CN104098655A - Polypeptides of mass spectrography internal standard used for synthesis of glatiramer acetate - Google Patents

Polypeptides of mass spectrography internal standard used for synthesis of glatiramer acetate Download PDF

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CN104098655A
CN104098655A CN201310120693.3A CN201310120693A CN104098655A CN 104098655 A CN104098655 A CN 104098655A CN 201310120693 A CN201310120693 A CN 201310120693A CN 104098655 A CN104098655 A CN 104098655A
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aak
yak
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CN104098655B (en
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李国弢
唐洋明
覃亮政
韦畅勇
马亚平
袁建成
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Hybio Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal

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Abstract

The invention relates to polypeptides of a mass spectrography internal standard used for synthesis of glatiramer acetate and a preparation method and application of the internal standard.

Description

For the synthesis of acetic acid lattice, draw for target polypeptide in the mass spectrum of thunder
Technical field
The present invention relates to draw for target polypeptide in the mass spectrum of thunder (claiming again copolymer-1), Preparation Method And The Use for the synthesis of acetic acid lattice.Particularly, the present invention relates to for drawing for thunder building-up process at acetic acid lattice, as the interior target polypeptide of intermediate molecular weight determination, Preparation Method And The Use.
Background technology
Autoimmunization class disease refers to, the immunity system of living organism is used as the tissue of some bodies itself as the phenomenon that " exotic " attacked.Common this disease can, by hindering living organism T cell and the reaction of B cell to body autologous tissue, be eased.These early stage immune responses are attached on major histocompatibility antigen mixture (MHC) molecule and are promoted by antigen, and by T cell expressing out.Autoimmunization class disease is exactly that tissue and the protein of body itself is taken as " from antigen ", by body immune system, is attacked.For example: multiple sclerosis is exactly the disease of the myelin of immune system attack isolation and protection nerve.This disease progression, to losing myelin, will bring neurone and motorius afunction.
A lot of drug developments are out used for the treatment of autoimmunization class disease, comprise multiple sclerosis.It is the mixture of a polypeptide being comprised of L-Ala, L-glutamic acid, Methionin and tyrosine that acetic acid lattice draw for thunder.Its amino acid molar ratio is approximately 0.392～0.462:0.129～0.153:0.300～0.374:0.086～0.100, about 5000～9000 dalton of molecular-weight average.Molecular-weight average optimum range and the synthesis technique of copolymer-1 and its terpolymer (mixture of the polypeptide of any three kinds of compositions in L-Ala, L-glutamic acid, Methionin and tyrosine) have been described in US5800808.
The earliest; Teitelbaum etc. are at Eur.J.Immun.; 1; 242; the method of having described synthetic copolymer-1 in 1971 is by being dissolved in the N-carboxylic acid anhydride of the ALANINE of Isosorbide-5-Nitrae-dioxane, prepared by the N-carboxylic acid anhydride of TYR, the N-carboxylic acid anhydride of gamma-glutamic acid benzyl ester and the N-carboxylic acid anhydride of trifluoroacetyl group Methionin the acetic acid lattice of protection draw for thunder (copolymer-1 of protection).Then with the acetic acid solution of 33% hydrogen bromide, remove the γ-benzyl protecting group on glutaminic acid residue, the peptide chain of the cut-out polypeptide of part, obtains simultaneously trifluoroacetyl group acetic acid lattice draw for thunder (trifluoroacetyl group copolymer-1).Then with the piperidines aqueous solution, remove the trifluoroacetyl group protecting group on lysine residue, obtain thick copolymer-1.Finally, through dialysis, by solution freeze-drying, obtain copolymer-1 bulk drug.
For copolymer-1 or the terpolymer that can guarantee to obtain has drug effect, just must know accurately that the molecular-weight average of polypeptide mixture in building-up process distributes and change.Existing ordinary method can obtain the molecular-weight average of copolymer-1, but with regard to the process control of whole technique, these class methods are not suitable for.Because multipolymer and the trifluoroacetyl base co-polymer of protection are all water insoluble, and copolymer-1 is soluble in water, ordinary method is also in aqueous systems, to measure the molecular-weight average of copolymer-1.And also can there is certain error because of differences such as the structure of product to be measured, compositions in the column chromatography that is generally used for determining molecular weight.
Therefore; in this area, still need in copolymer-1 building-up process, to measure more exactly the molecular weight of intermediate; especially the multipolymer of protecting and the molecular weight of trifluoroacetyl base co-polymer; correctly obtain the rule that in copolymer-1 building-up process, molecular-weight average and molecular weight distribution change, thereby strengthen the Quality control of intermediates in copolymer-1 building-up process.
Summary of the invention
The present invention is exactly in the process of synthetic copolymer-1, and strengthening process is controlled, and finds relation and Changing Pattern between copolymer-1, trifluoroacetyl group copolymer-1 and the molecular weight of copolymer-1 of protection.So the molecular-weight average of the multipolymer of measuring and protecting and trifluoroacetyl base co-polymer and molecular weight distribution are just particularly important accurately.In the present invention, utilized the interior mark of conduct of two serial synthetic polypeptide; determined the correction coefficient of chromatographic column; obtained molecular weight more accurately, for finding relation and Changing Pattern between multipolymer, trifluoroacetyl base co-polymer and the molecular weight of copolymer-1 of protection that reliable Data support is provided.
In one embodiment, the invention provides a kind of interior mark product 1 that synthesize for thunder that draw for acetic acid lattice, comprise and be selected from following 3-7 bar polypeptide:
Table 1. internal standard peptide series (wherein TFA represents trifluoroacetyl group, and Bn represents benzyl, and NEt2 represents that carbon teminal is N, N-diethylamide)
In one embodiment, the invention provides a kind of interior mark product 2 that synthesize for thunder that draw for acetic acid lattice, comprise and be selected from following 3-7 bar polypeptide:
Table 2. internal standard peptide series two (wherein TFA represents trifluoroacetyl group)
In one embodiment, described internal standard peptide is all comprised of L-amino acid.
In one embodiment, described internal standard peptide is all comprised of D-amino acid.
In one embodiment, the invention provides described interior mark product 1 as the molecular-weight average of the copolymer-1 of measuring and protecting and the interior target purposes of molecular weight distribution in copolymer-1 building-up process.
In one embodiment, the invention provides described interior mark product 2 as measure the molecular-weight average of trifluoroacetyl group copolymer-1 and the interior target purposes of molecular weight distribution in copolymer-1 building-up process.
In one embodiment, the invention provides a kind ofly for the molecular-weight average of copolymer-1 at copolymer-1 building-up process measuring and protecting and the method for molecular weight distribution, wherein use aforesaid interior mark product 1 as interior mark.
In one embodiment, the invention provides a kind ofly for measure the molecular-weight average of trifluoroacetyl group copolymer-1 and the method for molecular weight distribution in copolymer-1 building-up process, wherein use aforesaid interior mark product 2 as interior mark.
In one embodiment, described molecular weight is to use column chromatography to measure.
Accompanying drawing explanation
Logarithm and the retention time linear graph (transverse axis is RT, and the longitudinal axis is the logarithm of molecular weight) of the molecular weight of mark product 1 in Fig. 1
Logarithm and the relative retention time linear graph (transverse axis is the logarithm of molecular weight, and the longitudinal axis is RRT) of mark product 1 molecular weight in Fig. 2
Logarithm and the retention time linear graph (transverse axis is RT, and the longitudinal axis is the logarithm of molecular weight) of mark product 2 molecular weight in Fig. 3
Logarithm and the relative retention time linear graph (transverse axis is the logarithm of molecular weight, and the longitudinal axis is RRT) of mark product 2 molecular weight in Fig. 4
Embodiment
The solid phase synthesis of the internal standard peptide of the interior mark product 1 of embodiment 1.
Article seven, molecular weight synthesizes (in Table 3 and 4) from the daltonian molecular weight internal standard peptide of 5400-17000 by Shenzhen client of Han Yu medicine company limited-liability company peptide service department.These internal standard peptides are numbered TV-##-PR, and wherein ## is amino-acid residue number (for example: TV-35-PR is exactly the peptide chain that contains 35 amino-acid residues).Amino acid whose composition conforms to the eigenwert of copolymer-1, be that the amino acid molar ratio that L-Ala, L-glutamic acid, Methionin and tyrosine form is 0.392～0.462:0.129～0.153:0.300～0.374:0.086～0.100, about 5000～9000 dalton of molecular-weight average.
TV-35-PR synthetic method: adopt substitution degree 0.5mmol/g2-CTC resin 20g, put into solid state reaction post, DMF swelling 30 minutes, takes out DMF; Take Fmoc-Ala-OH6.2g, with 30ml DMF, dissolve, add 6.9ml DIPEA activation 3 minutes, add reaction column, react 2 hours, add 50ml20% piperidines/DMF solution, remove Fmoc protecting group, DMF washing 6 times, triketohydrindene hydrate detects color.
Take Fmoc-Glu(Bn)-OH9.3g, HObt2.97g, with 30ml DMF, dissolve, add 3.4ml DIC activation 3 minutes, add reaction column, react 2 hours, triketohydrindene hydrate detects water white transparency.According to TV-35-PR peptide order, coupling one by one, obtains TV-35-PR peptide resin 64.8g; the peptide resin obtaining adopts trifluoroethanol: methylene dichloride=1:4(V:V) 650ml cracking is 3 hours, filters out resin, adds ether sedimentation; centrifugal, the dry TV-35-PR deammoniation protection peptide 44.1g that obtains.
The full guard peptide 44g obtaining is joined in 2L flask, with 200ml tetrahydrofuran (THF), dissolve, add 200ml20% diethylamine tetrahydrofuran solution, react 5 hours, concentrated, HPLC purifying obtains TV-35-PR.
Other several interior marks are adopted and are used the same method.
Peptide order and the molecular weight of table 3 internal standard peptide
In table 4. effect, each internal standard peptide amino acid of mark product 1 forms
In embodiment 2. is used, mark product 1 are proofreaied and correct chromatographic column
Adopt TSKgel HHR(7.8 * 3000mm, 5 μ m) take DMF as moving phase, flow velocity 0.5ml/min, wavelength 275nm, sample introduction concentration is 10mg/ml, sample size is 20ul, carries out the correction of these seven internal standard peptides of interior mark.These internal standard peptides and the logarithm of retention time corresponding molecular weight with it of the multipolymer of protection are certain dependency.Its formula is:
LogMw=A+B * RT or Mw=10 (A+B * RT)
Wherein Mw is molecular weight, and RT is retention time, and A and B are respectively intercept and slope.In order to calculate more accurately the molecular weight of each component, we have adopted acetone to calculate as interior target relative retention time, and wherein the retention time of acetone is 39.8 minutes.
The chromatographic column of the interior mark product 1 of table 5. is proofreaied and correct result (Fig. 1,2)
* RRT=RT/RT (acetone);
Formula logMw=A+B * RT calculation result for *;
Formula RRT=B1+B2 * logMw calculation result for * *;
The solid phase synthesis of the internal standard peptide of the interior mark product 2 of embodiment 3.
Article seven, molecular weight synthesizes (in Table 3 and 5) from the daltonian molecular weight internal standard peptide of 4900-15400 by Shenzhen client of Han Yu medicine company limited-liability company peptide service department.These internal standard peptides are numbered TV-##-TFA, and wherein ## is amino-acid residue number (for example: TV-35-TFA is exactly the peptide chain that contains 35 amino-acid residues).Amino acid whose composition conforms to the eigenwert of copolymer-1.
TV-35-TFA synthetic method: adopt substitution degree 0.5mmol/g2-CTC resin 20g, put into solid state reaction post, DMF swelling 30 minutes, takes out DMF; Take Fmoc-Ala-OH6.2g, with 30ml DMF, dissolve, add 6.9ml DIPEA activation 3 minutes, add reaction column, react 2 hours, add 50ml20% piperidines/DMF solution, remove Fmoc protecting group, DMF washing 6 times, triketohydrindene hydrate detects color.
Take Fmoc-Glu(Bn)-OH9.3g, HObt2.97g, with 30ml DMF, dissolve, add 3.4ml DIC activation 3 minutes, add reaction column, react 2 hours, triketohydrindene hydrate detects water white transparency.According to TV-35-TFA peptide order, coupling one by one, obtains TV-35-TFA peptide resin 64.8g; the peptide resin obtaining adopts trifluoroethanol: methylene dichloride=1:4(V:V) 650ml cracking is 3 hours, filters out resin, adds ether sedimentation; centrifugal, the dry TV-35-TFA deammoniation protection peptide 44.1g that obtains.
Other several interior marks are adopted and are used the same method.
Table 6. forms as each internal standard peptide amino acid of interior mark product 2
In embodiment 4. is used, mark product 2 are proofreaied and correct chromatographic column
Adopt TSKgel HHR(7.8 * 3000mm, 5 μ m), take DMF as moving phase, flow velocity 0.5ml/min, wavelength 275nm, sample introduction concentration is 10mg/ml, sample size is 20ul, carries out the correction of these seven internal standard peptides of interior mark.These internal standard peptides and the logarithm of retention time corresponding molecular weight with it of the multipolymer of protection are certain dependency.Its formula is:
LogMw=A+B * RT or Mw=10 (A+B * RT)
Wherein Mw is molecular weight, and RT is retention time, and A and B are respectively intercept and slope.In order to calculate more accurately the molecular weight of each component, we have adopted acetone to calculate as interior target relative retention time.
The chromatographic column of the interior mark product 2 of table 7. is proofreaied and correct result (Fig. 3,4)
*rRT=RT/RT (acetone);
*by formula logMw=A+B * RT calculation result;
* *by formula RRT=B1+B2 * logMw calculation result.

Claims (10)

1. for acetic acid lattice, draw for the synthetic interior mark product of thunder, comprise and be selected from following 3-7 bar polypeptide:
TV-35-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)E(Bn)K(TFA)AAK(TFA)K(TFA )AYK(TFA)K(TFA)E(Bn)AK(TFA)AK(TFA)AAE(Bn)AAAK(TFA)E (Bn)AAYE(Bn)A-NEt2
TV-45-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)AK(TFA)AE(Bn)K(TFA)AK(TF A)K(TFA)AYK(TFA)AAE(Bn)AK(TFA)K(TFA)AAK(TFA)YE(Bn)K (TFA)AAAE(Bn)K(TFA)AAAK(TFA)E(Bn)AAYE(Bn)A-NEt2
TV-56-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)E(Bn)K(TFA)AYAK(TFA)K(TF A)AE(Bn)K(TFA)AAK(TFA)K(TFA)AE(Bn)AK(TFA)AYK(TFA)AA E(Bn)AK(TFA)K(TFA)K(TFA)AE(Bn)AK(TFA)YK(TFA)AE(Bn)AA K(TFA)AAAK(TFA)E(Bn)AAYE(Bn)A-NEt2
TV-66-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)E(Bn)K(TFA)AYAK(TFA)AK(T FA)K(TFA)AE(Bn)AK(TFA)AAK(TFA)K(TFA)AK(TFA)AE(Bn)AK (TFA)K(TFA)YAK(TFA)AAK(TFA)AE(Bn)K(TFA)K(TFA)E(Bn)YA AAE(Bn)AK(TFA)YK(TFA)AE(Bn)AAK(TFA)AAAK(TFA)E(Bn)A AYE(Bn)A-NEt2
TV-77-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)E(Bn)K(TFA)AYAK(TFA)K(TF A)AE(Bn)K(TFA)AAK(TFA)K(TFA)AE(Bn)AK(TFA)AYK(TFA)AA E(Bn)AK(TFA)K(TFA)K(TFA)AK(TFA)AE(Bn)AK(TFA)K(TFA)YA K(TFA)AAK(TFA)AE(Bn)K(TFA)K(TFA)E(Bn)YAAAE(Bn)AK(TF A)YK(TFA)AE(Bn)AAK(TFA)AAAK(TFA)E(Bn)AAYE(Bn)A-NEt2
TV-86-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)E(Bn)K(TFA)AYAK(TFA)K(TF A)AE(Bn)K(TFA)AAK(TFA)K(TFA)AE(Bn)AK(TFA)AYK(TFA)AA E(Bn)AK(TFA)K(TFA)K(TFA)AK(TFA)AE(Bn)AK(TFA)K(TFA)YA K(TFA)AAK(TFA)AE(Bn)K(TFA)K(TFA)E(Bn)YAAAE(Bn)AK(TF A)YK(TFA)AE(Bn)AAK(TFA)K(TFA)AYK(TFA)AE(Bn)AAK(TFA) AAAK(TFA)E(Bn)AAYE(Bn)A-NEt2
With
TV-109-PR AK(TFA)K(TFA)YAK(TFA)K(TFA)AE(Bn)K(TFA)AYAK(TFA)K(T FA)AK(TFA)AAK(TFA)E(Bn)K(TFA)K(TFA)AYAK(TFA)K(TFA)E (Bn)AK(TFA)AYK(TFA)AAE(Bn)AK(TFA)K(TFA)K(TFA)AK(TFA )AE(Bn)AK(TFA)K(TFA)YAK(TFA)E(Bn)AAK(TFA)AK(TFA)K(TF A)E(Bn)AYK(TFA)AE(Bn)AK(TFA)K(TFA)YAK(TFA)AAK(TFA)A E(Bn)K(TFA)K(TFA)E(Bn)YAAAE(Bn)AK(TFA)K(TFA)AE(Bn)AA K(TFA)AYK(TFA)AE(Bn)AAK(TFA)AAAK(TFA)E(Bn)AAYE(Bn) A-NEt2
Wherein TFA represents trifluoroacetyl group, and Bn represents benzyl, and NEt2 represents that carbon teminal is N, N-diethylamide.
2. for acetic acid lattice, draw for the synthetic interior mark product of thunder, comprise and be selected from following 3-7 bar polypeptide:
TV-35-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)EK(TFA)AAK(TFA)K(TFA)A YK(TFA)K(TFA)EAK(TFA)AK(TFA)AAEAAAK(TFA)EAAYEA- OH
TV-45-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)AK(TFA)AEK(TFA)AK(TFA) K(TFA)AYK(TFA)AAEAK(TFA)K(TFA)AAK(TFA)YEK(TFA)A AAEK(TFA)AAAK(TFA)EAAYEA-OH
TV-56-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)EK(TFA)AYAK(TFA)K(TFA) AEK(TFA)AAK(TFA)K(TFA)AEAK(TFA)AYK(TFA)AAEAK(TF A)K(TFA)K(TFA)AEAK(TFA)YK(TFA)AEAAK(TFA)AAAK(TF A)EAAYEA-OH
TV-66-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)EK(TFA)AYAK(TFA)AK(TF A)K(TFA)AEAK(TFA)AAK(TFA)K(TFA)AK(TFA)AEAK(TFA)K (TFA)YAK(TFA)AAK(TFA)AEK(TFA)K(TFA)EYAAAEAK(TFA )YK(TFA)AEAAK(TFA)AAAK(TFA)EAAYEA-OH
TV-77-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)EK(TFA)AYAK(TFA)K(TFA) AEK(TFA)AAK(TFA)K(TFA)AEAK(TFA)AYK(TFA)AAEAK(TF A)K(TFA)K(TFA)AK(TFA)AEAK(TFA)K(TFA)YAK(TFA)AAK( TFA)AEK(TFA)K(TFA)EYAAAEAK(TFA)YK(TFA)AEAAK(TFA )AAAK(TFA)EAAYEA-OH
TV-86-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)EK(TFA)AYAK(TFA)K(TFA) AEK(TFA)AAK(TFA)K(TFA)AEAK(TFA)AYK(TFA)AAEAK(TF A)K(TFA)K(TFA)AK(TFA)AEAK(TFA)K(TFA)YAK(TFA)AAK( TFA)AEK(TFA)K(TFA)EYAAAEAK(TFA)YK(TFA)AEAAK(TFA )K(TFA)AYK(TFA)AEAAK(TFA)AAAK(TFA)EAAYEA-OH
With
TV-109-TFA AK(TFA)K(TFA)YAK(TFA)K(TFA)AEK(TFA)AYAK(TFA)K(TF A)AK(TFA)AAK(TFA)EK(TFA)K(TFA)AYAK(TFA)K(TFA)EAK (TFA)AYK(TFA)AAEAK(TFA)K(TFA)K(TFA)AK(TFA)AEAK(T FA)K(TFA)YAK(TFA)EAAK(TFA)AK(TFA)K(TFA)EAYK(TFA) AEAK(TFA)K(TFA)YAK(TFA)AAK(TFA)AEK(TFA)K(TFA)EY AAAEAK(TFA)K(TFA)AEAAK(TFA)AYK(TFA)AEAAK(TFA)A AAK(TFA)EAAYEA-OH。
3. interior mark product claimed in claim 1, wherein said polypeptide is all formed or all D-amino acid, is consisted of by L-amino acid.
4. interior mark product claimed in claim 2, wherein said polypeptide is all formed or all D-amino acid, is consisted of by L-amino acid.
5. the interior mark product described in claim 1 or 3 draw for the molecular-weight average of thunder and the interior target purposes of molecular weight distribution as the acetic acid lattice of measuring and protecting in acetic acid lattice draw for thunder building-up process.
6. the interior mark product described in claim 2 or 4 draw for the molecular-weight average of thunder and the interior target purposes of molecular weight distribution as measure trifluoroacetyl group acetic acid lattice in acetic acid lattice draw for thunder building-up process.
7. for draw the acetic acid lattice for thunder building-up process measuring and protecting to draw for the molecular-weight average of thunder and a method for molecular weight distribution at acetic acid lattice, wherein the interior mark product described in right to use requirement 1 or 3 are as interior mark.
8. for drawing at acetic acid lattice for thunder building-up process measurement trifluoroacetyl group acetic acid lattice, draw for the molecular-weight average of thunder and a method for molecular weight distribution, wherein the interior mark product described in right to use requirement 2 or 4 are as interior mark.
9. the purposes described in claim 5 or 6, wherein said molecular weight is to use column chromatography to measure.
10. method claimed in claim 7, wherein said molecular weight is to use column chromatography to measure.
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CN112649538A (en) * 2015-04-28 2021-04-13 深圳翰宇药业股份有限公司 High performance liquid chromatography analysis method for polypeptide mixture
CN112649537A (en) * 2015-04-28 2021-04-13 深圳翰宇药业股份有限公司 High performance liquid chromatography analysis method for polypeptide mixture
CN112649538B (en) * 2015-04-28 2024-03-29 深圳翰宇药业股份有限公司 High performance liquid chromatography analysis method for polypeptide mixture
CN112649537B (en) * 2015-04-28 2024-03-29 深圳翰宇药业股份有限公司 High performance liquid chromatography analysis method for polypeptide mixture
CN106932459A (en) * 2015-12-31 2017-07-07 深圳翰宇药业股份有限公司 A kind of polypeptide polymer capillary isoelectric focusing analysis method

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