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CN105651848A - Protective-agent-containing capillary gel electrophoresis detection kit - Google Patents

Protective-agent-containing capillary gel electrophoresis detection kit Download PDF

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Publication number
CN105651848A
CN105651848A CN201410640495.4A CN201410640495A CN105651848A CN 105651848 A CN105651848 A CN 105651848A CN 201410640495 A CN201410640495 A CN 201410640495A CN 105651848 A CN105651848 A CN 105651848A
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China
Prior art keywords
buffer
acid
damping fluid
preferable
test kit
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Pending
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CN201410640495.4A
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Chinese (zh)
Inventor
方伟杰
王海彬
黄春辉
王宗襄
张中伟
白骅
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Zhejiang Haizheng Borui Biopharmaceutical Co., Ltd.
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Zhejiang Hisun Pharmaceutical Co Ltd
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Priority to CN201410640495.4A priority Critical patent/CN105651848A/en
Publication of CN105651848A publication Critical patent/CN105651848A/en
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Abstract

The present invention relates to a protective-agent-containing capillary gel electrophoresis detection kit, a protective agent is selected from white sugar, a polyol or an amino acid. The kit can improve the accuracy of capillary electrophoresis detection of the purity of biopharmaceuticals such as antibodies, reduces the detection deviation generated in an actual experimental operation process and can better reflect actual contents of various isomers, especially debris, of antibody molecules contained in a sample itself, and antibodies include recombinant anti-HER2 humanized monoclonal antibodies, recombinant human-mouse chimeric anti-tumor-necrosis-factor-alpha monoclonal antibodies, recombinant anti-interleukin-6 receptor humanized monoclonal antibodies, T-DM1, recombinant anti-tumor-necrosis-factor-alpha fully-humanized monoclonal antibody injections and recombinant anti-CD52 humanized monoclonal antibodies.

Description

A kind of containing protectant capillary gel electrophoresis detection kit
Technical field
The invention belongs to biological technical field, particularly relate to and a kind of can accurately detect bio-pharmaceuticals such as the capillary gel electrophoresis detection kit of antibody purity.
Background technology
Capillary electrophoresis (capillaryelectrophoresis, CE) technology is developed by conventional gel electrophoresis (gelelectrophoresis), it is taking the highfield of high voltage electric generation as motivating force, taking the quartz capillary of microbore as split tunnel. compare traditional gel electrophoresis, CE have automatization, fast, the accurate advantage such as quantitative, high resolving power, a lot of biomolecules, such as protein, polysaccharide and nucleic acid etc. can use capillary electrophoresis to analyze. sodium laurylsulfonate capillary electrophoresis (CE-SDS) technology adds linear gel in kapillary dissociating buffer to form molecular sieve, add anion surfactant sodium laurylsulfonate (sodiumdodecylsulfate, SDS) damping fluid to sample makes protein denaturation, and with protein is combined by the mass ratio of 1: 1.4, owing to this kind of protein-bonded mass-to-charge ratio of SDS is identical, the electrical forces that this associated proteins is subject to when carrying out kapillary and be separated is also identical, but owing to the size of differing molecular is different, thus in linear gel molecular sieves, the resistance size being subject to is moved different, make rate of migration different, thus the molecule realizing apparent upper molecular weight different is separated. non-reduced type CE-SDS iodo-acid amide processes, main different fragments, weight chain and the covalently bound polymer impurity detected in monoclonal antibody sample.
U.S.'s Beckman company PA800+ type capillary electrophoresis apparatus and supporting detection kit contain following component: sample buffer (samplebuffer), gel buffer liquid (gelbuffer), scavenging solution (0.1MNaOH, 0.1MHCl), interior mark (interstandard), reference standards (controlstandard), wherein sample buffer (samplebuffer) is containing 100mMTris-HCl, 1%SDS and 12.5mM iodo-acid amide. we use the sample buffer in this test kit the anti-HER 2 humanized monoclonal antibody of restructuring (mAbl) carried out the different structure of non-reduced type CE-SDS molecular size analyze time, find under the testing conditions (70 DEG C are heated 10 minutes) of Beckman manufacturer's recommended, the shive content (as containing a light chain of antibody L and the fragment containing heavy chain of antibody-heavy chain-light chain HHL) obtained is (see Fig. 1, CE-SDS main peak content 89.5%, shive content 10.5%) obviously higher than the content using additive method to obtain such as size exclusive chromatography (SEC-HPLC) (see Fig. 2, SEC-HPLC main peak content > 99%, shive content < 0.5%).By document (the .Suppressionofsodiumdodecylsulfate-polyacrylamidegelelec trophoresissamplepreparationaritfactsforanalysisofIgG4ha lf-antibodies.AnalyticalBiochemistry such as TaylorFR, 2006,353,204-208.) and our data analysis, these fragment impurity increased may be combines due to antibody and SDS and carry out the degraded product of formation in heat-treatment process. The iodo-acid amide of manufacturer's recommended is used can obviously to reduce shive content as inhibitor; but shive content is still obviously higher (as antibody mAbl purity made containing 12.5mM iodo-acid amide to be increased to 89.5% from 81% as protective material, but continue to increase iodo-acid amide concentration and purity can only be made to be increased to 90.6% to 37.5mM). Therefore, how to find a kind of formation that can obviously reduce fragment, thus to improve bio-pharmaceuticals such as the method for antibody test accuracy be problem demanding prompt solution.
Such as, it has been found that containing, on the basis of iodo-acid amide, the protective material adding other types can also improve antibody further, the CE-SDS of mAbl detects purity, namely can more accurately reflect the quality of antibody drug, thus complete the present invention.
Summary of the invention
The actual technical problem to be solved of the present invention; it it is exactly the deficiency for existing non-reduced type CE-SDS detection method; providing a kind of containing protectant capillary gel electrophoresis detection kit, this test kit can improve the accuracy that non-reduced type CE-SDS detects antibody molecule size isomer.
The technical solution used in the present invention is:
A kind of capillary gel electrophoresis detection kit containing sample buffer; containing a kind of protective material in described sample buffer (samplebuffer); described protective material concentration is 10-5000mM, it is preferable to 100-2000mM, it is more preferable to 500-1000mM.
Wherein, protective material of the present invention can suppress biological medicament if monoclonal antibody owing to occurring degraded to form fragment (fragmentation) in sample handling processes, and this protective material is selected from sugar, polyvalent alcohol or amino acid. In the present invention, amino acid is the wide in range implication of this area, comprises the form of its pharmacologically acceptable salt. Wherein, described sugar can be selected from fructose, glucose, sucrose, trehalose, seminose, lactose, maltose, sorbose, dextran, dextrin, cyclodextrin, hydroxyethylamyle or its combination, it is preferable that glucose, sucrose, trehalose or seminose. Described polyvalent alcohol can be selected from N.F,USP MANNITOL, glycerine, sorbyl alcohol, Saccharum lactis, maltose alcohol, Xylitol, propylene glycol, polyoxyethylene glycol or its combination, it is preferable that N.F,USP MANNITOL. Amino acid can be selected from L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, ��-amino-isovaleric acid or its combination.
Wherein, sample buffer of the present invention is further containing damping fluid, described damping fluid is selected from Tris damping fluid, phosphoric acid buffer, acetate buffer, succinate buffer, grape acid buffer, citrate buffer solution, ascorbate buffer, tartaric acid buffer, maleic acid damping fluid, lactic acid buffer, carbonic acid buffer, phenylformic acid damping fluid, imidazole buffer or buffered with amino acid liquid, preferred Tris damping fluid, it is more preferable to Tris-HCl damping fluid.The content of this damping fluid makes the pH value of described sample buffer between 3.0-10.0.
Wherein, sample buffer of the present invention is further containing sodium laurylsulfonate (SDS), and its content is between 0.1-5% (w/w), it is preferable that 0.5-2% (w/w), it is more preferable to 1% (w/w).
Wherein, test kit of the present invention is further containing iodo-acid amide, and its content is between 1-200mM, it is preferable that between 5-50mM, it is more preferable to 10-30mM. Wherein other components in iodo-acid amide and test kit separately store separately, it may also be useful to front configure mixing.
Preferably, the sample buffer in test kit of the present invention is containing, for example lower component:
(1) Tris-HCl damping fluid 100mM
(2) sodium laurylsulfonate (SDS) 1% (w/w)
(3) glucose or sucrose or trehalose or N.F,USP MANNITOL 500mM
PH value is 9.0.
Invention further provides the preparation method of sample buffer as above; comprise: by the damping fluid of recipe quantity, it is preferable that Tris damping fluid, the sodium laurylsulfonate of recipe quantity and the protective material of recipe quantity; preferred glucose; sucrose, trehalose or N.F,USP MANNITOL, be dissolved in appropriate deionized water; with salt acid for adjusting pH value to 3.0-10.0; preferably 9.0, then it is settled to 500mL with deionized water, obtains sample buffer. During use, the iodo-acid amide of recipe quantity is dissolved in deionized water, it is mixed with the iodo-acid amide solution of 250mM, then testing sample sample buffer is diluted to 1mg/ml, mix according to the ratio of 95 �� L: 5 �� L with iodo-acid amide solution again, carry out the purity detecting of the non-reduced type CE-SDS of antibody molecule size isomer afterwards again.
Invention further provides test kit as above when antagonist molecule carries out non-reduced type CE-SDS detection, suppress the purposes in antibody molecule cracking, wherein, described antibody molecule is preferably recombinated anti-HER 2 humanized monoclonal antibody, the chimeric anti-TNF-�� monoclonal antibody of recombinant human mouse, anti-interleukin-6 acceptor Humanized monoclonal antibodies of recombinating, T-DM1, restructuring anti-tumor necrosis factor-�� total man's resource monoclonal antibody or anti-CD52 Humanized monoclonal antibodies of recombinating, it is more preferable to be anti-HER 2 humanized monoclonal antibody of recombinating.
The useful effect of the present invention is:
Use the present invention with the addition of protectant capillary gel electrophoresis detection kit; according to the sample treatment (70 DEG C are heated 10 minutes) that Beckman company of CE manufacturer is recommended; capillary electrophoresis detection bio-pharmaceuticals can be improved such as the accuracy of antibody purity; reduce the detection error owing to experimental implementation process produces, more can reflect the actual content of antibody molecule size isomer that sample itself contains particularly fragment.
Accompanying drawing explanation
Fig. 1 does not add the non-reduced type CE-SDS collection of illustrative plates of the anti-HER 2 humanized monoclonal antibody of protectant restructuring (mAb1)
Fig. 2 recombinates the SEC-HPLC collection of illustrative plates of anti-HER 2 humanized monoclonal antibody (mAb1)
In Fig. 3 embodiment 2, dissimilar protective material is on the non-reduced type CE-SDS purity impact of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1)
In Fig. 4 embodiment 3, dissimilar protective material is on the non-reduced type CE-SDS purity impact of restructuring anti-tumor necrosis factor-�� people's mouse chimeric mAb (mAb2)
In Fig. 5 embodiment 4, different sucrose is on the non-reduced type CE-SDS purity impact of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1)
In Fig. 6 embodiment 5, sucrose is on the non-reduced type CE-SDS purity impact of different monoclonal antibody or antibody-small molecules conjugate
Embodiment
Below by way of specific embodiment, the present invention is described further.It must be noted that following examples are for illustration of the present invention, instead of limitation of the present invention.
The non-reduced type CE-SDS purity detecting of antibody in following embodiment all uses the method that Beckman company recommends.
The preparation of embodiment 1 one typical agents boxes
Take Tris6.06g, SDS5.0g, sucrose 75g, with 420mL deionized water dissolving to clear, then with 3M salt acid for adjusting pH to 9.0, then it is settled to 500mL with deionized water, obtains sample buffer.
The iodo-acid amide of 46mg is dissolved in 1mL deionized water, is mixed with the iodo-acid amide solution of 250mM.
By recombinating, anti-HER 2 humanized monoclonal antibody (mAb1) is diluted to 1mg/mL with the above-mentioned sample buffer prepared, then mixes in the ratio of 95 �� L: 5 �� L with the above-mentioned iodo-acid amide solution prepared.
The dissimilar protective material of embodiment 2 is on the non-reduced type CE-SDS purity impact of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1)
Compare dissimilar protective material to the impact of the non-reduced type CE-SDS purity of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1). The results are shown in Table 1 and Fig. 3. Wherein, the effect of sucrose and trehalose is best, and CE-SDS purity is increased to 92.1% from 89.5%; The effect of N.F,USP MANNITOL is taken second place, and purity is 91.6%; And the effect of glycine is worst, it is only 89.7%.
Table 1. is containing different protectant reagent constituents and on restructuring anti-HER 2 humanized monoclonal antibody (mAb1) CE-SDS main peak content impact
Note: #1 is the test kit of Beckman company, and all the other are for the addition of all kinds of protectant test kit on this basis, and various protectant concentration is 500mM.
The dissimilar protective material of embodiment 3 is on the non-reduced type CE-SDS purity impact of restructuring anti-tumor necrosis factor-�� people's mouse chimeric mAb (mAb2)
Compare dissimilar protective material to the impact of the non-reduced type CE-SDS purity of restructuring anti-tumor necrosis factor-�� people's mouse chimeric mAb (mAb2). The results are shown in Table 2 and Fig. 4. Wherein, glucose, trehalose and sucrose effect are best, and CE-SDS purity is increased to 91.6%, 91.3% and 91.0% respectively from 88.9%; The effect of sorbyl alcohol, glycine and N.F,USP MANNITOL is taken second place, and purity is respectively 90.2%, 90.2% and 90.0%; And the effect of glycerine is worst, it is only 89.1%.
Table 2. is containing different protectant reagent constituents and on restructuring anti-tumor necrosis factor-�� people's mouse chimeric mAb (mAb2) CE-SDS main peak content impact
Note: #6 is the test kit of Beckman company, and all the other are for the addition of all kinds of protectant test kit on this basis, and various protectant concentration is 500mM.
Embodiment 4 different sucrose is on the non-reduced type CE-SDS purity impact of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1)
The sucrose comparing different concns, on the impact of the purity of the non-reduced type CE-SDS of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1), the results are shown in Table 3 and Fig. 5. In sensing range (146-1020mM), the protected effect of sucrose and its concentration are directly related, and concentration is more high, and protected effect is more obvious.
The reagent component of table 3. containing different sucrose is to the CE-SDS main peak content balance of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1)
# Sucrose concentration (mM) Non-reduced type CE-SDS main peak (%)
1 0 89.5
14 146 91.3
15 292 91.6
16 439 91.8
17 731 92.0
18 877 92.2
19 1020 92.5
Embodiment 5 sucrose (500mM) is on the non-reduced type CE-SDS purity impact of different monoclonal antibody
Compare sucrose (500mM) to different monoclonal antibody or antibody-small molecules conjugate, comprise the impact of the non-reduced type CE-SDS purity of the anti-HER 2 humanized monoclonal antibody of restructuring (mAb1), the chimeric anti-TNF-�� monoclonal antibody (mAb2) of recombinant human mouse, anti-interleukin-6 acceptor Humanized monoclonal antibodies (mAb3) of recombinating, T-DM1 (mAb4), restructuring anti-tumor necrosis factor-�� total man's resource monoclonal antibody injection liquid (mAb5) and the anti-CD52 Humanized monoclonal antibodies (mAb6) of restructuring.The results are shown in Table 4 and Fig. 6. Due to the difference of antibody character, the stability shown in CE-SDS sample use process is different, and the protected effect of sucrose is also different. The protected effect that generally speaking more stable antibody (such as mAb5-mAb6) sucrose itself embodies is less, and the provide protection of the antibody of less stable (such as mAb1-mAb4) sucrose own is just clearly (main peak purity difference is 2.5-3.6%).
Table 4. sucrose (500mM) is on the impact of different monoclonal antibody or the non-reduced type CE-SDS main peak purity of antibody-small molecules conjugate

Claims (10)

1. one kind contains the capillary gel electrophoresis detection kit of sample buffer, it is characterised in that, containing a kind of protective material in described sample buffer.
2. test kit according to claim 1, it is characterised in that, described protectant concentration is 10-5000mM, it is preferable to 100-2000mM, it is more preferable to 500-1000mM.
3. test kit according to claim 1 and 2, it is characterised in that, described protective material is selected from sugar, polyvalent alcohol or amino acid.
4. test kit according to claim 3, it is characterized in that, described sugar is selected from fructose, glucose, sucrose, trehalose, seminose, lactose, maltose, sorbose, dextran, dextrin, cyclodextrin, hydroxyethylamyle or its combination, it is preferable to glucose, sucrose, trehalose or seminose; Described polyvalent alcohol is selected from N.F,USP MANNITOL, glycerine, sorbyl alcohol, Saccharum lactis, maltose alcohol, Xylitol, propylene glycol, polyoxyethylene glycol or its combination, it is preferable that N.F,USP MANNITOL; Described amino acid is selected from L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, ��-amino-isovaleric acid or its combination.
5. test kit according to the arbitrary item of claim 1-4, it is characterized in that, described sample buffer is further containing damping fluid, preferably, described damping fluid is selected from Tris damping fluid, phosphoric acid buffer, acetate buffer, succinate buffer, grape acid buffer, citrate buffer solution, ascorbate buffer, tartaric acid buffer, maleic acid damping fluid, lactic acid buffer, carbonic acid buffer, phenylformic acid damping fluid, imidazole buffer or buffered with amino acid liquid, preferred Tris damping fluid, more preferably Tris-HCl damping fluid, the content of described damping fluid makes the pH value of described sample buffer between 3.0-10.0.
6. test kit according to the arbitrary item of claim 1-5, it is characterized in that, described sample buffer is further containing sodium laurylsulfonate, and its content is between 0.1-5% (w/w), preferred 0.5-2% (w/w), it is more preferable to 1% (w/w).
7. test kit according to the arbitrary item of claim 1-6, it is characterised in that, described test kit is further containing iodo-acid amide, and its content is between 1-200mM, it is preferable that between 5-50mM, it is more preferable to 10-30mM.
8. test kit according to the arbitrary item of claim 1-7, it is characterised in that, described sample buffer is containing, for example lower component:
(1) Tris-HCl damping fluid 100mM
(2) sodium laurylsulfonate 1% (w/w)
(3) glucose or sucrose or trehalose or N.F,USP MANNITOL 500mM
PH value is 9.0.
9. prepare the method for the sample buffer in the claim 1-8 described test kit of arbitrary item for one kind, it is characterised in that, described method comprises:
By the damping fluid of recipe quantity; preferred Tris damping fluid; the sodium laurylsulfonate of recipe quantity and the protective material of recipe quantity, it is preferable that glucose, sucrose; trehalose or N.F,USP MANNITOL; it is dissolved in appropriate deionized water, with salt acid for adjusting pH value to 3.0-10.0, it is preferable that 9.0; then it is settled to 500mL with deionized water, obtains sample buffer.
10. test kit described in the arbitrary item of claim 1-8 antagonist molecule carry out non-reduced type CE-SDS detect time, suppress the purposes in antibody molecule cracking, wherein, described antibody molecule is preferably recombinated anti-HER 2 humanized monoclonal antibody, the chimeric anti-TNF-�� monoclonal antibody of recombinant human mouse, anti-interleukin-6 acceptor Humanized monoclonal antibodies of recombinating, T-DM1, restructuring anti-tumor necrosis factor-�� total man's resource monoclonal antibody or anti-CD52 Humanized monoclonal antibodies of recombinating, it is more preferable to be anti-HER 2 humanized monoclonal antibody of recombinating.
CN201410640495.4A 2014-11-13 2014-11-13 Protective-agent-containing capillary gel electrophoresis detection kit Pending CN105651848A (en)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN113189184A (en) * 2021-04-28 2021-07-30 浙江大学 Capillary gel electrophoresis sample buffer solution containing cysteine
CN118348098A (en) * 2024-03-31 2024-07-16 珠海联邦生物医药有限公司 A gel separation buffer for capillary electrophoresis detection

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CN104132992A (en) * 2000-09-25 2014-11-05 应用生物系统有限公司 High speed, high resolution compositions, methods, and kits for capillary electrophoresis
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113189184A (en) * 2021-04-28 2021-07-30 浙江大学 Capillary gel electrophoresis sample buffer solution containing cysteine
CN113189184B (en) * 2021-04-28 2022-09-09 浙江大学 Capillary gel electrophoresis sample buffer containing cysteine
WO2022227363A1 (en) * 2021-04-28 2022-11-03 浙江大学 Capillary gel electrophoresis sample buffer containing cysteine
CN118348098A (en) * 2024-03-31 2024-07-16 珠海联邦生物医药有限公司 A gel separation buffer for capillary electrophoresis detection

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