CN102507960A - Method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) for injection, and metabolin thereof - Google Patents
Method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) for injection, and metabolin thereof Download PDFInfo
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- CN102507960A CN102507960A CN201110352049XA CN201110352049A CN102507960A CN 102507960 A CN102507960 A CN 102507960A CN 201110352049X A CN201110352049X A CN 201110352049XA CN 201110352049 A CN201110352049 A CN 201110352049A CN 102507960 A CN102507960 A CN 102507960A
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Abstract
The invention discloses a method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) (1-84) for injection, and metabolin rhPTH (1-34) thereof, and belongs to the technical field of determination of plasma concentration of PTH. According to the method, solid phase extraction is combined with radioimmunoassay (RIA), and a plasma sample is extracted by a solid phase extraction column StyreScreen H2P, and rhPTH (1-84) is determined by thyroxine radioimmunoassay kit enzyme-linked immunosorbent assay (ELSA)-PTH; and the method comprises the following steps of: adding an rhPTH (1-84) standard substance or a treated plasma sample into ELSA TUBES, adding 125I-PTH, testing by using a compound tube, and measuring count per minute (CPM) by a gamma-counter; drawing by taking CPM value as a Y coordinate and rhPTH (1-84) concentration as an X coordinate to obtain a regression equation Y=19.791X; determining the rhPTH (1-34) by using a thyroxine radioimmunoassay kit RK-055-08 (PTH1-34); adding samples and antibodies into double compound tubes; incubating; after 125I-PTH is added into each tube, uniformly oscillating and incubating; adding goat anti-rabbit and normal rabbit serum and incubating; and measuring count B by using the gamma-counter, and drawing according to a formula B0=TB-NSB by taking B/B0 as the Y coordinate and rhPTH (1-34) concentration logarithm as the X coordinate to obtain a functional equation. By the method, the repeatability and stability of the RIA detection are improved, and reliability of determination data is improved.
Description
Technical field
The assay method of injection recombinant human parathyroid hormone rhPTH (1-84) and metabolic product rhPTH (1-34) blood concentration thereof belongs to the assay method technical field of PTH PC.
Background technology
PTH is the natural parathyroid of pth secretion, and what wherein have physiologically active is the polypeptide hormone that contains 84 amino acid residues, and it can stimulate new bone formation and balance and the phosphorus metabolism of keeping normal calcium.Injection recombinant human parathyroid hormone (1-84) (Recombinant human parathyroid hormone (1-84) for injection; Be abbreviated as rhPTH (1-84)) be a kind of recombinant human parathyroid hormone of the PTH of containing total length preface; By U.S. NPS-Allelix company research and development, be used to treat women's osteosporosis after menopause disease by the FDA approval in 2006.RhPTH (1-84) can partly be degraded to rhPTH (1-34) in vivo; 34 consensus amino acid sequences of the N of rhPTH (1-34) structure and rhPTH (1-84) end, rhPTH (1-34) can increase bone density, strengthen sclerotin and improve the bone framework and become the main treatment means of osteoporosis.RhPTH (1-34) is by the research and development of U.S. Eli Lilly company, and FDA approval listing in 2002 has good curative effect aspect treatment PMO and the part osteoporosis in aged males.RhPTH (1-84) has better security, and both are important anti-osteoporosis disease medicine, but since every day must take make price too costliness limited its clinical practice.Because social senilization; The osteoporosis incidence of disease raises year by year; RhPTH (1-84) is because of more effectively having good potential applicability in clinical practice to the increase effect of bone amount than the other treatment means of present clinical practice, and domestic have how tame biopharmaceutical company in research and development.
RhPTH (1-84) can be degraded to the peptide chain of different length in vivo; Because rhPTH (1-34) has higher efficacy; Therefore measure the concentration and the medicine dynamics research of metabolic product rhPTH (1-34) in blood plasma of rhPTH (1-84), significant to pharmacodynamics, mechanism of action and the clinical rational drug use of understanding injection rhPTH (1-84).
The assay method of PTH PC have radiommunoassay (Radioimmunoassay, RIA), enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatogram-Mass Spectrometer Method method (HPLC-MS) etc.The HPLC-MS method needs the special detection device, and use costs an arm and a leg; ELISA and RIA analytic approach method are easy, operation is convenient, but because the complicacy of plasma fraction causes defectives such as measuring result's repeatability and poor stability.
Summary of the invention
The object of the invention provides the assay method of a kind of injection recombinant human parathyroid hormone rhPTH (1-84) and metabolic product rhPTH (1-34) blood concentration thereof, adopts SPE to combine RIA to detect and measures the concentration of rhPTH (1-84) in blood plasma and the variation and the pharmacokinetic studies of metabolic product rhPTH (1-34) PC simultaneously.Improve the repeated and stable of RIA detection, increased the reliability of determination data.
The assay method of technical scheme of the present invention: a kind of injection recombinant human parathyroid hormone rhPTH (1-84) and metabolic product rhPTH (1-34) blood concentration thereof: plasma sample adopts solid-phase extraction column Styre Screen H2P extraction, and rhPTH (1-84) adopts thyroxine radioimmunological kit ELSA-PTH to measure: rhPTH (1-34) adopts thyroxine radioimmunological kit RK-055-08 (PTH 1-34) to measure.
1, standard solution preparation
The preparation of rhPTH (1-84) standard solution
Get rhPTH (1-84) standard items, with deionized water dissolving vibration, compound concentration is respectively 0,24,55,177,520pgmL
-1Standard solution; It is 46 pgmL that interior Quality Control (PC) is mixed with rhPTH (1-84) concentration
-1Solution; It is 25,250,500 pgmL that outer Quality Control (QC) preparation contains rhPTH (1-84) standard items concentration
-1Three kinds of standard solution are sub-packed in the centrifuge tube, and-80 ℃ of preservations are for use;
The preparation of rhPTH (1-34) standard solution
Get rhPTH (1-34) standard items and be diluted to 10,20 with damping fluid, 40,80,160,320,640,1280 pgmL
-1Standard solution;
The QC quality-control sample: precision takes by weighing rhPTH (1-34) standard, contains rhPTH (1-34) concentration with the damping fluid preparation and is respectively 40.00,160.00,640.00 pgmL
-1
Said damping fluid is the damping fluid that thyroxine radioimmunological kit RK-055-08 (PTH 1-34) is provided; Down together.
2, plasma sample is handled
With solid-phase extraction column Styre Screen H2P with 1mL ethanol, the activation of 1mL0.1% trifluoroacetic acid deionized water; Draw the 0.5mL plasma sample,, with the drip washing of 1mL deionized water, drain behind the example enrichment with the speed adding solid-phase extraction column of 1 ~ 2mL/min; With ethanol-0.1% trifluoroacetic acid water (V/V, 60:40) eluant solution is drained; Use the dry 15min of SpeedVac, remove organic solvent; Through the freeze-dryer concentrate drying, be diluted to suitable concentration with damping fluid during mensuration;
3, operating process
RhPTH (1-84) measurement operation flow process:
The plasma sample of getting after 200 μ L rhPTH (1-84) standard items or SPE are handled adds ELSA TUBES (the ELSA test tube that solid-phase coating is good, thyroxine radioimmunological kit ELSA-PTH provides), adds 100 μ L respectively
125I-PTH vibration 3 minutes, multiple pipe test is hatched 18 ± 2h for 25 ℃, abandons supernatant, adds the 3mL polysorbas20, abandons supernatant after 5 minutes, repeated washing 4 times, γ-count surveys counting (CPM);
RhPTH (1-34) measurement operation flow process:
Operate with thyroxine radioimmunological kit RK-055-08 (PTH 1-34), polystyrene tube is numbered: house steward (TC), non-specific binding (NSB), total combine (TB), standard A～H, positive control PC, sample S, two multiple pipes add sample and add antibody; Behind the vibration mixing, seal 4 ℃ and hatch 16～24h; Every pipe adds 100 μ L
125Behind the I-PTH, hatch 16～24h for 4 ℃ behind the vibration mixing; Add goat anti-rabbit igg (GAR), positive rabbit anteserum (NRS), vibration incubated at room 90 minutes; Except that the every pipe of TC pipe adds 500 μ L damping fluids vibration mixing, centrifugal 20 minutes of 3000rpm (4 ℃) inhales and abandons supernatant (completion in 15～30 minutes), residue γ-counter measure B
X, and calculate B=B
X-NSB.
4, computing method
RhPTH (1-84) concentration is calculated:
To the mapping of rhPTH (1-84) concentration, get regression equation y=ax (Y=19.791X) with CPM.From equation, try to achieve rhPTH in the plasma sample (1-84) concentration, with DAS 2.0 computed in software pharmacokinetic parameters.
RhPTH (1-34) concentration is calculated:
γ-counter records counting by B
0=TB-NSB, B/B
0(%)=(B
X-NSB)/B
0* 100% calculates, with B/B
0Be ordinate, rhPTH (1-34) concentration logarithm is the horizontal ordinate mapping, and curve fitting gets functional equation, from equation, tries to achieve rhPTH in the plasma sample (1-34) concentration, with DAS 2.0 computed in software pharmacokinetic parameters.
Beneficial effect of the present invention: the present invention adopts number of ways such as adding Aprotinin, SPE to reduce the RIA influence factor; Investigated the extraction efficiency of different filler solid phase pillars; And methodology investigated, improved the repeatability of measuring the result and stable.The research of rhPTH (1-34) is more, but does not appear in the newspapers as metabolic product its blood concentration of research and the pharmacokinetics of rhPTH (1-84), and the present invention is that rhPTH (1-84) entering clinical practice provides accurate clinical testing detection method.
Description of drawings
Fig. 1 rhPTH (1-84) typical curve.
Fig. 2 rhPTH (1-34) typical curve.
Fig. 3 different batches rhPTH (1-84) stability.
Fig. 4 rhPTH (1-84) long term storage stability.
Fig. 5 rhPTH (1-84) and rhPTH (1-34) blood concentration-time plot.
Embodiment
Embodiment 1 range of linearity
RhPTH (1-84) range of linearity:
Getting concentration is 0,24,55,177,520 pgmL
-1RhPTH (1-84) standard solution; Press the operation of thyroxine radioimmunological kit ELSA-PTH operating process, the gained typical curve is seen Fig. 1.Adopt straight-line regression at 24～520 pgmL
-1Scope internal linear relation is good, equation of linear regression: Y=19.791X (R
2=0.9989, Fig. 1 b).Investigated many linear relationships of different batches respectively, PC, QC accompany and are used for investigating mensuration degree of accuracy (seeing table 1) in typical curve, and each item quality control standard measured value relative error is all less than 10%.
Getting rhPTH (1-34) standard items concentration is 10,20,40,80,160,320,640,1280 pgmL
-1Standard solution, operate by thyroxine radioimmunological kit RK-055-08 (PTH 1-34) radioimmunology analysis operating process, set up typical curve.Logarithm with rhPTH (1-34) normal concentration is a horizontal ordinate, B/B
0Be ordinate, adopt four parameter l ogistic regressing calculations, get equation:
，A
1=0.8462，A
2=-28.1523，x
0=13.8682，p=2.7849，R
2=0.9961。
Get the QC sample, exempt from operating process operation, each Quality Control concentration replication 5 times by putting; 3 days METHOD FOR CONTINUOUS DETERMINATION totally 3 analyze batch; Calculate in a few days and day to day precision and accuracy (table 1), precision representes that with relative standard deviation (RSD%) accuracy is represented with relative error (RE%).
Precision and accuracy (n=5) in the table 1 methodology conclusive evidence
Accurate preparation contains the plasma sample of rhPTH (1-84) and rhPTH (1-34) and is diluted to 10 pgmL
-1, to operate by the radioimmunology analysis operating process, replication 5 times calculates precision and degree of accuracy.RhPTH under this concentration (1-84) records mean concentration and is (8.70 ± 0.57) pgmL
-1, relative error RE is 13.0%, relative standard deviation RSD is 6.54%; RhPTH (1-34) records mean concentration and is (8.82 ± 0.31) pgmL
-1, relative error RE is 11.9%, relative standard deviation RSD is 3.51%.
The selection of embodiment 4, solid-phase extraction column
Adopt Sepax-BRC18, Styre Screen H2P and common ODS pillar respectively, pillar is by 2.0 plasma sample activation processing, and preparation contains rhPTH (1-84) 25.0,250.0,500.0 pgmL
-1RhPTH (1-34) 40.0,160.0,640.0 pgmL
-1The plasma sample of high, medium and low concentration, blood plasma are exempted from operating process mensuration by putting after SPE.
Extraction efficiency=C measured value/C standard value * 100%.Adopt Styre Screen H2P extraction, efficient is greater than 85%.
The different solid-phase extraction column blood plasma of table 2 extraction efficiency is measured result (n=3)
The investigation of embodiment 5, Aprotinin consumption
Collect the healthy subjects blank plasma in EDTA anti-freezing test tube, be transferred to then and contain in the variable concentrations Aprotinin centrifuge tube, shake up 4 ℃ of 3000 rmin
-1Centrifugal 10 min obtain blood plasma.The accurate high, medium and low concentration rhPTH of preparation (1-84) exempts from operating process mensuration (table 3) in the plasma sample of variable concentrations Aprotinin by putting.Adopt the Aprotinin of 0.5 (TIU/mL) in the blood plasma.
Table 3 Aprotinin consumption is to result's influence
Embodiment 6, rhPTH (1-84) plasma stability are investigated
The plasma sample that preparation contains the basic, normal, high concentration of rhPTH (1-84) is sub-packed in several the centrifuge tubes, places 6 h respectively in 4 ° of C, room temperature; 3 times ,-80 ° C of-80 ° of C multigelations stored 80 days, operated by the radioimmunology analysis operating process, measured the content of rhPTH (1-84) in the blood plasma.
Room temperature is placed and is surpassed 6 h, and rhPTH (1-84) density loss in blood plasma arrives 75.2% ~ 77.6% of original content; 4 ° of C place more than 6 h, freeze-thaw was stored 80 days for 3 times repeatedly ,-80 ℃, and rhPTH (1-84) concentration in blood plasma maintains between 90% ~ 110%, shows good stability; Fig. 3 is the basic, normal, high QC sample of rhPTH (1-84) changes of contents figure (n=60) of a plurality of batches in blood plasma, and Fig. 4 shows rhPTH (1-84) QC plasma sample long term storage stability.
Embodiment 7, pharmacokinetic studies
10 healthy premenopausal volunteers hypodermic injection 2.0 μ gkg
-1RhPTH (1-84), respectively at 5 min before the administration, after the administration 5,15,30,60,90,120,180,240,360,480,600 min gather venous blood 3 mL and place and contain EDTA anti-freezing test tube, 4 ℃ of 3000 rmin
-1Centrifugal 10 min obtain blood plasma, exempt from the concentration that working specification is measured rhPTH (1-84) and rhPTH (1-34) in the blood plasma by putting.Measured PC medication is handled for dynamics software DAS 2.0, and blood medicine change curve is in time seen Fig. 5.
Calculate gained main medicine kinetic parameter rhPTH (1-84): AUC0 – ∞ is (614.3 ± 158.7) pghmL
-1, Cmax is (131.2 ± 33.5) pgmL
-1, Tmax is (0.68h ± 0.1) h.RhPTH (1-34): AUC0 – ∞ is (245.7 ± 139.5) pghmL
-1, Cmax is (58.0 ± 10.0) pgmL
-1, Tmax is (0.38h ± 0.02) h.
Claims (1)
1. the assay method of an injection recombinant human parathyroid hormone rhPTH (1-84) and metabolic product rhPTH (1-34) blood concentration thereof; It is characterized in that: plasma sample adopts solid-phase extraction column Styre Screen H2P extraction, and rhPTH (1-84) adopts thyroxine radioimmunological kit ELSA-PTH to measure: rhPTH (1-34) adopts thyroxine radioimmunological kit RK-055-08 (PTH 1-34) to measure; Step is:
(1), standard solution preparation
The preparation of rhPTH (1-84) standard solution
Get rhPTH (1-84) standard items, with deionized water dissolving vibration, compound concentration is respectively 0,24,55,177,520pgmL
-1Standard solution; It is 46 pgmL that interior Quality Control PC is mixed with rhPTH (1-84) concentration
-1Solution; It is 25,250,500 pgmL that outer Quality Control QC preparation contains rhPTH (1-84) standard items concentration
-1Three kinds of standard solution are sub-packed in the centrifuge tube, and-80 ℃ of preservations are for use;
The preparation of rhPTH (1-34) standard solution
Get rhPTH (1-34) standard items and be diluted to 10,20 with damping fluid, 40,80,160,320,640,1280 pgmL
-1Standard solution;
The QC quality-control sample: precision takes by weighing rhPTH (1-34) standard, contains rhPTH (1-34) concentration with the damping fluid preparation and is respectively 40.00,160.00,640.00 pgmL
-1
Said damping fluid is the damping fluid that thyroxine radioimmunological kit RK-055-08 (PTH 1-34) is provided;
(2), plasma sample is handled
Solid-phase extraction column Styre Screen H2P is contained 0.1% trifluoroacetic acid deionized water activation with 1mL ethanol, 1mL successively; Draw the 0.5mL plasma sample,, with the drip washing of 1mL deionized water, drain behind the example enrichment with the speed adding solid-phase extraction column of 1 ~ 2mL/min; Use the ethanol-0.1% trifluoroacetic acid deionized water solution wash-out of V/V, drain as 60:40; Use the dry 15min of SpeedVac, remove organic solvent; Through the freeze-dryer concentrate drying, be diluted to suitable concentration with damping fluid during mensuration;
(3), operating process
RhPTH (1-84) measurement operation flow process:
Get 200 μ L rhPTH (1-84) standard items or handle after plasma sample add ELSA TUBES, add 100 μ L respectively
125I-PTH vibration 3 minutes, multiple pipe test is hatched 18 ± 2h for 25 ℃, abandons supernatant, adds the 3mL polysorbas20, abandons supernatant after 5 minutes, and repeated washing 4 times, γ-count are surveyed counting CPM;
Said ELSA TUBES is the good ELSA test tube of solid-phase coating that thyroxine radioimmunological kit ELSA-PTH provides;
RhPTH (1-34) measurement operation flow process:
Polystyrene tube is numbered: house steward TC, non-specific binding NSB always combines TB, standard A～H, positive control PC, sample S, two multiple pipes add sample and add antibody; Behind the vibration mixing, seal 4 ℃ and hatch 16～24h; Every pipe adds 100 μ L
125Behind the I-PTH, hatch 16～24h for 4 ℃ behind the vibration mixing; Adding goat anti-rabbit igg is GAR, positive rabbit anteserum NRS, vibration incubated at room 90 minutes; Except that the every pipe of TC pipe adds 500 μ L damping fluids vibration mixing, centrifugal 20 minutes of 4 ℃ of 3000rpm, supernatant is abandoned in suction in 15～30 minutes, residue γ-counter measure B
X, and calculate B=B
X-NSB;
(4), computing method
RhPTH (1-84) concentration is calculated:
With CPM rhPTH (1-84) concentration is mapped; With the CPM value is ordinate, and rhPTH (1-84) concentration is the horizontal ordinate mapping, gets regression equation Y=19.791X; From equation, try to achieve rhPTH in the plasma sample (1-84) concentration, with DAS 2.0 computed in software pharmacokinetic parameters;
RhPTH (1-34) concentration is calculated:
γ-counter records counting by B
0=TB-NSB, B/B
0(%)=(B
X-NSB)/B
0* 100% calculates, with B/B
0Be ordinate, rhPTH (1-34) concentration logarithm is the horizontal ordinate mapping, and curve fitting gets functional equation, from equation, tries to achieve rhPTH in the plasma sample (1-34) concentration, with DAS 2.0 computed in software pharmacokinetic parameters.
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113841052A (en) * | 2018-10-18 | 2021-12-24 | 费森尤斯医疗保健控股公司 | Techniques for modeling parathyroid function and calcimimetic drug activity |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4369138A (en) * | 1980-07-15 | 1983-01-18 | Immuno Nuclear Corporation | Parathyroid radioimmunoassay |
| WO2005018413A2 (en) * | 2003-07-10 | 2005-03-03 | Scantibodies Laboratory, Inc. | Methods, kits and antibodies for detecting parathyroid hormone |
-
2011
- 2011-11-09 CN CN201110352049.XA patent/CN102507960B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4369138A (en) * | 1980-07-15 | 1983-01-18 | Immuno Nuclear Corporation | Parathyroid radioimmunoassay |
| WO2005018413A2 (en) * | 2003-07-10 | 2005-03-03 | Scantibodies Laboratory, Inc. | Methods, kits and antibodies for detecting parathyroid hormone |
Non-Patent Citations (4)
| Title |
|---|
| ANNE DENIZOT: "Normocalcemia with elevated parathyroid hormone levels after surgical treatment of primary hyperparathyroidism", 《THE AMERICAN JOURNAL OF SURGERY》 * |
| 何子强: "用放免法测定血中PTH的基础研究", 《国外医学临床生物化学与检验学分册》 * |
| 孙恒德: "人血清PTH的放免测定", 《天津医科大学学报》 * |
| 李克坚: "重组人甲状旁腺激素效价测定方法的研究", 《药物生物技术》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113841052A (en) * | 2018-10-18 | 2021-12-24 | 费森尤斯医疗保健控股公司 | Techniques for modeling parathyroid function and calcimimetic drug activity |
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