ' . ' phospholipid concentration (measured as inorganic phosphate).. The tissue.
factor.
based PT reagent was also coated and dried into a test strip format, The ISI
acid ' ' ' ' ~ . ~ MNPT of each multiple tissue~factor-based ~PT reagent' lots were then deteimiried by testing test strips with at last 100 whole blood capillary samples; at least 20 of which . . , ' : . were from noamal patients and at lcas~t 80 of which were ~frorn,patients on oral . anticoagulant therapy. The ISI value was obtained by plotting, on a logarithmic scale, '.
the prothrombin time obtained with a lot irt question versus the prothrombin, time .
values~obtained with a standardized lot of thromboplastiri feared on a reference instrument. 'The ISI value is the slope ofthe resulting line multiplied by the ISI oftlie, .
reference thmmboplastin. The M,NPT is the mean prothrornbin tune of at~ least twenty .. , ' normal donors. ' ~~ . . ~ . .
[00035) Table 2 shows the ISI and MNPT for tissue factor-based PT reagent Iota ~ ~ .
., ; r . ~ . manufactured using methods that encompassed a relatively widz range of added~itn'F ' .
and that utilized a resin that was nvt prescreened. An .acceptable range for 1SI and . ' . . . ~ .
y ' MIfPZ' is 1.0 -1.3 and 7.0 to 9.0 seconds, respectively. ?'he data in 'Table .2 indicate ' ' ~t certain combinations of TF and phospholipid cause the lots to fail ~tbe ISI
at~d : ;
MNPT release criteria. For. example, when both a low TF. and a low phospholipid 7 _ ' .,~ w concentration are used to make 1?T reagent, the Tesulting ISI is above the acceptable , .~ . range. ' ~ .:
= Table 2: Summary of ISI and MNPT for nI'F-based PT Rea,Bent Lots ;
Manufactured Using Resin that lhad not been Prescreened.
RTF- , .. . ' based ~ ConcentrationInorganic . ' ~
. 'T (I,1/Lj in Phosphate . . .
p the ReagentTF/phospholipidConcentrationISI (seconds)pass/Fail, Lot mixture . (mlVJ7 , Number , ; , I 9 250 1.93 1.37' '8.88F
.
2 9 250 2.56 ' . 9.72 F ' ~ . ' .
l .f~
3 9 250 2.78 I.2'.i8.52 p . . . . ' 4 9 218 3.02 1,113'8.17 P, ' . 5 ~ 9,248 3.80 1.238.36 P ' . .
6 9 000 5 .20 Z 8..03 ' P : . ' .10 7 11 500 2.42 1.258.62 ' P~ ~ .
8 ~ ~ 13 250 3.00 1.23.7.95 p. ~. .
9 13,856 3.40 1.088.12 P
10 13 500 3.60 1.117.66 P . .
I 1 I 3 s o0 3:90 1.1 . 7.s4 'p . ~
. , . 12, 7 000 I .92 I.32.7.92 ~ F
I3 1 ~ 244 3.60 1.097.39 P ~ ~~
.
14 ' 18,000 3.80 1.12 7.42 . . p ' . , 100036y Table 3 summarises the ISI and MNPT
results fox tissue factor-based PT ' ~ ~ .
reagent lots manufactured according to an exert~plary pYOCess (described above) and . . ' ,. adjusted to provide various t'I~, and phospholipid concentrations:
The results in. Table v.
3 indicate that tissue'factor-based PT reagent lots manufactured.accordit~~
to the. , .~ , present invention include consistept (1.e., reproducible) amounts of rTF and phospholipid and lave more reproducible and suitable ISI
and MNpT.vaJues than lots ~ . ' manufactured using conventional methods.
None ofthe lots ofTabl~
3 failed the'ISI
' . ' .
arid 31~VPT criteria:
, . ' .. .
...
' ' :..
~- . . . . - , -.:..
.
based'rZ'F ConceniratiotiInorganic ' . , ' .: - '. ~ pr ' NIL) in ' Phosphate MNPT ' . .. ' ' the ISI pa r ' . ~gen TF/PhospholipidConcentration (seconds)~ _ , .
y .
. , L,ot mixture (~ . ' .: . .
. . _.
' . . . . .
b 15 9 056 2.5 1.18 ?.93 ;p . .
. 16 . . 9 056 2.5 ' 1.15 ' .7.80 p . , _ , .
.
. ' . ;
'. . ~ 17 ~ 9 056 2.S 1.15 ' 8.07 ~ ~ .
' ~ p I S '13 500 . 3.9 i.11 ?.39 p ' . ; "
~ ~
19 . 13 500 3.2 1.16 7:42 ' p ~ ~ , . ~ .
' 20 13 500 3.9 1.09 7.44 p ' ' , :' ~
~ ' ' .
, ' . . 2a is sa0 3.9 1.11. , 7.ss ' ~ . ~ ' .
.
p .
' ~ 22 i3 500 3.6 1.14 7.59 .p ' ~ v13 500 3.6 '1.11 . ~ ?.7l' ~ ~ . ' ..
, ' P .
..' ' ' 24 I3 500 '3.2 1.19 7.72 . ~ ~ ' ~' p .~
. . . . a5 18 000 3.s 1.10 ' 7.48: P
~
. . 26 13 s0o . 3.2 1.15 ' 7:64 . ~. . . . ' : ~ p . "
27 13 500 3.7 '' 1.14 ?.93 p _ y ~ ;
' ~
28 ' 13 500 . 3.9 1.11 7.58. ~ ' .
' . p . 29 I3 300 3.4 1-.13 . 7.65 p ~ ~ : ~ ~ .' ' ' [0p03?3 Based.
on the data of Table 3, suitable r'I~ activity concentrations are in the range of9;000 to 13,500, while suitable phospholipid concentrations are in the range .~~ . ' ' .
of 2.5 to 3.9 mM. Various ratios of added fIF
activity and phosphate conce~ation . : ~ ' .
that racy be of interest to those skilled in the art can be routinely derived ~6rcim the. data .
of.Tables 2 and 3. Horwover, is believed that employing predeterminod concentration ~
~ . ' . . . ranges for added TF activity and phospholipid is sufficient in terms.of reproducibly ; ~ ' . .
.
.
.
: .
.. .
yielding a suitably sensitive TF-based PT reagent. ~
[00038 She effect of the phospholipid concentration (measured ~as inorgat:ic phosphate) on ISI and MNPT'at s constant conce~tiat'ton of iTF are ~~
yFIC3s:.2. .
. .
and 3, respectively.
FIG. 2 dernonstrntes that when the inorganic phosghate _ . .
concentration is in the range of approximately 2.5 and 3.9 mM, the ISI is within the ~ ~ .
' ' ' desired range of .1.0 to 1.3. As shown -in F1Q. 3, the same inorganic pho~phatc ~ .
~ ~ v .' .
Table 3: Summary of ISI and MN'fT for rT:E-based pr Reagent Lots '.
Manufactured Using Exemplary Methods According to the Present Invention ...... _.._.__ _._.._.~_~...*...~,~ ~~.»3~,,_., m..._......_~_._..~__ ._.....~. ~_...*.,.~.~.~_ ..,._
Claims (16)
1. A method for manufacturing a tissue factor-based prothrombin time (PT) reagent, the method comprising:
combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture, wherein the predetermined quantity of TF is based on a measured TF activity thereof; and removing the detergent from the TF/phospholipid mixture to create an essentially detergent-free TF/phospholipid vesicle mixture, thereby creating a tissue factor-based PT reagent.
combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture, wherein the predetermined quantity of TF is based on a measured TF activity thereof; and removing the detergent from the TF/phospholipid mixture to create an essentially detergent-free TF/phospholipid vesicle mixture, thereby creating a tissue factor-based PT reagent.
2. The method of claim 1, wherein the combining step includes adding the predetermined quantity of TF to a mixture of phospholipid and detergent-containing buffer.
3. The method of claim 1, wherein the combining step includes adding a predetermined quantity of recombinant tissue factor (rTF).
4. The method of claim 1, wherein the removing step is accomplished using a technique that includes:
adding a hydrophobic resin to the TF/phospholipid mixture;
incubating the TF/phospholipid mixture and the added resin until the TF/phospholipid mixture is essentially free of detergent; and removing said hydrophobic resin from the TF/phospholipid mixture.
adding a hydrophobic resin to the TF/phospholipid mixture;
incubating the TF/phospholipid mixture and the added resin until the TF/phospholipid mixture is essentially free of detergent; and removing said hydrophobic resin from the TF/phospholipid mixture.
5. The method of claim 4, wherein the removing step is accomplished using a technique that further includes the step of prescreening the hydrophobic resin prior to the adding step.
6. The method of claim 5, wherein the prescreening includes prescreening the hydrophobic resin based,on solubilized phospholipid recovery.
7. The method of claim 5, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery in the range of 75%
to 95%.
to 95%.
8. The method of claim 1 further comprising the steps of:
measuring the total phospholipid concentration of the essentially detergent-free TF/phospholipid vesicle mixture; and determining acceptability of the essentially detergent-free TF/phospholipid vesicle mixture for use as a tissue factor-based PT reagent based on the measured total.
phospholipid concentration.
measuring the total phospholipid concentration of the essentially detergent-free TF/phospholipid vesicle mixture; and determining acceptability of the essentially detergent-free TF/phospholipid vesicle mixture for use as a tissue factor-based PT reagent based on the measured total.
phospholipid concentration.
9. The method of claim 8, wherein the measuring step measures the phospholipid concentration in terms of inorganic phosphate concentration and the determining step includes accepting for use tissue factor-based PT reagent with a measured total phospholipid concentration in the range of 2.5 to 5.0 mM in terms of inorganic phosphate concentration.
10. The method of claim 1, wherein the TF activity is measured by a fluorogenic TF activity assay.
11.The method of claim 10, wherein the fluorogenic TF activity assay utilizes a 6-peptidylamino-1-naphthalenesulfonamide fluorogenic substrate.
12.The method of claim 11, wherein the combining step combines a predetermined quantity of TF such that a TF activity of the TF/phospholipid mixture is in the range of 9,000 units to 18,000 units per liter.
13. A method for manufacturing a recombinant tissue factor-based prothrombin time (PT) reagent, the method comprising:
combining predetermined quantities of recombinant tissue factor (rTF), phospholipid and detergent-containing buffer to create a rTF/phospholipid mixture, by adding the predetermined quantity of rTF to a mixture of phospholipid and detergent-containing buffer, wherein:
the predetermined quantity of rTF is based on a measured rTF activity thereof the rTF activity having been measured using a fluorogenic-based rTF
assay employing a 6-peptidylamino-1-naphthalene sulforlamide fluorogenic substrate;
adding a prescreened hydrophobic resin to the rTF/phospholipid mixture;
incubating the rTF/phospholipid mixture and the added prescreened resin until the rTF/phospholipid mixture is essentially free of detergent; and removing said hydrophobic resin from the rTF/phospholipid mixture, thereby providing an essentially detergent-free rTF/phospholipid vesicle mixture;
measuring the total phospholipid concentration of the essentially detergent-free rTF/phospholipid vesicle mixture; and determining acceptability of the essentially detergent-free rTF/phospholipid mixture for use as a recombinant tissue factor-based PT reagent based on the measured total phospholipid concentration.
combining predetermined quantities of recombinant tissue factor (rTF), phospholipid and detergent-containing buffer to create a rTF/phospholipid mixture, by adding the predetermined quantity of rTF to a mixture of phospholipid and detergent-containing buffer, wherein:
the predetermined quantity of rTF is based on a measured rTF activity thereof the rTF activity having been measured using a fluorogenic-based rTF
assay employing a 6-peptidylamino-1-naphthalene sulforlamide fluorogenic substrate;
adding a prescreened hydrophobic resin to the rTF/phospholipid mixture;
incubating the rTF/phospholipid mixture and the added prescreened resin until the rTF/phospholipid mixture is essentially free of detergent; and removing said hydrophobic resin from the rTF/phospholipid mixture, thereby providing an essentially detergent-free rTF/phospholipid vesicle mixture;
measuring the total phospholipid concentration of the essentially detergent-free rTF/phospholipid vesicle mixture; and determining acceptability of the essentially detergent-free rTF/phospholipid mixture for use as a recombinant tissue factor-based PT reagent based on the measured total phospholipid concentration.
14. The method of claim 13, wherein the combining step combines a predetermined quantity of rTF such that the rTF activity of the rTF/phospholipid mixture is in the range of 9,000 units to 18,000 units per liter.
15. The method of claim 13, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery.
16. The method of claim 14, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery in the range of 75% to 95%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10/288,249 | 2002-11-05 | ||
| US10/288,249 US7049087B2 (en) | 2002-11-05 | 2002-11-05 | Method for manufacturing a tissue factor-based prothrombin time reagent |
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| CA002448188A Abandoned CA2448188A1 (en) | 2002-11-05 | 2003-11-05 | Method for manufacturing a tissue factor-based prothrombin time reagent |
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| Country | Link |
|---|---|
| US (1) | US7049087B2 (en) |
| EP (1) | EP1418435A1 (en) |
| JP (1) | JP2004157122A (en) |
| KR (1) | KR20040040384A (en) |
| CN (1) | CN1523358A (en) |
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| DE19749259A1 (en) | 1997-11-07 | 1999-05-12 | Dade Behring Marburg Gmbh | Process for the production of functional recombinant tissue factor |
| DE19811016A1 (en) * | 1998-03-13 | 1999-09-16 | Dade Behring Marburg Gmbh | Ready-to-use prothrombin time reagent based on recombinant tissue factor |
| US6521182B1 (en) | 1998-07-20 | 2003-02-18 | Lifescan, Inc. | Fluidic device for medical diagnostics |
| US6183979B1 (en) | 1999-03-24 | 2001-02-06 | International Technidyne Corporation | Preparation of dried synthetic prothrombin time reagents |
-
2002
- 2002-11-05 US US10/288,249 patent/US7049087B2/en not_active Expired - Lifetime
-
2003
- 2003-10-30 IL IL15869203A patent/IL158692A0/en unknown
- 2003-11-03 SG SG200306499A patent/SG129263A1/en unknown
- 2003-11-04 TW TW092130764A patent/TW200502552A/en unknown
- 2003-11-04 EP EP03256966A patent/EP1418435A1/en not_active Withdrawn
- 2003-11-04 JP JP2003374727A patent/JP2004157122A/en active Pending
- 2003-11-05 CN CNA2003101204633A patent/CN1523358A/en active Pending
- 2003-11-05 KR KR1020030078000A patent/KR20040040384A/en not_active Application Discontinuation
- 2003-11-05 CA CA002448188A patent/CA2448188A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JP2004157122A (en) | 2004-06-03 |
| CN1523358A (en) | 2004-08-25 |
| IL158692A0 (en) | 2004-05-12 |
| EP1418435A1 (en) | 2004-05-12 |
| TW200502552A (en) | 2005-01-16 |
| US20040086953A1 (en) | 2004-05-06 |
| KR20040040384A (en) | 2004-05-12 |
| SG129263A1 (en) | 2007-02-26 |
| US7049087B2 (en) | 2006-05-23 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |