CN102197142B - The quality control test method containing FXIII sample - Google Patents
The quality control test method containing FXIII sample Download PDFInfo
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- CN102197142B CN102197142B CN200980142104.3A CN200980142104A CN102197142B CN 102197142 B CN102197142 B CN 102197142B CN 200980142104 A CN200980142104 A CN 200980142104A CN 102197142 B CN102197142 B CN 102197142B
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- 238000003908 quality control method Methods 0.000 title claims abstract description 12
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 46
- 239000011780 sodium chloride Substances 0.000 claims description 25
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 22
- 238000005259 measurement Methods 0.000 claims description 22
- NKCXQMYPWXSLIZ-PSRDDEIFSA-N (2S)-2-[[(2S)-1-[(2S)-5-amino-2-[[2-[[(2S)-6-amino-2-[[2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-3-hydroxybutanoyl]amino]propanoyl]amino]-4-oxobutanoyl]amino]-3-m Chemical compound O=C([C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](N)[C@@H](C)O)[C@@H](C)O)C(C)C)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O NKCXQMYPWXSLIZ-PSRDDEIFSA-N 0.000 claims description 21
- 108090000190 Thrombin Proteins 0.000 claims description 20
- 229960004072 thrombin Drugs 0.000 claims description 20
- 230000000694 effects Effects 0.000 claims description 18
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical group OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 14
- 239000004094 surface-active agent Substances 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 238000010521 absorption reaction Methods 0.000 claims description 7
- 238000007421 fluorometric assay Methods 0.000 claims description 4
- 101700024603 ANNU Proteins 0.000 claims description 3
- 101700034322 TGAS Proteins 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 3
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- 239000010695 polyglycol Substances 0.000 claims description 3
- 101700012968 tgl Proteins 0.000 claims description 3
- 238000000151 deposition Methods 0.000 claims 1
- 238000005571 anion exchange chromatography Methods 0.000 abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 230000004913 activation Effects 0.000 description 38
- 239000003795 chemical substances by application Substances 0.000 description 33
- 238000004587 chromatography analysis Methods 0.000 description 15
- 239000012149 elution buffer Substances 0.000 description 13
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 6
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- 238000005349 anion exchange Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 4
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000023555 blood coagulation Effects 0.000 description 4
- 239000003114 blood coagulation factor Substances 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000002797 proteolythic Effects 0.000 description 4
- 210000004369 Blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L cacl2 Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- NTNZTEQNFHNYBC-UHFFFAOYSA-N ethyl 2-aminoacetate Chemical compound CCOC(=O)CN NTNZTEQNFHNYBC-UHFFFAOYSA-N 0.000 description 3
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- 229940088598 Enzyme Drugs 0.000 description 2
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- 150000001412 amines Chemical class 0.000 description 2
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium monoxide Chemical compound [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
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- 238000010828 elution Methods 0.000 description 2
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- 238000005497 microtitration Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
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- AXCZMVOFGPJBDE-UHFFFAOYSA-L Calcium hydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 229950003499 FIBRIN Drugs 0.000 description 1
- 108010000196 Factor XIIIa Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
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- 239000012190 activator Substances 0.000 description 1
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- 239000000292 calcium oxide Substances 0.000 description 1
- -1 calcium.Cause This Chemical class 0.000 description 1
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Abstract
The present invention relates to the quality control test method containing FXIII (FXIII) sample, this includes detecting pre-activate FXIII(FXIIIa in described sampleO) existence and/or measure pre-activate FXIII(FXIIIa in described sampleO) the step of concentration, and relate to measure the quality control reagents box of the quality containing FXIII (FXIII) sample.Preferably, anion-exchange chromatography post and fluorogenic substrate Abz NE(Cad Dnp are used) EQVSPLTLLK OH.
Description
Technical field
The present invention relates to the quality control test method containing FXIII (FXIII) sample, this includes detecting described sample
Middle pre-activate FXIII(FXIIIaO) existence and/or measure pre-activate FXIII(FXIIIa in described sampleO) the step of concentration
And relate to measure the quality control reagents box of quality containing FXIII (FXIII) sample suddenly,.
Background technology
Blood coagulation is by the complicated process formed that interacts of various blood constitutents (or factor), and described complexity is mutual
Effect produces fibrin clot.Usually, participation has been referred to as the albumen that the blood constitutent of solidification " cascade " is enzymatic inactivation
Matter (preferment or proenzyme), it is converted to proteolytic enzyme by the effect of activator (himself being activation coagulation factors).?
The coagulation factors going through this type of conversion is commonly referred to as " active factors ", and orders by adding letter " a " to coagulation factors title
Name (such as factor XIIIa).
FXIII preponderate with the FXIII(FXIII-A that recombinates2), plasma F XIII(FXIII-A2B2) or intracellular FXIII
(FXIII-A2) zymogen forms find.In blood plasma, in Blood Coagulation Process, activated by thrombin approach is preponderated.Blood coagulation
Enzyme cuts the activated peptide of 37 amino acid residues from the N-terminal part of each FXIII-A subunit, and at Ca2+In the presence of
Generate activity form FXIIIa*.On the contrary, intracellular FXIII is unapproachable for thrombin, and has observed that non-protein
Hydrolytic activation occurs under given conditions, to obtain activity FXIII kind (FXIIIaO), it contains in each FXIII-A subunit
There is all 1-731 aminoacid, and therefore can not be by cut (Polgar et al. (1990) Biochem. of activated thrombin
J. 267,557-560).
For having the patient of Severe haemophilic, use blooc coagulation factor such as FXIII to help Blood Coagulation Process.
FXIII uses with its inactivation/zymogen forms, and this is the most such as the most native activating when lacerated wound occurs.Make
For the mass parameter of product, need to measure pre-activate FXIII(FXIIIaO) content, and keep minima.But, exist
In pharmaceutical preparation, the FXIII of specified quantitative can activate into FXIIIa with non-proteolyticOProbability, therefore this will increase non-spy
The danger of opposite sex solidification.
Therefore the method being highly desirable to measure the quality containing FXIII preparation, to guarantee the safety of FXIII product and to guarantee blood
The sick treatment of friend obtains optimization.
Summary of the invention
According to the first aspect of the invention, it is provided that the quality control test method containing FXIII (FXIII) sample,
This includes detecting pre-activate FXIII(FXIIIa in described sampleO) existence and/or measure pre-activate FXIII in described sample
(FXIIIaO) the step of concentration.
According to the second aspect of the invention, it is provided that for measuring the matter of the quality containing FXIII (FXIII) sample
Amount controls test kit, and this comprises pre-activate FXIII(FXIIIaO) detect and/or measure component, and according to side as defined herein
Method uses the description of described test kit.
Accompanying drawing explanation
Fig. 1 shows the Ca in various amounts2+In the presence of measure FXIII titer and %FXIIIaO.Left figure shows %
FXIIIaO, right figure shows relative potency.Upper drawing shows from 0.05 mM to 5 mM Ca2+, and bottom panel show from 1 mM
To 50 mM Ca2+；
Fig. 2 shows the FXIII titer and %FXIIIa measured in the presence of the NaCl of various amountsO.Left figure shows
%FXIIIaO, right figure shows the relative potency being standardized for 150 mM NaCl；
Fig. 3 shows the titer according to pH measurement, FXIIIaOAnd %FXIIIaO.The picture left above is with RFU/ second/nM
FXIIIaOActivity, lower-left figure is the relative potency according to pH, and bottom-right graph is obtained %FXIIIaO.Relative potency pin
PH 7.4 is standardized；
Fig. 4 shows that buffer components is to titer, FXIIIaOAnd %FXIIIaOImpact.The picture left above confirm with the RFU/ second/
The FXIIIa of nMOActivity, lower-left figure confirms according to buffer agent and Ca2+Relative potency, and obtained by top right plot confirms
%FXIIIaO.As indicated, all mensuration are at buffer components and 1 or 5 mM Ca2+In perform pH 7.4 times.Effect relatively
Valency is for having 1 mM CaCl2Hepes be standardized；
Fig. 5 shows and uses proenzyme FXIII-A2(Fig. 5 A) and activation FXIIIaOThe anion exchange H PLC chromatography of (Fig. 5 B)；
With
Fig. 6 shows that display is former from the mensuration of FXIII+ thrombin and the signal in the case of there is not thrombin
Reason.In the case of there is not FXIII, elapse the increase being not detected by fluorescence over time.
Detailed Description Of The Invention
According to the first aspect of the invention, it is provided that the quality control test method containing FXIII (FXIII) sample,
This includes detecting pre-activate FXIII(FXIIIa in described sampleO) existence and/or measure pre-activate FXIII in described sample
(FXIIIaO) the step of concentration.
The method that present invention provide for measuring the FXIII activity in the case of there is not thrombin, thus sample
In FXIIIaOSignal will be produced with established FXIIIa*.This activity is referred to as FXIIIa, because activation mechanism will not
It is distinguished.It practice, easily detect FXIIIa*, because it has different from FXIII by standard HPLC and MS method
Quality.Additionally, easily avoid the generation of FXIIIa* in medicine FXIII preparation.By contrast, FXIIIaOHave with
The quality of FXIII equivalent, and be therefore more difficult to be detected by standard method.In the case of there is not FXIIIa*,
Enzymatic assay method measures FXIIIaOLevel.
The invention provides with regard to the remarkable advantage accurately and for sensitive determination pre-activate FXIII, it has been found that this be for
Assess and guarantee that the quality containing FXIII sample is required.Especially, it has been found that the method for the present invention can detect at proenzyme
Trace pre-activate FXIII in FXIII, its sensitivity is about 0.1%.This invention therefore provides and give containing FXIII preparation
Quality and the Accurate Determining of effectiveness, this is directly related with the pre-activate FXIII amount existed in preparation.For example, it is envisioned that such as
Fruit come the sample of self-preparing agent containing have more than pre-activate FXIII(of particular percentile such as more than 0.3,0.6,1 or 2%), then
The batch containing FXIII preparation should be discarded.
" pre-activate FXIII " mentioned above or " FXIIIaO" each refer in the case of there is not thrombin by non-egg
The FXIII of white hydrolytic activation." activation FXIII " mentioned above refers in the presence of thrombin by proteolytic activations
FXIII。
In one embodiment, by chromatography detection FXIIIa in described sampleOExistence.
A further aspect according to the present invention, it is provided that detection pre-activate FXIII(FXIIIaO) existence and/or
Measure pre-activate FXIII(FXIIIaO) the method for concentration, this includes chromatography step.
In one embodiment, chromatography comprises anion exchange HPLC.
Have been found that proenzyme FXIII and FXIIIaOThere is different elution profile and retention time.Such as, can see in Figure 5
Go out proenzyme FXIII and there is the retention time (Fig. 5 A) of 7.1 minutes, and FXIIIaOThere is the retention time (figure of 10.4 minutes
5B).Anion exchange methods can detect FXIII and FXIIIa as 2 baseline resolved peaksO.Collection derives from Fig. 5 B aobvious
The eluting fraction at the peak shown, and according to mensuration described herein with regard to FXIIIaOActivity is tested, and it was found that contain
100%FXIIIaO。FXIIIaODetectable limit the most advantageously turns out to be few to 0.1-0.2%FXIIIaO, this points out chromatography
Technology is sensitive and quick method of quality control.Additionally, chromatography detection method also is able to measure FXIIIaOAnd without from any
Measure the interference of component, thus eliminate the probability of in-situ activation during analyzing.
In an embodiment of chromatography, it is cloudy that anion exchange HPLC comprises anion-exchange column such as DEAE-
Ion or the use of Q-anion column.In a further embodiment, anion-exchange column comprises DEAE-anion column
(such as TSKgel DEAE-NPR(Tosoh Bioscience LLC)).
For it is obvious to the skilled person that chromatography step will perform according to known procedure, such as using balance
With elution buffer agent.
It is to be understood that balance and elution buffer agent will hopefully select based on forming insoluble salt such as calcium.Cause
This, in an embodiment of chromatography, balance and elution buffer agent comprise with 20-100mM(such as 50mM) concentration
Tris.
In an embodiment of chromatography, balance and elution buffer agent are buffered to pH 7 8(such as 7.5).
In an embodiment of chromatography, balance and elution buffer agent contain at least 2mM Ca2+.One is entered at one
In the embodiment of step, balance and elution buffer agent contain 3mM-5mM Ca2+。
In an embodiment of chromatography, elution step performs by increasing ionic strength, and this can be by increasing
The concentration adding selected salt obtains, such as 200mM-700mM(such as 500mM) NaCl.
In one embodiment, pre-activate FXIII(FXIIIa during the method comprises additionally in the described sample of detectionO) deposit
With measure pre-activate FXIII(FXIIIa in described sampleO) the step of concentration.
In one embodiment, pre-activate FXIII(FXIIIa is measuredO) the step of concentration include measuring at thrombin
Existence and in the absence of FXIII activity.
A further aspect according to the present invention, it is provided that measure pre-activate FXIII(FXIIIaO) the step of concentration
Suddenly, this include measure thrombin existence and in the absence of FXIII activity.
It is total FXIII percent activity that this aspect of the present invention allows pre-activate FXIII percentage calculation.Adding
The gross activity measured after thrombin comprises by adding already present pre-activate (if present) in sample via activated by thrombin
The activity generated.Such as:
In one embodiment, the step measuring activation/pre-activate FXIII is included in the presence of FXIII substrate
Analyze, i.e. fluorescence or non-fluorescence (i.e. light absorption) is analyzed.
In one embodiment, it is glimmering that the step measuring activation/pre-activate FXIII is included in the presence of fluorogenic substrate
Light measurement is analyzed.
In a further embodiment, fluorogenic substrate can be by the substrate of transglutamin-ase 9 cleavage.?
One further in embodiment, fluorogenic substrate is Abz-NE(Cad-Dnp) EQVSPLTLLK(ZEDIRA GmbH,
Roesslerstrasse 83, D-64293 Darmstadt, Germany production code member A101).According to the present invention one
Further aspect, it is provided that Abz-NE(Cad-Dnp) EQVSPLTLLK is in pre-activate FXIII(FXIIIaO) purposes in measurement.
Without being bound by theory, it is believed that in the presence of glycine-ethyl ester, FXIII cuts from Abz-NE(Cad-Dnp)
The quencher (Cad-Dnp) of EQVSPLTLLK side chain and mix described ester group.
In a further embodiment, according to WO 2006/018164(N-Zyme Biotec GmBH) described in
Method measure activation/pre-activate FXIII, described measuring method is incorporated herein by reference.
In one embodiment, activation/pre-activate FXIII is at 0.5-10mM Ca2+In the presence of measure.One
In individual further embodiment, activation/pre-activate FXIII is at 1-2mM Ca2+In the presence of measure.Have been found that hope dimension
Hold calcium concentration and be less than 2mM, because %FXIIIaOMensuration depend on calcium concentration (in this analysis that can show from Fig. 1
Go out).In a further embodiment, activation/pre-activate FXIII is at 2mM Ca2+In the presence of measure.Select
2mM Ca2+Benefit be this value close to physiological values, and therefore situation in best simulation body.
In one embodiment, Ca2+Will be as atomic absorption standard Ca2+(this can be purchased from Fluka or Sigma-
Aldrich) exist.Use atomic absorption standard Ca2+Advantage be Ca2+Concentration the most accurately monitored and at acid condition
Lower holding is under control.By contrast, alkalescence condition undesirably causes the formation of insoluble calcium hydroxide and calcium oxide.This
Outward, atomic absorption standard Ca2+Not there is the shortcoming confirmed by calcium chloride powder.Such as, calcium chloride powder typically will capture from
The moisture content of environment, and quickly become inaccurate under the hydration level of calcium chloride salt, thus hinder Ca2+The standard of given mole
Really weigh.
It is to be understood that and should drop at sample and the time span measured between component incubation and activation/pre-activate FXIII measurement
To minimum, in order to make the non-proteolytic activation danger of FXIII be preferably minimized.Therefore, in one embodiment, sample with
Measure the time span between component incubation and activation/pre-activate FXIII measurement less than 1 hour.
In one embodiment, activation/pre-activate FXIII measures in the presence of 50-500mM NaCl.One
In individual further embodiment, activation/pre-activate FXIII measures in the presence of 100-200mM NaCl.Have been found that
Wish to control the NaCl concentration in measuring, because %FXIIIaOAffected by the NaCl concentration fluctuated that (this can show from Fig. 2
Analysis in find out).In one further embodiment, FXIII concentration is surveyed in the presence of 150mM NaCl
Amount.Select the benefit of 150mM NaCl be this value close to physiological values, and therefore situation in best simulation body.
In one embodiment, activation/pre-activate FXIII measures under pH 6.5-10.At one further
Embodiment in, activation/pre-activate FXIII measures under pH 7-8.Have been found that and wish to control the pH in measuring, because of
For having been found that pH affects titer and %FXIIIaO(this analysis that can show from Fig. 3 is found out).For instance, it has been found that
The avtive spot of FXIII* is affected by pH.In one further embodiment, activation/pre-activate FXIII is at pH 7.4
Under measure.Select the benefit of pH 7.4 be this value close to physiological values, and therefore situation in best simulation body.
Being to be understood that activation/pre-activate FXIII typically measures in the presence of buffer agent, described buffer agent can delay
Rush to pH 7 8(such as 7.4).In one embodiment, activation/pre-activate FXIII is in the presence of Hepes buffer agent
Measure.About Abz-NE(Cad-Dnp) the recommendation buffer agent of EQVSPLTLLK is to use hydrochloric acid to adjust to pH 7.5
50mM Tris.However it has been found that the selection of buffer agent is to titer and %FXIIIaOTool has a significant impact.Such as, for Hepes
Measure relatively low %FXIIIa surprisinglyOValue, this hint Tris may the new FXIIIa in situ of inductionO(seeing Fig. 4).This
Quality testing process is the most harmful, because falsity may cause the unnecessary of the batch containing FXIII preparation to discard.One
In individual further embodiment, activation/pre-activate FXIII is in the existence of 50-200mM Hepes buffer agent (such as 100mM)
Under measure.
In one embodiment, measure under activation/pre-activate FXIII is in the presence of surfactant.One
In individual further embodiment, surfactant is polyglycol surfactants (such as PEG 8000).When it is present, table
Face activating agent typically will be with 0.05-0.5%(such as 0.1%) amount exist.
According to the second aspect of the invention, it is provided that for measuring the matter of the quality containing FXIII (FXIII) sample
Amount controls test kit, and this comprises pre-activate FXIII(FXIIIaO) detect and/or measure component, and according to side as defined herein
Method uses the description of described test kit.
In one embodiment, detected components comprises anion exchange HPLC column, and such as DEAE-anion column is (such as
TSKgel DEAE-NPR).In a further embodiment, detected components additionally comprise balance as defined above and
Elution buffer agent.
In one embodiment, measure component and comprise fluorogenic substrate (such as Abz-NE(Cad-Dnp) as defined above
EQVSPLTLLK).In a further embodiment, measure component and additionally comprise Ca2+(such as 2mM atomic absorption standard
Ca2+).In one further embodiment, measure component and additionally comprise 100-200mM NaCl(such as 150mM
NaCl).In one further embodiment, measurement component additionally comprises and is buffered to pH 6.5-10(such as 7.4)
50-200mM Hepes buffer agent (such as 100mM).In one further embodiment, measure component and additionally comprise
0.05-0.5%(such as 0.1%) surfactant (such as PEG 8000).
The present invention is described referring now to following non-limiting example.
Embodiment
Embodiment 1
FXIIIaOHPLC detection
According to the condition described in table 1, can to containing FXIII sample implement Anion exchange HPLC analysis, with detection and
Quantitatively FXIIIaOExistence.
Table 1
| Post: | TSKgel DEAE-NPR 4.6 x 35 mm+protect 4.6 x 5 mm |
| Column temperature: | 25℃ |
| Flow velocity: | 1 ml/ minute |
| Buffer agent A: | 50 mM Tris, pH 7.5+3 mM CaCl2 |
| Buffer agent B: | 50 mM Tris, pH 7.5+500 mM NaCl+5 mM CaCl2 |
| Gradient: | T=0,1%B, T=2-17.2 minutes, from 1%B to 27%B, T=17.2 19.2 minute, isocratic under 27%B, T=19.2 19.7 minute, from 27%B to 70%B, T=19.7 1%B buffer agent within 21.7 minutes, is returned to from 70% to 90%B with when T=23.0 minute. |
| Automatic sampler Temperature: | Ambient temperature, ~ 21 DEG C |
| Sampling volume: | ~ 20 l or 10 20 g |
| Detection: | 215 nm |
| The operation time: | 32 minutes |
%FXIIIaOIt is determined as the FXIIIa of representative sample total mark areaOPeak area percent.Such as, as in Fig. 5
Display.
Embodiment 2
FXIIIaOThe fluorescence measurement of activity
FXIII activity in the presence of thrombin is by typically by mixed containing the 200 l reactions listing component in table 2
Compound adds 96 hole microtitration plates and measures.
Table 2
| Component | Finally measure concentration |
| rFXIII | 0-80 nM |
| Thrombin | 0.24 NIH/mL |
| 2.5 M glycine ethyl esters (amine donor) | 10 mM |
| Abz-NE(Cad-Dnp) EQVSPLTLLK(substrate) | 30 µM |
| PEG8000 | 0.1% |
| NaCl | 150 mM |
| Ca2+ | 2 mM |
| Hepes buffer agent, pH 7.4 | 100 mM |
Launch at 418nm subsequently and excite lower monitoring fluorescence with 313nm.Usually, SoftMaxPro software is used to read at plate
Read to monitor fluorescence on device (such as from the SpectraMax Gemini EM of Molecular Devices).
FXIII activity in the case of there is not thrombin is passed through general by containing 200 l listing component in table 3
Reactant mixture adds 96 hole microtitration plates and measures.Thrombin existence and in the absence of FXIII measurement guarantee
In same plate, to provide the most accurately comparing of fluorescence signal.
Table 3
| Component | Finally measure concentration |
| rFXIII | 0-10 000 nM |
| Thrombin | Nothing |
| 2.5 M glycine ethyl esters (amine donor) | 10 mM |
| Abz-NE(Cad-Dnp) EQVSPLTLLK(substrate) | 30 µM |
| PEG8000 | 0.1% |
| NaCl | 150 mM |
| Ca2+ | 2 mM |
| Hepes buffer agent, pH 7.4 | 100 mM |
Subsequently by above-described that similar in the way of monitor fluorescence, and result can be found out in figure 6.Subsequently
Relative fluorescence units with RFU/ second/nM FXIII(wherein RFU) measure thrombin existence and in the absence of activity, and
And %rFXIIIaOIt is calculated as the ratio measured 2 times.
All references cited herein includes that publications, patent applications and patents are incorporated by reference at this,
And its degree and each list of references indivedual and particularly point out be incorporated herein by reference and entirety illustrate in this article the same (extremely by
Allowed by law at utmost), it is unrelated that the concrete file carried out with elsewhere herein any is separately provided introducing.
Term " one " and " its " (" a " and " an " and " the ") and the similar use referred in the background describing the present invention
Should be interpreted that and contain odd number and plural number, unless otherwise indicated herein or context substantially conflicts.Such as, phrase " compound "
(" the compound ") is understood to mean various " compounds " (" compounds ") in terms of the present invention or specific description, removes
Non-it is otherwise noted.
Except as otherwise noted, all exact values provided herein represent corresponding approximation (such as, time suitable, with regard to concrete because of
The all precise example values provided for son or measurement can be considered the corresponding approximate measure also providing for being modified by " about ").
This paper uses term such as " to comprise " for one or more elements, " having ", " including " or " containing " description
Any one or more aspects of the present invention be intended to provide about " being made up of one or more that concrete elements ", " basic
On be made up of one or more that concrete elements " or the present invention one of " basically comprising one or more that concrete element "
Individual or the support of multiple similar aspect, except as otherwise noted or context substantially conflicts (such as described herein as comprising concrete unit
The compositions of part is interpreted as also describing the compositions being made up of that element, except as otherwise noted or context substantially conflicts).
The preferred feature of the present invention:
1. a quality control test method containing FXIII sample, it includes detecting pre-activate FXIII in described sample
(FXIIIaO) existence and/or measure pre-activate FXIII(FXIIIa in described sampleO) the step of concentration.
2. such as the method limited in clause 1, the FXIIIa in wherein said sampleOExist and examined by chromatography
Survey.
3., such as the method limited in clause 2, wherein said chromatography comprises anion exchange HPLC.
4., such as the method limited in clause 3, wherein said anion exchange HPLC comprises anion-exchange column such as
DEAE-anion or Q-anion column.
5., such as the method limited in clause 4, wherein said anion-exchange column comprises DEAE-anion column such as
TSKgel DEAE-NPR。
6. the method limited in as any one of clause 25, wherein said balance and elution buffer agent comprise with 20-
100mM(such as 50mM) Tris of concentration.
7. the method limited in as any one of clause 26, wherein said balance and elution buffer agent are buffered to pH 7
8 such as 7.5.
8. the method limited in as any one of clause 27, wherein said balance and elution buffer agent comprise at least
2mM Ca2+。
9., such as the method limited in clause 8, wherein said balance and elution buffer agent comprise 3mM-5mM Ca2+。
10. in as any one of clause 29 limit method, wherein said elution buffer agent additionally comprise 200mM-
700mM NaCl, such as 500mM.
The method limited in 11. such as clause 1, it includes detecting pre-activate FXIII(FXIIIa in described sampleO) deposit
With measure pre-activate FXIII(FXIIIa in described sampleO) the step of concentration.
The method limited in 12. such as clause 11, wherein said measurement pre-activate FXIII(FXIIIaO) the step of concentration
Active including existence (activation FXIII) and the FXIII not existed under (pre-activate FXIII) measured at thrombin.
The method limited in 13. such as clause 12, the step of wherein said measurement activation/pre-activate FXIII is included in fluorescence
Or the fluorometric assays in the presence of non-fluorescence FXIII substrate.
The method limited in 14. such as clause 12, the step of wherein said measurement activation/pre-activate FXIII is included in fluorescence
Fluorometric assays in the presence of FXIII substrate.
The method limited in 15. such as clause 14, wherein said fluorogenic substrate is can be by the end of transglutamin-ase 9 cleavage
Thing.
The method limited in 16. such as clause 15, wherein said fluorogenic substrate is Abz-NE(Cad-Dnp) EQVSPLTLLK.
17. as any one of clause 12 16 in limit method, wherein activation/pre-activate FXIII is at 0.5-10mM
Ca2+Such as 1-2mM Ca2+In the presence of measure,
The method limited in 18. such as clause 17, wherein activation/pre-activate FXIII is at 2mM Ca2+In the presence of survey
Amount.
The method limited in 19. such as clause 17, wherein Ca2+Source, Ca2+Concentration the most accurately monitored and in acid
Keep under control under the conditions of property.
20. such as clause 17 or clause 18 in limit method, wherein Ca2+As atomic absorption standard Ca2+Exist.
21. as any one of clause 12 20 in limit method, wherein said activation/pre-activate FXIII is at 50-
Measure in the presence of 500mM NaCl such as 100-200mM NaCl.
The method limited in 22. such as clause 21, wherein activation/pre-activate FXIII is carried out in the presence of 150mM NaCl
Measure.
23. as any one of clause 12 22 in the method that limits, wherein activation/pre-activate FXIII pH 6.5-
Measure under 10 such as 7-8.
The method limited in 24. such as clause 23, wherein activation/pre-activate FXIII measures for 7.4 times at pH.
25. as any one of clause 12 24 in limit method, wherein activation/pre-activate FXIII Hepes buffering
Measure in the presence of agent.
The method limited in 26. such as clause 25, wherein activation/pre-activate FXIII is in 50-200mM Hepes buffer agent example
Measure as in the presence of 100mM.
27. as any one of clause 12 26 in limit method, the concentration of wherein said FXIII is at surfactant
In the presence of measure.
In 28. such as clause 27 limit method, wherein said surfactant be polyglycol surfactants such as
PEG 8000。
29. such as clause 27 or clause 28 in limit method, wherein said surfactant is with 0.05-0.5% such as
The amount of 0.1% exists.
The quality control reagents box of 30. 1 kinds of quality containing FXIII (FXIII) sample for mensuration, it comprises pre-
Activation FXIII(FXIIIaO) detect and/or measure component, and according to as described in the method use limited in any aforementioned clause
The description of test kit.
The test kit limited in 31. such as clause 30, wherein said detected components comprises as limited in clause 4 or clause 5
Anion exchange HPLC column.
32. such as clause 30 or clause 31 in limit test kit, wherein said detected components additionally comprises such as clause 6-10
The balance limited in any one of and elution buffer agent.
The test kit limited in 33. such as clause 30, wherein said measurement component comprises as limited in clause 15 or clause 16
Fluorogenic substrate.
The test kit limited in 34. such as clause 33, wherein said measurement component additionally comprises Ca2+Such as 2mM Atomic Absorption
Standard Ca2+。
35. such as clause 33 or clause 34 in limit test kit, wherein said measurement component additionally comprises 100-200mM
NaCl such as 150mM NaCl.
36. as any one of clause 33-35 in limit test kit, wherein said measurement component additionally comprises 50-
200mM Hepes buffer agent such as 100mM Hepes buffer agent.
The test kit limited in 37. such as clause 36, wherein said Hepes buffer agent is buffered to pH 78 such as 7.4.
38. as any one of clause 33-37 in the test kit that limits, wherein said measurement component additionally comprises 0.05-
0.5% surfactant such as 0.1%PEG 8000.
39. Abz-NE(Cad-Dnp) EQVSPLTLLK measure pre-activate FXIII(FXIIIaOPurposes in).
40. 1 kinds of detection pre-activate FXIII(FXIIIaO) existence and/or measure pre-activate FXIII(FXIIIaO)
The method of concentration, it includes chromatography step.
Measure pre-activate FXIII(FXIIIa for 41. 1 kindsO) the method for concentration, it includes measuring the existence at thrombin
FXIII activity in the absence of with.
Claims (23)
1. a quality control test method containing FXIII sample, it is included in and there is 0.5-10mM Ca2+The lower described sample of detection
The existence of pre-activate FXIII and/or measure the step of the concentration of pre-activate FXIII in described sample in product.
2., such as the method limited in claim 1, it is described that it includes detecting the existence of pre-activate FXIII and measurement in described sample
The step of the concentration of pre-activate FXIII in sample.
3., such as the method limited in claim 2, the step of the concentration of wherein said measurement pre-activate FXIII includes measuring not
There is the FXIII activity under thrombin.
4., such as the method limited in claim 3, the step of wherein said measurement pre-activate FXIII is included in fluorescence or non-fluorescence
Fluorometric assays in the presence of FXIII substrate.
5., such as the method limited in claim 4, wherein said fluorogenic substrate is Abz-NE(Cad-Dnp) EQVSPLTLLK.
6. the method limited as any one of claim 4-5, the step of wherein said measurement pre-activate FXIII is included in fluorescence
Fluorometric assays in the presence of FXIII substrate.
7. the method limited as any one of claim 4-5, wherein said fluorogenic substrate is can be by transglutamin-ase 9 cleavage
Substrate.
8. the method limited as any one of claim 1-5, wherein there is 1-2mM Ca in pre-activate FXIII2+Lower measurement.
9., such as the method limited in claim 8, wherein there is 2mM Ca in pre-activate FXIII2+Lower measurement.
10., such as the method limited in claim 8, wherein accurately monitor described Ca2+Concentration and control are in acid condition.
The method limited in 11. such as claim 8, wherein Ca2+As atomic absorption standard Ca2+Exist.
12. methods limited as any one of claim 1-5, wherein in pre-activate FXIII under there is 50-500 mM NaCl
Measure.
The method limited in 13. such as claim 12, wherein pre-activate FXIII is measured under there is 100-200 mM NaCl.
The method limited in 14. such as claim 12, wherein pre-activate FXIII is measured under there are 150 mM NaCl.
15. methods limited as any one of claim 1-5, wherein pre-activate FXIII is measured under pH 6.5-10.
The method limited in 16. such as claim 15, wherein pre-activate FXIII is measured under pH 7-8.
The method limited in 17. such as claim 16, wherein pre-activate FXIII is measured for 7.4 times in pH.
18. methods limited as any one of claim 1-5, wherein pre-activate FXIII is surveyed under there is Hepes buffer
Amount.
The method limited in 19. such as claim 18, wherein in pre-activate FXIII under there is 50-200 mM Hepes buffer
Measure.
The method limited in 20. such as claim 19, wherein pre-activate FXIII is surveyed under there are 100 mM Hepes buffer
Amount.
21. as any one of claim 1-5 in limit method, concentration the depositing at surfactant of wherein said FXIII
Measure under.
The method limited in 22. such as claim 21, wherein said surfactant is polyglycol surfactants.
The method limited in 23. such as claim 21, wherein said surfactant exists with the amount of 0.05-0.5%.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP08167476 | 2008-10-24 | ||
| EP08167476.4 | 2008-10-24 | ||
| PCT/EP2009/063973 WO2010046468A1 (en) | 2008-10-24 | 2009-10-23 | Method of quality control testing a factor xiii containing sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102197142A CN102197142A (en) | 2011-09-21 |
| CN102197142B true CN102197142B (en) | 2016-11-30 |
Family
ID=
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006018164A2 (en) * | 2004-08-11 | 2006-02-23 | N-Zyme Biotec Gmbh | Fluorescence-based kinetic determination of transglutaminase activity |
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006018164A2 (en) * | 2004-08-11 | 2006-02-23 | N-Zyme Biotec Gmbh | Fluorescence-based kinetic determination of transglutaminase activity |
Non-Patent Citations (2)
| Title |
|---|
| Non-proteolytic activation of cellular protransglutaminase placenta macrophage factor XIII;Polgar J;《Biochem J》;19900415;557-560 * |
| Transformation of cellular factor XIII into an active zymogen transglutaminase in thrombin-stimulated platelets;Muszbek L,;《Thrombosis an haemostasis》;19951231;第173卷(第4期);702-705 * |
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