WO2016155620A1 - 一种蛋白质二硫键配对分析方法 - Google Patents

一种蛋白质二硫键配对分析方法 Download PDF

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WO2016155620A1
WO2016155620A1 PCT/CN2016/077810 CN2016077810W WO2016155620A1 WO 2016155620 A1 WO2016155620 A1 WO 2016155620A1 CN 2016077810 W CN2016077810 W CN 2016077810W WO 2016155620 A1 WO2016155620 A1 WO 2016155620A1
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protein
peptide
analysis
disulfide
fragment
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PCT/CN2016/077810
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English (en)
French (fr)
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吕锋华
谭青乔
张倩倩
环民霞
黄黎明
刘周阳
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三生国健药业（上海）股份有限公司
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Priority claimed from CN201510145272.5A external-priority patent/CN106153746B/zh
Priority claimed from CN201510145271.0A external-priority patent/CN106153745B/zh
Priority claimed from CN201510145067.9A external-priority patent/CN106153744B/zh
Priority claimed from CN201510146095.2A external-priority patent/CN106153747B/zh
Application filed by 三生国健药业（上海）股份有限公司 filed Critical 三生国健药业（上海）股份有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a protein disulfide bond pairing analysis method.
  • Disulfide bonds are a common post-translational modification of proteins that cross-link two cysteines within a chain or between chains to play a role in forming a stable protein space structure, maintaining a correct spatial conformation, and regulating biological activity. Very important role.
  • the disulfide bond is properly distributed to facilitate rapid folding of the peptide chain and form a tight, stable spatial structure, forming a local hydrophobic center, which prevents water molecules from entering the interior of the peptide to break hydrogen bonds and maintain a stable high-level structural region.
  • the disulfide bond has only relative stability, and its covalent bond is easily broken by reduction.
  • the disulfide bond is broken down to a mercapto group, the structure and conformation of the protein are inevitably changed, and the original or the part may be completely lost.
  • Biological function, antibody protein with high thiol content is often low in biological activity and thermal stability (Chaderjian WB, Chin ET, Harris RJ, et al. Effect of copper sulfateon performance of a serum-free CHO cell culture process and the level of Free thiol in the recombinant antibody expressed. Biotechnology Progress 2005; 21(2): 550-553; Lacy ER, Baker M, Brigham-Burke M.
  • the antibody protein Denosumab is a typical IgG2 type antibody with four pairs of disulfide bonds in its hinge region (Liu H, May K. Disulfide bond structures of IgG molecules. mAbs 2012; 4:1 17- 23), its own structure is easy to form disulfide bonds of different pairing methods.
  • monitoring the disulfide bond pairing can determine the maturity of antibody protein process development.
  • the existing technical methods are mostly combined with several specific enzymes to confirm the disulfide pairing form of Denosumab, which is very inconvenient.
  • Golimumab (golimumab, see WO 02/12502) is a fully human anti-TNF-a IgG1 ⁇ monoclonal antibody developed by Centocor, which has been approved by the United States, Japan and other countries for the treatment of moderate to severe activities. Rheumatoid arthritis, moderate to severe ulcerative colitis, psoriatic arthritis and ankylosing spondylitis. At present, the disulfide pairing analysis of Golimumab is mostly carried out by using a combination of several specific enzymes for analysis and confirmation, which is very inconvenient.
  • Cetuximab (Cetuximab, WO9640210 WO9640210 WO9640210 WO9640210WO9640210 WO9640210 WO9640210) is a chimeric antibody protein which has been approved for the treatment of colorectal cancer in many countries and is an important anticancer drug comprising 6 pairs of intrachain disulfide bonds, 1 pair of chains. An inter-disulfide bond and a pair of hinge region disulfide bonds.
  • the current cetuximab disulfide bond analysis requires a combination of multiple specific enzymes for enzymatic digestion to confirm, which is very inconvenient.
  • rhCTLA4-Ig is a modified Fc (hinge, CH2 and CH3 domain) that binds the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) to human immunoglobulin G1 (IgG1)
  • CTLA-4 cytotoxic T-lymphocyte-associated antigen 4
  • IgG1 human immunoglobulin G1
  • TNFR2 Fc fusion protein Etanercept is a recombinant human type II tumor necrosis factor receptor (TNFR2) fused to the Fc segment of IgG1 antibody. It is a recombinant protein dimer that is fused to two human p75sTNFR molecules.
  • the Fc fragment of IgG1 molecule contains 934 amino acids, the monomer contains 467 amino acids (235 of which are the extracellular part of p75sTNFR), and the remaining 232 amino acids are the Fc part of human immunoglobulin G1, including CH2, CH3 and hinge
  • TNF tumor necrosis factor
  • the TNFR2 Fc fusion protein contains a total of 58 cysteine residues, wherein the Fc segment contains 4 pairs of disulfide bonds, the hinge region has 3 pairs of disulfide bonds, and the Fab segment, the TNFR2 region, has up to 22 pairs of disulfide bonds, and
  • the amino acid sequence of this region contains a large number of spatially adjacent or adjacent cysteine residues, forming an extremely complex and compact disulfide bond core region, which is divided into four cysteine-rich regions according to its structural features (CRD). ), including cysteine knots, nested bridges, etc. (Mukai Y, Nakamura T, Yoshikawa M, et al.
  • the glycosylated part of the TNFR2 Fc fusion protein accounts for about 1/3 of the total molecular weight.
  • the sTNFR region also contains two complex N-saccharides. The basement site and a large number of O-glycosylation sites, complex glycosylation modifications undoubtedly increase the difficulty of disulfide pairing analysis (Houel S, Hilliard M, Yu Y, et al.
  • LC-MS/MS Liquid chromatography-tandem mass spectrometry
  • CID collision-induced dissociation
  • LC-MS/MS in a single CID fragmentation mode can resolve relatively simple IgG1/IgG2 antibodies by enzymatic cleavage of antibody proteins denatured under non-reducing conditions, comparison of disulfide peptides and corresponding free thiol-containing peptides after reduction. Disulfide bond pairing, but the sequence-specific, structurally complex, highly glycosylated TNFR2Fc fusion protein is beyond the reach.
  • Electron transfer dissociation is another fragmentation mode different from CID. It can also fragment the linear peptide backbone to produce c/z ions, but it is more inclined when analyzing disulfide-linked peptide ions.
  • the disulfide bond is homogenized to form a single peptide or a partially reduced peptide.
  • a method combining the advantages of both CID and ETD fragmentation modes has been successfully used for the characterization of protein disulfide bonds (Wu SL, Jiang H, Lu Q, et al. Mass spectrometric determination of disulfide linkages in recombinant therapeutic proteins using on-line LC -MS with electron transfer dissociation (ETD).
  • ETD electron transfer dissociation
  • the object of the present invention is to provide a method for protein disulfide pairing analysis, which can form a peptide fragment suitable for mass spectrometry by a specific protease according to the molecular structure of the protein to be analyzed, and can easily and accurately confirm disulfide. Key connection characterization.
  • the present inventors have found that the specific protease can be used to digest the target protein, which can be well avoided. Describe the shortcomings of the technology.
  • the optimum pH of most proteolytic enzymes is generally alkaline, but under alkaline conditions, it is easy to cause mismatches in disulfide bonds of structural protein samples rich in adjacent or similar cysteine and cysteine knots, nested bridges, etc.
  • Specific proteases that are active under acidic conditions are used. In addition to shielding the free sulfhydryl groups by alkylating agents, the probability of disulfide bond mismatches can also be reduced.
  • Common alkylating agents are sodium iodoacetate (IAA), iodoacetamide (IAM), N-ethylmaleimide (NEM) and the like.
  • IAA sodium iodoacetate
  • IAM iodoacetamide
  • NEM N-ethylmaleimide
  • the present inventors have skillfully integrated different proteolytic enzymes for fusion proteins rich in cysteine, highly glycosylated, cysteine knots, nested bridges, and the like, such as TNFR2 Fc fusion proteins.
  • the present invention has been completed by a method of cutting and a plurality of sample processing methods in combination with a plurality of tandem mass spectrometry methods to comprehensively analyze the disulfide bond pairing condition.
  • a protein disulfide paired analysis method comprising preparing a protein sample for analysis and performing mass peptide mapping analysis on the sample to obtain a disulfide linkage characterization of the protein, comprising the steps of:
  • step 2) determining a specific protease type according to the molecular structure of the protein to be analyzed, and digesting the denatured protein obtained in step 1) with the specific protease determined under non-reducing conditions;
  • the specific protease may be selected from one or several combinations of trypsin, chymotrypsin, elastase, intracellular protease Lys-C and endonuclease Glu-C. According to the characteristics of the protein molecule to be analyzed, it is confirmed whether it is necessary to remove the N-glycan (the N-glycan site containing the N-glycan site generally needs to remove the N-glycan), and can be further removed by using the peptide N-glycosidase F before the step 1). The step of the N-glycan of the protein.
  • step 1) protein denaturation can be carried out using guanidine hydrochloride, and the preferred concentration of guanidine hydrochloride is 5.0 to 7.6 M.
  • the present invention also provides specific disulfide pairing analysis methods for various protein molecules for different protein molecules.
  • a method for disulfide pairing analysis of an IgG2 type monoclonal antibody comprising:
  • step b) digesting the denatured protein obtained in step a) with trypsin under non-reducing conditions;
  • step d) The two samples obtained in step c) were subjected to enzymatic hydrolysis, and the mass peptide spectrum analysis was carried out using the ESI-Q-TOF MS method.
  • the IgG2 type monoclonal antibody is Denosumab.
  • the buffer used in the denaturation treatment is guanidine hydrochloride; in the preferred denatured sample system, the concentration of guanidine hydrochloride is from 5 M to 7.6 M.
  • the denaturation treatment uses a buffer of 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3.
  • the specific enzyme is trypsin; preferably trypsin is used in an amount of 1:5 to 12 (wt/wt, enzyme/protein); more preferably trypsin is used in an amount of 1:10 ( Wt/wt, enzyme/protein).
  • the specific enzyme is preferably selected from one or more of the group consisting of trypsin, chymotrypsin, elastase, and intracellular protease Lys-C; the amount of trypsin is preferably 1:25 (wt/wt, enzyme/protein); the amount of chymotrypsin is preferably 1:50 (wt/wt, enzyme/protein); the amount of elastase is preferably 1:50 (wt/wt, The amount of Lys-C enzyme is preferably 1:200 (wt/wt, enzyme/protein).
  • one of the samples is reduced using DTT in step c); preferably the concentration of DTT is 60 mM.
  • the formic acid (FA) is added to the step d) to terminate the enzymatic hydrolysis; preferably the formic acid is added to a final concentration of 0.1%.
  • step d the ESI-Q-TOF MS method is used for analysis, and the obtained data is processed and analyzed by BiopharmaLynx software.
  • a method for disulfide pairing analysis of golimumab comprising:
  • step i) enzymatically digesting the denatured protein obtained in step i) with trypsin and the intracellular protease Lys-C under non-reducing conditions;
  • step iii) The two samples obtained in step iii) were subjected to enzymatic hydrolysis, and the mass peptide spectrum analysis was carried out using the ESI-Q-TOF MS method.
  • the protein is denatured in step i) using guanidine hydrochloride (GuHCl); preferably, the concentration of guanidine hydrochloride in the denatured sample system is from 5.0 M to 7.6 M.
  • guanidine hydrochloride preferably, the concentration of guanidine hydrochloride in the denatured sample system is from 5.0 M to 7.6 M.
  • the specific enzyme in step ii) is trypsin and/or intracellular protease Lys-C; preferably trypsin is used in an amount of 1:25 (wt/wt, enzyme/protein); preferably intracellular protease Lys The amount of -C is 1:200 (wt/wt, enzyme/protein).
  • the trypsin is used in an amount of 1:25 (wt/wt, enzyme/protein) and the intracellular protease Lys-C is used in an amount of 1:200 (wt/wt, enzyme/protein).
  • one of the samples is reduced using DTT in step iii); preferably the DTT concentration in the sample system is 60 mM.
  • the formic acid FA is added to the step iv) to terminate the enzymatic hydrolysis; preferably, the final volume concentration of the FA is 0.1%.
  • one of the samples in step iv) is added with 0.1 M DTT and incubated in a 37 ° C water bath for 30 min; both samples are separately added with formic acid FA to a final volume concentration of 0.1% to terminate the enzymatic hydrolysis reaction. .
  • step iv) the disulfide pairing confirmation of the protein is analyzed by the ESI-Q-TOFMS method, and the obtained data is processed and analyzed by BiopharmaLynx software.
  • a method for disulfide pairing analysis of cetuximab comprising:
  • step III a denatured protein obtained by the enzymatic hydrolysis of step II) with trypsin and intracellular protease Lys-C under non-reducing conditions;
  • a method for disulfide pairing analysis of the fusion protein rhCTLA4-Ig comprising the steps of:
  • step II digesting the denatured protein obtained in step II) with trypsin and/or chymotrypsin under non-reducing conditions;
  • step E) The two samples obtained in the step D) were subjected to the enzymatic hydrolysis reaction, and the mass peptide spectrum analysis was carried out using the ESI-Q-TOF MS method.
  • the N-glycan of the fusion protein is removed using the peptide N-glycosidase F (PNGase F) in step A); preferably the pH of the solution is 7.0 to 8.0.
  • PNGase F peptide N-glycosidase F
  • the N-glycan removal of the fusion protein is achieved by taking a sample of the fusion protein, adding a solution such as 1% FA (volume concentration) or 1% NH 3 .H 2 O (volume concentration), The pH was adjusted to 7.0 to 8.0, the peptide N-glycosidase F was added, and after mixing, the mixture was incubated at 37 ° C for 24 hours.
  • the N-glycoside-removed fusion protein is denatured using a high-concentration guanidine hydrochloride solution; preferably, the guanidine hydrochloride (GuHCl) concentration is 6 M; more preferably, 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M is used.
  • the fusion protein was denatured by Tris, pH 8.3.
  • the specific enzyme in step C) comprises trypsin and/or chymotrypsin; preferably trypsin is used in an amount of 1:25 (wt/wt, enzyme/protein); preferably the amount of chymotrypsin is used. It is 1:50 (wt/wt, enzyme/protein).
  • the incubation temperature of the specific enzyme enzymatic fusion protein in step C) is 37 ° C
  • the incubation time is 2 to 4 hours
  • the incubation time is preferably 4 hours.
  • the amount of trypsin is 1:25 (wt/wt, enzyme/protein) and the amount of chymotrypsin is 1:50 (wt/wt, enzyme/protein).
  • the one sample is reduced using DTT in step D); preferably the concentration of DTT is 60 mM.
  • formic acid is added to step E) to terminate the enzymatic hydrolysis; preferably the formic acid is added to a final volume concentration of 0.1%.
  • step E the ESI-Q-TOFMS method is used for analysis, and the obtained data is processed and analyzed by BiopharmaLynx software.
  • a method for the disulfide pairing analysis of the TNFR2Fc fusion protein etanercept comprising:
  • the pH of the reaction system of steps 1 and 3 is 6.5.
  • the reaction system of step 3 contains an alkylating agent N-ethylmaleimide.
  • the denaturation means that under the action of a high concentration of salt or the like, secondary bonds such as hydrogen bonds between proteins are destroyed, and the protein molecules are changed from the originally ordered curly compact structure to the disordered loose stretch. Structure, but its primary structure has not changed.
  • the reduction means that the protein disulfide bond is broken by the reducing reagent.
  • the disulfide bond pairing is also basically the same, and the main difference between IgG1 and IgG2 is the difference in the number of disulfide bonds in the hinge region. Therefore, in the ESI-Q-TOF MS analysis mode, it is mainly shown that the variable region of the monoclonal antibody Fab segment has different amino acid sequence disulfide bond peptides and corresponding fragment ion maps containing free thiol peptides.
  • Figure 1 is a fragment diagram of an antibody protein disulfide peptide (1C/2C) fragment
  • Figure 2 is a fragment diagram of an antibody protein disulfide peptide (3C/4C) fragment
  • Figure 3 is a fragment diagram of the antibody protein disulfide peptide (5C/8C) fragment ion;
  • Figure 4 is a fragment diagram of the antibody protein disulfide bond peptide (6C/7C) fragment
  • Figure 5 is a fragment diagram of an antibody protein disulfide peptide (9C/10C) fragment
  • Figure 6 is a fragment diagram of the antibody protein disulfide peptide (11C+12C+13C+14C/11C+12C+13C+14C) fragment ion;
  • Figure 7 is a fragment diagram of an antibody protein disulfide peptide (15C/16C) fragment ion
  • Figure 8 is a fragment diagram of the antibody protein disulfide peptide (17C/18C) fragment ion
  • Figure 9 is a fragment diagram of a free thiol peptide (1C) fragment
  • Figure 10 is a fragment diagram of free thiol peptide (2C) fragment ions
  • Figure 11 is a fragment diagram of free thiol peptide (3C) fragment ions
  • Figure 12 is a fragment diagram of a free thiol peptide (4C) fragment
  • Figure 13 is a fragment diagram of free thiol peptide (6C) fragment ions
  • Figure 14 is a fragment diagram of free thiol peptide (7C) fragment ions
  • Figure 15 is a fragment diagram of free thiol peptide (8C) fragment ions
  • Figure 16 is a fragment diagram of free thiol peptide (9C) fragment ions
  • Figure 17 is a fragment diagram of a free thiol peptide (10C) fragment
  • Figure 18 is a fragment diagram of a free thiol peptide (11C+12C+13C+14C) fragment
  • Figure 19 is a fragment diagram of a free thiol peptide (15C) fragment
  • Figure 20 is a fragment diagram of free thiol peptide (17C) fragment ions
  • Figure 21 is a fragment diagram of a free thiol peptide (18C) fragment
  • Figure 22 is a fragment diagram of a monoclonal antibody disulfide bond peptide (1C/2C) fragment
  • Figure 23 is a fragment diagram of a monoclonal antibody disulfide bond peptide (5C/10C) fragment
  • Figure 24 is a fragment diagram of a monoclonal antibody disulfide peptide (6C/7C) fragment ion;
  • Figure 25 is a fragment diagram of a monoclonal antibody disulfide peptide (8C/9C) fragment ion;
  • Figure 26 is a fragment diagram of a monoclonal antibody disulfide peptide (11C+12C/11C+12C) fragment ion;
  • Figure 27 is a fragment diagram of free thiol peptide (1C) fragment ions
  • Figure 28 is a fragment diagram of free thiol peptide (2C) fragment ions
  • Figure 29 is a fragment diagram of free thiol peptide (5C) fragment ions
  • Figure 30 is a fragment diagram of free thiol peptide (6C) fragment ions
  • Figure 31 is a fragment diagram of a free thiol peptide (7C) fragment
  • Figure 32 is a fragment diagram of a free thiol peptide (8C) fragment
  • Figure 33 is a fragment diagram of a free thiol peptide (9C) fragment
  • Figure 34 is a fragment diagram of a free thiol peptide (11C+12C) fragment
  • Figure 35 is a fragment diagram of an antibody protein disulfide bond peptide (1C/2C) fragment
  • Figure 36 is a fragment diagram of the antibody protein disulfide peptide (6C/7C) fragment ion
  • Figure 37 is a fragment diagram of free thiol peptide (1C) fragment ions
  • Figure 38 is a fragment diagram of free thiol peptide (2C) fragment ions
  • Figure 39 is a fragment diagram of free thiol peptide (6C) fragment ions
  • Figure 40 is a fragment diagram of a free thiol peptide (7C) fragment
  • 41 to 52 are the results of disulfide pairing analysis of the fusion protein rhCTLA4-Ig in Example 4 of the present invention.
  • Figure 41 is a fragment diagram of a fusion protein disulfide bond peptide (1C/4C) fragment
  • Figure 42 is a fragment diagram of a fusion protein disulfide bond peptide (2C/3C) fragment
  • Figure 43 is a fragment diagram of free thiol peptide (1C) fragment ions
  • Figure 44 is a fragment diagram of free thiol peptide (4C) fragment ions
  • Figure 45 is a fragment diagram of free thiol peptide (2C) fragment ions
  • Figure 46 is a fragment diagram of free thiol peptide (3C) fragment ions
  • Figure 47 is a fragment diagram of a fusion protein disulfide bond peptide (5C/5C) fragment
  • Figure 48 is a fragment diagram of a fusion protein disulfide bond peptide (2C/3C) fragment
  • Figure 49 is a fragment diagram of a fusion protein disulfide bond peptide (6C/7C) fragment
  • Figure 50 is a fragment diagram of free thiol peptide (5C) fragment ions
  • Figure 51 is a fragment diagram of free thiol peptide (2C) fragment ions
  • Figure 52 is a fragment diagram of free thiol peptide (3C) fragment ions
  • 53-63 are the results of paired analysis of the TNFR2 Fc fusion protein Etanercept disulfide bond in Example 5 of the present invention.
  • FIG. 53A is a second fragment of the CID of the antibody protein disulfide bond peptide (1C/2C+3C/5C) according to an embodiment of the present invention.
  • Piece ion map Fig. 53B is a disulfide bond peptide (1C/2C+3C/5C) ETD secondary fragment ion map, and C is a partially reduced ⁇ + ⁇ peptide CID tertiary fragment ion map;
  • Figure 54A is a diagram showing the Cid secondary fragment ion of the antibody protein disulfide peptide (7C/8C+9C/11C) according to an embodiment of the present invention
  • Figure 54B is a disulfide peptide (7C/8C+9C/11C) ETD secondary. Fragment ion map
  • Figure 54C is a partially reduced ⁇ + ⁇ peptide CID tertiary fragment ion map
  • FIG. 55A is a diagram showing a Cid secondary fragment ion of an antibody protein disulfide peptide (10C/12C+13C/16C) according to an embodiment of the present invention
  • FIG. 55B is a disulfide peptide (10C/12C+13C/16C) ETD secondary.
  • Fragment ion map Figure 55C is a partially reduced ⁇ + ⁇ peptide CID tertiary fragment ion map;
  • Figure 56A is a first-order mass spectrum of an antibody protein disulfide peptide (17C/18C+19C+20C+21C/22C), and Figure 56B is a disulfide peptide (17C/18C+19C+20C+21C). /22C) ETD secondary fragment ion map, Figure 56C is a partially reduced ⁇ + ⁇ peptide CID tertiary fragment ion map;
  • FIG. 57A is a diagram showing a second fragment ion of an antibody protein disulfide peptide (4C/6C) CID according to an embodiment of the present invention
  • FIG. 57B is a second fragment ion diagram of a disulfide peptide (4C/6C) ETD;
  • FIG. 58A is a diagram showing a Cid secondary fragment ion of an antibody protein disulfide peptide (14C/15C) according to an embodiment of the present invention
  • FIG. 58B is a second fragment ion diagram of a disulfide peptide (14C/15C) ETD;
  • FIG. 59A is a diagram showing a Cid secondary fragment ion of an antibody protein disulfide peptide (17C/18C) according to an embodiment of the present invention
  • FIG. 59B is a second fragment ion diagram of a disulfide peptide (17C/18C) ETD;
  • FIG. 60A is a diagram showing a Cid secondary fragment ion of an antibody protein disulfide peptide (23C/23C) according to an embodiment of the present invention
  • FIG. 60B is a second fragment ion diagram of a disulfide peptide (23C/23C) ETD;
  • FIG. 61A is a diagram showing a Cid secondary fragment ion of an antibody protein disulfide peptide (24C+25C/24C+25C) according to an embodiment of the present invention, and FIG. 61B is a second fragment ion diagram of a disulfide peptide (26C/27C) ETD;
  • FIG. 62A is a diagram showing a Cid secondary fragment ion of an antibody protein disulfide peptide (26C/27C) according to an embodiment of the present invention
  • FIG. 62B is a second fragment ion diagram of a disulfide peptide (26C/27C) ETD;
  • FIG. 63A is a diagram showing a Cid secondary fragment ion map of an antibody protein disulfide bond peptide (28C/29C) according to an embodiment of the present invention
  • FIG. 63B is a second fragment ion map of a disulfide bond peptide (28C/29C) ETD;
  • Each of the dotted lines between the amino acids in the peptides of Figures 1 to 52 indicates a fragment ion (b ion or y ion) of the detected peptide, y ion is from the carboxy terminus of the peptide, and b ion is from the peptide.
  • the amino terminus of the segment begins.
  • Figures 53 to 56 designate three peptides as ⁇ , ⁇ , ⁇ , respectively, in the order of the amino acid sequence of the peptide.
  • the more fragment ions are richer, indicating that the peptide is consistent with the theoretical peptide, and the fragment ions are less, which is not necessarily consistent with the theoretical peptide.
  • the length of the segment is related to the fragmentation mode. For peptide fragments that are too short or too long, the fragment ions are generally less in the induced collision ionization fragmentation mode.
  • the invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
  • the experimental methods in the following examples, which do not specify the specific conditions, were tested according to the conventional conditions or conditions recommended by the manufacturer.
  • the intracellular protease Lys-C enzyme used in the examples was purchased from Roche, and the remaining proteases were purchased from Promega unless otherwise stated.
  • denaturation refers to the destruction of secondary bonds such as hydrogen bonds between protein molecules under the action of high concentration of salt, etc., and the protein molecules change from the tight structure of the original order to the loose stretch of disorder. Structure, but its primary structure has not changed.
  • the definition of reduction means that the protein disulfide bond is broken by the reducing reagent.
  • the antibody protein used was Denosumab (supplied by Shanghai National Institute of Antibody Engineering).
  • the light protein of the antibody protein Denosumab contains five cysteine Cys, which are Cys23, Cys89, Cys135, Cys195 and Cys215, respectively.
  • the heavy chain contains 13 cysteine Cys, which are Cys22 and Cys96, respectively.
  • the ends are labeled as 6C-18C, and the theoretical paired chain disulfide bonds are 1C/2C, 3C/4C, 6C/7C, 9C/10C, 15C/16C, 17C/18C, and the interchain disulfide bond is 5C/8C.
  • the disulfide bond in the hinge region is 11C+12C+13C+14C/11C+12C+13C+14C.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to a 50 mM NH4HCO3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • Typical parameters of the ESI-Q-TOF MS analysis include mass spectrometry parameters and liquid phase conditions as shown in Table 1:
  • Mobile phase A 0.1% FA-H2O
  • mobile phase B 0.1% FA-ACN
  • mass spectrometry cleaning solution 50% CAN
  • mass spectrometer IntelliStart valve cleaning solution 50% MeOH
  • column temperature 45 ° C
  • detection wavelength 214 nm
  • Sample volume 5 ⁇ L or 10 ⁇ L
  • sample chamber temperature 10 ° C
  • liquid phase gradient elution conditions mobile phase B from 1% to 36% in 80 minutes.
  • Tables 2 to 3 are non-reducing condition antibody protein disulfide peptide segment molecular weight and theoretical molecular weight comparison table; Table 3 is a non-reducing condition antibody protein of Example 1 of the present invention. Comparison of molecular weight and theoretical molecular weight of free thiol peptides:
  • the disulfide peptide (1C/2C) fragment ion diagram is shown in Figure 1.
  • the disulfide bond peptide (3C/4C) fragment ion diagram is shown in Figure 2
  • the disulfide bond peptide (5C/8C) fragment ion map is shown in Figure 3
  • the disulfide bond peptide (6C/7C) fragment ion map is shown in Figure 4
  • the disulfide bond peptide (9C/10C) fragment ion diagram is shown in Figure 5
  • the disulfide bond peptide (11C + 12C + 13C + 14C / 11C + 12C + 13C + 14C) fragment ion diagram see the attached figure 6
  • disulfide bond peptide (15C / 16C) fragment ion diagram see Figure 7
  • disulfide bond peptide (17C / 18C) fragment ion diagram see Figure 8
  • the fragment ion diagram is shown in Figure 16.
  • the free thiol peptide (10C) fragment ion map is shown in Figure 17, and the free thiol peptide (11C+12C+13C+14C) fragment ion map is shown in Figure 18, free thiol peptide (15C).
  • the fragment ion map is shown in Figure 19, and the free thiol peptide (17C) fragment ion map is attached.
  • Figure 20 is a fragment diagram of the free thiol peptide (18C) fragment ion.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • the present invention can directly confirm all disulfide bond pairing manners of the antibody protein Denosumab under a non-reducing condition by a single enzyme trypsin, and can exclude the space of the special amino acid sequence of the antibody protein on the enzymatic protein.
  • the steric hindrance effect, all disulfide bond pairing can be confirmed by a single enzyme digestion method, and the method is simple, convenient, effective and reliable. At the same time, this method can detect partially mismatched disulfide peptides.
  • the monoclonal antibody golimumab light chain of the present invention contains five cysteine Cys, which are Cys23, Cys88, Cys135, Cys 195 and Cys215, respectively, and the heavy chain contains 11 cysteine Cys, respectively Cys22 and Cys96.
  • the monoclonal antibody used in the following examples was golimumab (supplied by Shanghai National Institute of Antibody Drug Engineering).
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • Table 4 is a comparison table of the molecular weight of the disulfide bond of the monoclonal antibody in non-reducing conditions and the theoretical molecular weight
  • Table 5 is a comparison table of the molecular weight of the monoclonal antibody free thiol peptide and the theoretical molecular weight:
  • the ion map of the disulfide bond peptide (1C/2C) fragment is shown in Figure 22, and the ion map of the disulfide bond peptide (5C/8C) fragment is shown in Figure 23.
  • Disulfide The bond peptide (6C/7C) fragment ion map is shown in Figure 24, and the disulfide bond peptide (9C/10C) fragment ion map is shown in Figure 25.
  • Disulfide bond peptide segment (11C+12C/11C+12C) fragment ion Figure 26 free thiol peptide (1C) fragment ion diagram see Figure 27, free thiol peptide (2C) fragment ion diagram see Figure 28, free thiol peptide (5C) fragment ion diagram see Figure 29
  • the free thiol peptide (6C) fragment ion map is shown in Figure 30
  • the free thiol peptide (7C) fragment ion map is shown in Figure 31
  • the free thiol peptide (8C) fragment ion map is shown in Figure 32
  • free thiol peptide (9C) Fragment ion diagram is shown in Figure 33
  • free thiol peptide (11C+12C) fragment ion diagram is shown in Figure 34.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 2.1.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 2.1.
  • the present invention can directly confirm all disulfide bond pairing methods of Golimumab by trypsin combined with intracellular protease Lys-C digestion method, and can exclude monoclonal antibody special.
  • the steric hindrance of the amino acid sequence to enzymatically dissociate the protein, all disulfide bond pairing can be confirmed by only one enzyme digestion method, and the method is simple, convenient, effective and reliable.
  • the Cetuximab light chain contains five cysteine Cys, Cys23, Cys88, Cys134, Cys194, Cys214, and the heavy chain contains 11 cysteine Cys, which are Cys22, Cys95, Cys146, respectively.
  • Cys 202, Cys222, Cys228, Cys231, Cys263, Cys323, Cys369, Cys427 are labeled as 1C to 5C from the N-terminus to the C-terminus of the light chain, and 6C from the N-terminus to the C-terminus of the heavy chain.
  • the theoretical paired chain disulfide bond is 1C/2C, 3C/4C, 6C/7C, 8C/9C, 13C/14C, 15C/16C
  • the interchain disulfide bond is 5C/10C
  • the disulfide bond in the hinge region is 11C+12C/11C+12C.
  • the experimental results show that the steric hindrance of the specific enzyme in the enzymatic cleavage of the protein can be ruled out by removing the antibody protein N-glycan by using the specific deglycosidase and then performing protein denaturation and then enzymatic hydrolysis.
  • the antibody protein used in the following examples was cetuximab (available from Shanghai National Institute of Antibody Engineering).
  • Example 1.1 The ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in Example 1.1.
  • Experimental results The experimental results are shown in the following table, wherein Table 6 is a non-reducing condition antibody protein disulfide bond peptide molecular weight and theoretical molecular weight comparison table; Table 7 is a non-reducing condition antibody protein free thiol peptide molecular weight and theoretical molecular weight comparison table.
  • the ion chromatogram of the antibody protein disulfide peptide (1C/2C) fragment is shown in Figure 35
  • the ion map of the disulfide peptide (6C/7C) fragment is shown in Figure 36.
  • the free thiol peptide (1C) fragment ion map is shown in Figure 37
  • the free thiol peptide (2C) fragment ion map is shown in Figure 38
  • the free thiol peptide (6C) fragment ion map is shown in Figure 39
  • free thiol peptide ( 7C) The fragment ion diagram is shown in Figure 40.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 3.1.
  • 0.5 mg of the protein sample was added to 380 ⁇ l of denaturing buffer (8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3), and incubated at 37 ° C for 60 minutes. Ultrafiltration was centrifuged or desalted to 50 mM NH 4 HCO 3 (pH 8.0) buffer via a desalting column.
  • denaturing buffer 8 M guanidine hydrochloride + 5 mM EDTA + 0.5 M Tris, pH 8.3
  • ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 3.1.
  • the present invention removes N-glycan by a specific deglycosidase, and can eliminate the steric hindrance effect of the specific protein N-glycan on the enzymatically cleavable protein, and reduce the N-sugar. Inhibition of the ionization efficiency of the peptide, the disulfide bond pairing method of the antibody protein (such as cetuximab) can be confirmed by trypsin combined with the intracellular protease Lys-C. The method is simple, convenient, effective and reliable. .
  • Example 4 Fusion of fusion protein (rhCTLA4-Ig) disulfide bond analysis
  • the fusion protein rhCTLA4-Ig single strand has 9 cysteine Cys, which are Cys21, Cys48, Cys 66, Cys 92, Cys120, Cys171, Cys231, Cys277, Cys335, respectively, from the N-terminus to the C-terminus.
  • 1C ⁇ 9C the theoretical pairing is a single-chain intrachain disulfide bond: 1C/4C, 2C/3C, 6C/7C, 8C/9C, interchain disulfide bond 5C/5C.
  • the protein is denatured by removing the fusion protein N-glycan by using a specific deglycosidase, and the 2C/3C pair of disulfide bond peptides containing N-glycan sites can be confirmed by digestion with trypsin and/or chymotrypsin. .
  • the experimental results also showed that the paired disulfide bond pairing modes of 2C/3C and 6C/7C after N-glycan removal of the fusion protein were confirmed by the digestion conditions of trypsin and/or chymotrypsin.
  • the fusion protein used in the following examples was rhCTLA4-Ig (supplied by Shanghai National Institute of Antibody Research).
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • Table 8 is a comparison table of molecular weight and theoretical molecular weight of the non-reducing condition fusion protein disulfide bond peptide of Example 1 of the present invention
  • Table 9 is a non-reducing condition fusion protein free sulfhydryl group of Example 1 of the present invention. Comparison of molecular weight of peptides with theoretical molecular weight.
  • the ion map of the disulfide bond peptide (1C/4C) fragment is shown in Figure 41, and the ion map of the disulfide bond peptide (2C/3C) fragment is shown in Figure 42.
  • the peptide (1C) fragment ion map is shown in Figure 43
  • the free thiol peptide (4C) fragment ion map is shown in Figure 44
  • the free thiol peptide (2C + 3C) fragment ion map is shown in Figure 45
  • free thiol peptide ( 6C) The fragment ion diagram is shown in Figure 46.
  • the ESI-Q-TOF MS analysis typical parameters including mass spectrometry parameters and liquid phase conditions are preferably the same as in 1.1.
  • Table 10 is a non-reducing condition fusion protein disulfide peptide segment molecular weight and theoretical molecular weight of the embodiment of the present invention
  • Table 11 is a non-reducing example 2 of the present invention
  • the disulfide bond peptide (5C/5C) fragment ion map is shown in Figure 47
  • the disulfide bond peptide (2C/3C) fragment ion map is shown in Figure 48
  • the key peptide (6C/7C) fragment ion diagram is shown in Figure 49
  • the free thiol peptide (5C) fragment ion map is shown in Figure 50
  • the free thiol peptide (2C) fragment ion map is shown in Figure 51
  • free thiol peptide (3C) The fragment ion diagram is shown in Figure 52.
  • the present invention removes N-glycan by a specific deglycosidase, and can eliminate the steric hindrance effect of the specific protein N-glycan on the enzymatically cleavable protein, and reduce the N-sugar. Inhibition of the ionization efficiency of the peptide, the N-glycan site disulfide bond peptide can be confirmed only by trypsin and/or chymotrypsin digestion, and the fragment ion information is rich, and all disulfide bond pairs are also It can be confirmed by only two kinds of enzyme digestion methods.
  • TNFR2 Fc fusion protein Etanercept single chain contains 29 cysteine Cys, Cys18, Cys31, Cys32, Cys35, Cys45, Cys53, Cys56, Cys71, Cys74, Cys78, Cys88, Cys96, Cys98 , Cys104, Cys112, Cys115, Cys121, Cys139, Cys142, Cys157, Cys163, Cys178, Cys240, Cys246, Cys249, Cys281, Cys341, Cys387, Cys445, from the N-terminus to the C-terminus, respectively, labeled 1C-29C, chain
  • the theoretical disulfide linkage of the inner Fab segment corresponds to: 1C/2C, 3C/5C, 4C/6C, 7C/8C, 9C/11C, 10C/12C, 13C/16C, 14C/15C, 17C/18C, 19C/20C 21C/22C; the disulfide bond in the hinge region is 23
  • the present invention is based on the amino acid sequence of the TNFR2 Fc fusion protein, preferably a combination of Trypsin, Lys-C and Glu-C, by reducing the pH of the reaction system to 6.5 while adding an alkylating agent to the reaction system.
  • N-ethylmaleimide (NEM) which shields the free sulfhydryl group, reduces the probability of disulfide bond mismatch and accurately confirms all disulfide bond pairs of the fusion protein.
  • the sample used was: TNFR2 Fc fusion protein Etanercept LC/MS (LC-MS/MS): Thermo Fisher Scientific Thermo Orbitrap Elite mass spectrometer with: ESI source, Orbitrap mass analyzer, and ETD source.
  • Liquid phase The system is Dionex's Ultimate 3000 and is equipped with: LPG-3400 (RS) pump, WPS-3000 (RS) injector, TCC-3000 (RS) column oven, VWD-3000 (RS) detector.
  • the data was collected using the software ThermoXcalibur, data processing and analysis software: Pepfinder, Protome Discoverer, pLabel.
  • a 1 mg antibody protein sample was dissolved in a 50 mM Tris-HCl (pH 6.5) system, 1 ⁇ L of PNGase F enzyme was added, and NEM was added to a final concentration of 2 mM, mixed, and incubated at 37 ° C for 18 hours.
  • Step two protein denaturation treatment:
  • guanidine hydrochloride denaturing solution 8M guanidine hydrochloride, 5 mM EDTA, 0.5 M Tris, pH 8.3
  • water to make guanidine hydrochloride concentration 6 M
  • N-ethyl Malay The imide (NEM) was mixed at a final concentration of 2 mM and incubated at 37 ° C for 60 minutes.
  • the denatured sample was desalted by ultrafiltration into a 100 mM Tris-HCl (pH 6.5) solution.
  • the digested samples were separated on an Acquity UPLC BEH300 C18 (1.7 ⁇ m 2.1 ⁇ 150 mm, Waters) column and then subjected to Thermo LTQ-Orbitrap Elite mass spectrometry, which was analyzed by various fragmentation modes, first obtaining the same precursor ion.
  • the CID secondary spectrum and the ETD secondary spectrum are then subjected to CID three-stage fragmentation of the first three largest abundance ions in the ETD spectrum.
  • liquid phase conditions are preferably as follows:
  • Mobile phase A 0.1% FA-H2O
  • mobile phase B 0.1% FA-ACN
  • column temperature 45 ° C
  • detection wavelength 214 nm
  • injection volume 10 ⁇ g
  • sample chamber temperature 10 ° C
  • flow rate 0.15 mL / min
  • Mobile phase B is from 3% to 20% in 60 minutes, and after 3 minutes to 35%.
  • the data were analyzed by Pepfinder and pLabel analysis software.
  • the basic pairing method of antibody protein disulfide bond can be obtained by CID secondary spectrum.
  • the artificial binding of ETD mass spectrometry can further confirm the connection between peptides.
  • pass ETD level 2 The CID tertiary spectrum of some of the ions in the spectrum can ultimately determine the way in which the two cysteines are linked.
  • the disulfide bond peptides confirmed by the present invention can be classified into three types according to their attachment characteristics.
  • the first class is the linkage of three peptides containing an interchain disulfide bond, including: 1C/2C+3C/5C, 7C/8C+9C/11C, 10C/12C+13C/16C, see Figures 53-55.
  • Pepfinder software can only match two peptide ions as a modification of another peptide when performing CID secondary spectral matching (see Figure 53A, Figure 54A and Figure 55A), although the match is good.
  • the second type of ion is a complex peptide ion containing both interchain and inter-/intra-peptide bonds: 17C/18C+19C+20C+21C/22C.
  • the current Pepfinder software cannot perform CID secondary spectral matching and requires manual de-split.
  • a rough calculation is carried out, and the possible peptide combination is presumed.
  • the single peptide and the partial reduction peptide are matched, and finally, the partially reduced peptide ⁇ + ⁇ is used.
  • the level spectrum determines the connection status of cysteine, see Figure 56.
  • FIG. 56A is a first-order mass spectrum of an antibody protein disulfide peptide (17C/18C+19C+20C+21C/22C), and FIG. 56B is a disulfide peptide (17C/18C+19C+20C+). 21C/22C) ETD secondary fragment ion map, Figure 56C is a partially reduced ⁇ + ⁇ peptide CID tertiary fragment ion map.
  • the third type is the ligation of two peptides containing inter-peptide bonds, including: 4C/6C, 14C/15C, 17C/18C, 23C/23C, 24C+25C/ 24C+25C, 26C/27C, 28C/29C
  • the invention of the present invention confirms the formation of a single peptide segment after the disulfide bond cleavage in the b/y fragment ion and the ETD secondary fragment ion map in the CID secondary fragment ion map.
  • 57A is a diagram showing an antibody protein disulfide peptide (4C/6C) CID secondary fragment ion map
  • FIG. 57B is a disulfide bond peptide (4C/6C) ETD secondary fragment ion map
  • FIG. 58A is In the embodiment of the present invention, the antibody protein disulfide bond peptide segment (14C/15C) CID secondary fragment ion map
  • FIG. 58B is the disulfide bond peptide segment (14C/15C) ETD secondary fragment ion map
  • FIG. 58A is In the embodiment of the present invention, the antibody protein disulfide bond peptide segment (14C/15C) CID secondary fragment ion map
  • FIG. 58B is the disulfide bond peptide segment (14C/15C) ETD secondary fragment ion map
  • FIG. 59A is an embodiment of the present invention Antibody protein disulfide peptide (17C/18C) CID secondary fragment ion map
  • Figure 59B is disulfide bond peptide (17C/18C) ETD secondary fragment ion map
  • Figure 60A is an antibody protein disulfide of the present invention Key peptide (23C/23C) CID secondary fragment ion map
  • FIG. 60B is a disulfide bond peptide (23C/23C) ETD secondary fragment ion map
  • 61A is an antibody protein disulfide bond peptide of the present invention (24C+25C/24C+25C) CID secondary fragment ion map
  • Figure 61B is a disulfide bond peptide (26C/27C) ETD secondary fragment ion map
  • Figure 62A is an antibody protein disulfide peptide (26C/27C) CID secondary fragment of the present invention. Ionogram, FIG. 62B is a disulfide bond peptide (26C/27C) ETD secondary fragment ion map;
  • 63A is a diagram showing the antibody protein disulfide peptide (28C/29C) CID secondary fragment ion diagram of the embodiment of the present invention
  • 63B is a disulfide bond peptide (28C/29C) ETD secondary fragment ion map.
  • the protein disulfide paired analysis method of the present invention can analyze the disulfide bond in the protein molecule simply, efficiently, and accurately, and has the advantages of good repeatability and simple spectrum analysis, and the invention is used.
  • the method can accurately and comprehensively confirm the disulfide-linked peptides in the protein molecule.
  • the process of the invention is suitable for quality monitoring of larger batches in industrial production.

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Abstract

一种蛋白质二硫键配对分析方法，根据待分析的蛋白质分子结构，以特异性蛋白酶酶解蛋白质，生成适于进行质谱分析的肽片段，可简单高效准确地确认二硫键连接表征。该方法重复性好，谱图解析简单，可准确全面地确证蛋白质分子中的二硫键连接的肽段，适于工业生产中较大批次的质量监控。

Description

一种蛋白质二硫键配对分析方法 技术领域
本发明属于生物技术领域，具体涉及一种蛋白质二硫键配对分析方法。
背景技术
二硫键是一种常见的蛋白质翻译后修饰，交联多肽链内或链间的两个半胱氨酸，在形成稳定的蛋白质空间结构、保持正确的空间构象、调节生物学活性方面起着非常重要的作用。二硫键正确配利于肽链迅速折叠并形成紧密的、稳定的空间结构，形成局部疏水中心，能阻止水分子进入肽的内部破坏氢键，维持稳定的高级结构区域。
二硫键只具有相对的稳定性，其共价键容易被还原而断裂，当二硫键被断开还原为巯基基团时，蛋白质结构及构象必然发生改变，可能完全或部分丧失原有的生物学功能，巯基含量较高的抗体蛋白往往生物活性和热稳定性较低(Chaderjian WB，Chin ET，Harris RJ，et al.Effect of copper sulfateon performance of a serum-free CHO cell culture process andthe level of free thiol in the recombinant antibody expressed.Biotechnology Progress 2005；21(2):550-553；Lacy ER,Baker M,Brigham-Burke M.Free sulfhydryl measurement as an indicator of antibody stability.Analytical Biochemistry 2008；382:66-8)。正确的二硫键配对方式对抗体蛋白药物生物学活性至关重要，错配或不能完全形成二硫键往往意味活性的降低(Ouellette D,Alessandri L,Chin A,et al.Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule.Analytical Biochemistry 2010；397:37-47)。
抗体蛋白狄迪诺塞麦(Denosumab)是一种典型的IgG2类型抗体，其铰链区有4对二硫键(Liu H，May K.Disulfide bond structures of IgG molecules.mAbs 2012；4:1 17-23)，其本身结构容易形成不同配对方式的二硫键。在细胞株克隆筛选、纯化工艺确认、制剂配方筛选、产品稳定性确认等研发周期过程中，监控二硫键配对可以判断抗体蛋白工艺开发的成熟度。现有的技术方法多是采用几种特异性酶进行联合酶解来确认Denosumab的二硫键配对形式，非常不方便。
Golimumab(戈利木单抗，参见WO 02/12502)是由Centocor公司开发的一种全人抗TNF-a的IgG1κ单克隆抗体，目前已被美国、日本等多国批准用于治疗中至重度活动型类风湿性关节炎、中度至重度溃疡性结肠炎、银屑病关节炎及强直性脊柱炎等多种疾病。目前Golimumab的二硫键配对分析，多是采用几种特异性酶进行联合酶解方法来进行分析确认，非常不方便。
西妥昔单抗(Cetuximab，
Figure PCTCN2016077810-appb-000001
WO9640210 WO9640210 WO9640210  WO9640210WO9640210)是一种嵌合型抗体蛋白，目前已在多个国家批准用于治疗结直肠癌，是一种重要的抗癌药物，其包括6对链内二硫键，1对链间二硫键和1对铰链区二硫键。但是，目前对西妥昔单抗二硫键分析需要采用多种特异性酶进行联合酶解才能确认，非常不方便。
现有技术对二硫键配对的确认，可以采用多种酶切方式结合MS/MS模式确认5对二硫键配对(K·J·莱斯特等人，组合物和用于生产组合物的方法，中国专利公开号：CN101448852)，但该方法存在酶切种类相对较多，且碎片离子不丰富等缺点。
rhCTLA4-Ig是将人细胞毒性T-淋巴细胞-联合抗原4(CTLA-4)的细胞外功能域连接到人免疫球蛋白G1(IgG1)的修饰Fc(铰合部，CH2和CH3功能域)上而成的一种融合蛋白，BMS公司已将其开发用于治疗类风湿性关节炎等疾病(通用名abatacept，商品名
Figure PCTCN2016077810-appb-000002
)，目前对rhCTLA4-Ig等融合蛋白的二硫键配分析方法往往需通过多种酶切才能确认，非常不方便。
TNFR2 Fc融合蛋白依那西普(Etanercept)是一种重组人II型肿瘤坏死因子受体(TNFR2)与IgG1抗体Fc段融合的蛋白，是重组蛋白二聚体，由两个p75sTNFR分子融合到人IgG1分子的Fc片段，含934个氨基酸，单体含467个氨基酸(其中235个氨基酸为p75sTNFR的胞外部分)，余下的232个氨基酸为人免疫球蛋白G1的Fc部分，包括CH2、CH3和铰链区域，能特异性结合人肿瘤坏死因子(TNF)α与β，目前主要用于治疗风湿性关节炎、银屑病和强制性脊柱炎。TNFR2 Fc融合蛋白共含有58个半胱氨酸残基，其中Fc段含4对二硫键，铰链区3对二硫键，而Fab段，即TNFR2区域，则高达22对二硫键，并且该区的氨基酸序列中含有大量空间相近或相邻的半胱氨酸残基，形成了极其复杂紧凑的二硫键核心区域，根据其结构特征被分成四个半胱氨酸富集区(CRD)，包括cysteine knots，nested bridges等结构(Mukai Y,Nakamura T,Yoshikawa M,et al.Solution of the Structure of the TNF-TNFR2 Complex.Sci Signal.2010；3(148):ra83)。与一般单克隆抗体不一样，TNFR2 Fc融合蛋白糖基化部分约占整个分子重量的1/3，除了Fc CH2区域的N-糖基化修饰外，sTNFR区域还含有2个复杂的N-糖基化位点和大量的O-糖基化修饰位点，复杂糖基化修饰无疑加大了二硫键配对分析的难度(Houel S,Hilliard M,Yu Y,et al.N-and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality.Anal.Chem.2014,86,576-584)。
液相色谱串联质谱(LC-MS/MS)因其分析效率快、准确度高、样品用量少等特点，已经成为抗体蛋白二硫键连接表征的重要方式(Goyder MS,Rebeaud F,Pfeifer ME,et al.Strategies in mass spectrometry for the assignment of Cys-Cys  disulfide connectivities in proteins.Expert Rev Proteomics.2013,10(5):489-501)。当前，LC-MS/MS分析中使用最普遍的是碰撞诱导解离(CID)模式，倾向于沿着多肽骨架断开肽键，生成b/y离子，但不易断开二硫键，因此在表征二硫键连接的肽段时往往为非典型的CID碎裂(Loo JA,Edmonds CG,Udseth，et al.Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra.1990,62(7):693-698)。通过酶切非还原条件下变性的抗体蛋白，对比二硫键肽段及还原后对应的含自由巯基肽段，单一CID碎裂模式下的LC-MS/MS能解析相对简单的IgG1/IgG2抗体二硫键配对，但对序列特异、结构复杂、高度糖基化的TNFR2Fc融合蛋白则力所不及。电子转移解离(ETD)是不同于CID的另外一种碎裂模式，也可以碎裂线性肽段骨架，产生c/z离子，但在分析二硫键连接的肽段离子时，更倾向于均裂二硫键，形成单独的肽段或部分还原的肽段。结合CID和ETD两种碎裂模式优点的方法已经成功用于蛋白二硫键的表征(Wu SL,Jiang H,Lu Q,et al.Mass spectrometric determination of disulfide linkages in recombinant therapeutic proteins using on-line LC-MS with electron transfer dissociation(ETD).Anal Chem.2009,8(1):112–122；Wu SL,Jiang H,Hancock WS,et al.Identification of the unpaired cysteine status and complete mapping the 17 disulfides of recombinant tissue plasminogen activator using LC-MS with ETD/CID.Anal Chem.2010,82(12):5296–5303；Wang Y,Lu Q,Wu SL,et al.Characterization and comparison of disulfide linkages and scrambling patterns in therapeutic monoclonal antibodies using LC-MS with electron transfer dissociation.Anal Chem.2011,83(8):3133–3140)。
尽管化学裂解的方法被报道较适用于含有大量空间相邻或相近半胱氨酸残基的蛋白二硫键配对分析，但也有反应程度难控制，特异性差，谱图复杂，解析困难，重复性差等缺陷。
由于高序列特异性、高度异质性、结构复杂等原因导致TNFR2 Fc融合蛋白的二硫键表征难度极大，目前对于其二硫键的表征仍然是基于X-ray的方法，但晶体衍射法难度大，周期长，不适用于工业生产中较大批次的质量监控。因此，简单、高效的单抗和融合蛋白二硫键配对分析方法仍是目前急需解决的技术问题。
发明内容
本发明的目的在于提供一种蛋白质二硫键配对分析方法，根据待分析的蛋白质分子结构，以特异性蛋白酶酶解蛋白质，生成适于进行质谱分析的肽片段，可简单高效准确地确认二硫键连接表征。
本发明人发现，采用特异性蛋白酶对目标蛋白进行酶切，能很好地避免上 述技术的缺陷。大部分蛋白水解酶的最适pH值一般为碱性，但碱性条件下容易造成富含相邻或相近半胱氨酸及cysteine knots、nested bridges等结构蛋白样品二硫键的错配，因此需使用酸性条件下具有活性的特异性蛋白酶。除通过烷基化试剂屏蔽自由巯基，也可以降低二硫键错配的几率。常见的烷基化试剂有碘乙酸钠(IAA)，碘乙酰胺(IAM)，N-乙基马来酰亚胺(NEM)等。针对富含半胱氨酸、高度糖基化、含cysteine knots、nested bridges等复杂二硫键结构的融合蛋白(例如TNFR2 Fc融合蛋白)，本发明人正是通过巧妙地综合不同蛋白水解酶酶切方法以及多种样品处理方法，并结合多种串联质谱方法才全面解析二硫键配对状况，从而完成了本发明。
一种蛋白质二硫键配对分析方法，包括制备用于分析的蛋白样品和将所述样品进行质量肽谱图分析，得到所述蛋白的二硫键连接表征，包括下述步骤：
1)对蛋白进行变性处理；
2)根据待分析蛋白质的分子结构确定特异性蛋白酶类型，在非还原条件下用所确定的特异性蛋白酶酶解步骤1)获得的变性的蛋白；
3)终止酶解反应得到用于分析的蛋白样品；和
4)分析步骤3)的蛋白样品获得所述蛋白的二硫键配对信息。
在本发明方法中，特异性蛋白酶可选自胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、胞内蛋白酶Lys-C和蛋白内切酶Glu-C的一种或几种组合。根据待分析的蛋白分子特点确认是否需要去除N-糖(含N-糖位点的二硫键肽段一般需去除N-糖)，可以进一步在步骤1)之前使用肽N-糖苷酶F去除所述蛋白质的N-糖的步骤。
步骤1)中可使用盐酸胍进行蛋白变性，优选的盐酸胍浓度为5.0～7.6M。
针对不同的蛋白质分子，在上述方法的基础上，本发明也提供了具体的各种蛋白质分子的二硫键配对分析方法。
在本发明的一个优选实施方案中，提供了IgG2型单克隆抗体的二硫键配对分析方法，所述方法包括：
a)用盐酸胍对蛋白进行变性处理；
b)在非还原条件下用胰蛋白酶酶解步骤a)获得的变性蛋白；
c)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
d)将步骤c)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
较佳的，所述IgG2型单抗如狄迪诺塞麦(Denosumab)。
较佳的，步骤a)中，所述变性处理使用的缓冲液为盐酸胍；优选的变性样品体系中盐酸胍浓度为5M～7.6M。
在一具体实施例中，所述变性处理使用的缓冲液为8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3。
较佳的，步骤b)中，所述特异性酶为胰蛋白酶；优选胰蛋白酶的用量为1﹕5～12(wt/wt，酶/蛋白)；更优选胰蛋白酶的用量为1﹕10(wt/wt，酶/蛋白)。
在其它优选实施例中，所述特异性酶较佳地选自：胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶以及胞内蛋白酶Lys-C中的一种或多种；胰蛋白酶的用量优选地为1﹕25(wt/wt，酶/蛋白)；胰凝乳蛋白酶的用量优选地为1:50(wt/wt，酶/蛋白)；弹性蛋白酶的用量优选地为1:50(wt/wt，酶/蛋白)；Lys-C酶的用量优选地为1:200(wt/wt，酶/蛋白)。
较佳的，步骤c)中使用DTT对所述其中一份样品进行还原；优选DTT的浓度为60mM。
较佳的，步骤d)中加入甲酸(FA)终止酶解；优选加入所述甲酸至体积终浓度0.1％。
较佳的，步骤d)中使用ESI-Q-TOF MS方法进行分析，所得数据采用BiopharmaLynx软件进行处理分析。
在另一具体实施例中，所述ESI-Q-TOF MS分析采用以下方法实现：将抗体蛋白肽段样品用50mM NH4HCO3(pH8.0)溶液稀释至1.5-2mg/mL(以酶解前蛋白浓度计)；然后Waters Acquity UPLC BEH300 C18 1.7μm 2.1×150mm色谱柱分离；进行Q-TOF质谱分析。
在本发明的一个优选实施方案中，提供了戈利木单抗的二硫键配对分析方法，所述方法包括：
i)用盐酸胍对蛋白进行变性处理；
ii)在非还原条件下用胰蛋白酶和胞内蛋白酶Lys-C联合酶解步骤i)获得的变性蛋白；
iii)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
iv)将步骤iii)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
较佳的，步骤i)中使用盐酸胍(GuHCl)对所述蛋白质进行变性处理；优选变性样品体系中盐酸胍浓度为5.0M～7.6M。
较佳的，步骤ii)中所述特异性酶为胰蛋白酶和/或胞内蛋白酶Lys-C；优选胰蛋白酶的用量为1﹕25(wt/wt，酶/蛋白)；优选胞内蛋白酶Lys-C的用量为1﹕200(wt/wt，酶/蛋白)。
在一具体实施例中，所述胰蛋白酶的用量为1﹕25(wt/wt，酶/蛋白)，胞内蛋白酶Lys-C的用量为1﹕200(wt/wt，酶/蛋白)。
较佳的，步骤iii)中使用DTT对所述其中一份样品进行还原；优选样品体系中DTT浓度为60mM。
较佳的，步骤iv)中加入甲酸FA终止酶解；优选FA终体积浓度为0.1％。
在另一具体实施例中，对步骤iv)中所述其中一份样品加入0.1M DTT，于37℃水浴孵育30min；两份样品均分别加入甲酸FA至终体积浓度0.1％，终止酶解反应。
较佳的，步骤iv)中，所述蛋白质的二硫键配对确认是通过ESI-Q-TOFMS方法进行分析，所得数据采用BiopharmaLynx软件进行处理分析。
在另一具体实施例中，ESI-Q-TOF MS分析采用以下方法实现：将蛋白质用50mM NH4HCO3(pH8.0)溶液稀释至1.5-2mg/mL(以酶解前蛋白浓度计)；然后Waters Acquity UPLC BEH300 C18 1.7μm 2.1×150mm色谱柱分离；进行Q-TOF质谱分析。
在本发明的一个优选实施方案中，提供了西妥昔单抗的二硫键配对分析方法，所述方法包括：
I)用肽N-糖苷酶F去除蛋白质的N-糖；
II)用盐酸胍对蛋白进行变性处理；
III)在非还原条件下用胰蛋白酶和胞内蛋白酶Lys-C联合酶解步骤II)获得的变性蛋白；
IV)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
V)将步骤IV)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
在本发明的一个优选实施方案中，提供了融合蛋白rhCTLA4-Ig的二硫键配对分析方法，所述方法包括下述步骤：
A)用肽N-糖苷酶F去除蛋白质的N-糖；
B)用盐酸胍对蛋白进行变性处理；
C)在非还原条件下用胰蛋白酶和/或胰凝乳蛋白酶酶解步骤II)获得的变性蛋白；
D)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
E)将步骤D)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
较佳的，步骤A)中使用肽N-糖苷酶F(PNGase F)去除所述融合蛋白的N-糖；优选溶液pH值为7.0～8.0。
在一具体实施方式中，所述融合蛋白的N-糖去除通过以下方法实现：取融 合蛋白样品，加入溶液如1％FA(体积浓度)或1％NH3.H2O(体积浓度)，调节pH值至7.0～8.0，加入肽N-糖苷酶F，混匀后，37℃孵育24小时。
较佳的，步骤B)中使用高浓度盐酸胍溶液对去除了N-糖的融合蛋白进行变性处理；优选盐酸胍的(GuHCl)浓度为6M；更优选使用8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3对融合蛋白进行变性处理。
较佳的，步骤C)中所述特异性酶包括胰蛋白酶和/或胰凝乳蛋白酶；优选胰蛋白酶的用量为1﹕25(wt/wt，酶/蛋白)；优选胰凝乳蛋白酶的用量为1﹕50(wt/wt，酶/蛋白)。
较佳的，步骤C)中所述特异性酶酶解融合蛋白孵育温度为37℃，孵育时间为2～4小时，优选孵育时间为4小时。
在另一具体实施方式中，所述胰蛋白酶的用量为1﹕25(wt/wt，酶/蛋白)，胰凝乳蛋白酶用量为1﹕50(wt/wt，酶/蛋白)。
较佳的，步骤D)中使用DTT对所述一份样品进行还原；优选DTT的浓度为60mM。
较佳的，步骤E)中加入甲酸(FA)终止酶解；优选加入所述甲酸至终体积浓度0.1％。
较佳的，步骤E)中使用ESI-Q-TOFMS方法进行分析，所得数据采用BiopharmaLynx软件进行处理分析。
在另一具体实施例中，所述ESI-Q-TOF MS分析采用以下方法实现：将融合蛋白用50mM NH4HCO3(pH8.0)溶液稀释至1.5-2mg/mL(以酶解前蛋白浓度计)；然后Waters Acquity UPLC BEH300 C18 1.7μm 2.1×150mm色谱柱分离，进行Q-TOF质谱分析。
在本发明的一个优选实施方案中，提供了TNFR2Fc融合蛋白依那西普的二硫键配对分析方法，所述方法包括：
①在微酸性条件下对蛋白质进行脱N-糖处理；
②用盐酸胍对步骤①处理后的蛋白质进行变性处理；
③在非还原条件下使用胰蛋白酶、胞内蛋白酶Lys-C和蛋白内切酶Glu-C组合酶酶解步骤②变性处理后的蛋白质；
④终止酶解后获得的肽段样品进行LTQ-Orbitrap Elite质谱分析。
较佳地，步骤①和步骤③的反应体系pH为6.5。
较佳地，步骤③的反应体系中含有烷基化试剂N-乙基马来酰亚胺。
本发明中，所述变性是指在高浓度盐等作用下，蛋白质分子间氢键等次级键被破坏，蛋白质分子就从原来有序的卷曲的紧密结构变为无序的松散的伸展状结构，但其一级结构并未改变。
本发明中，所述还原是指蛋白质二硫键在还原试剂作用下断开。
附图说明
由于大部分单抗氨基酸序列差异主要在于Fab段可变区，而恒定区氨基酸序列一致，二硫键配对也基本一致，IgG1和IgG2主要差异在于铰链区肽段二硫键个数差异。因此ESI-Q-TOF MS分析模式下，主要展示单抗Fab段可变区具有不同氨基酸序列二硫键肽段和对应含自由巯基肽段的碎片离子图。
图1～21为本发明实施例1中Denosumab单抗的二硫键配对分析结果；
图1为抗体蛋白二硫键肽段(1C/2C)碎片离子图；
图2为抗体蛋白二硫键肽段(3C/4C)碎片离子图；
图3为抗体蛋白二硫键肽段(5C/8C)碎片离子图；
图4为抗体蛋白二硫键肽段(6C/7C)碎片离子图；
图5为抗体蛋白二硫键肽段(9C/10C)碎片离子图；
图6为抗体蛋白二硫键肽段(11C+12C+13C+14C/11C+12C+13C+14C)碎片离子图；
图7为抗体蛋白二硫键肽段(15C/16C)碎片离子图；
图8为抗体蛋白二硫键肽段(17C/18C)碎片离子图；
图9为自由巯基肽段(1C)碎片离子图；
图10为自由巯基肽段(2C)碎片离子图；
图11为自由巯基肽段(3C)碎片离子图；
图12为自由巯基肽段(4C)碎片离子图；
图13为自由巯基肽段(6C)碎片离子图；
图14为自由巯基肽段(7C)碎片离子图；
图15为自由巯基肽段(8C)碎片离子图；
图16为自由巯基肽段(9C)碎片离子图；
图17为自由巯基肽段(10C)碎片离子图；
图18为自由巯基肽段(11C+12C+13C+14C)碎片离子图；
图19为自由巯基肽段(15C)碎片离子图；
图20为自由巯基肽段(17C)碎片离子图；
图21为自由巯基肽段(18C)碎片离子图；
图22～34为本发明实施例2中Golimumab单抗的二硫键配对分析结果；
图22为单克隆抗体二硫键肽段(1C/2C)碎片离子图；
图23为单克隆抗体二硫键肽段(5C/10C)碎片离子图；
图24为单克隆抗体二硫键肽段(6C/7C)碎片离子图；
图25为单克隆抗体二硫键肽段(8C/9C)碎片离子图；
图26为单克隆抗体二硫键肽段(11C+12C/11C+12C)碎片离子图；
图27为自由巯基肽段(1C)碎片离子图；
图28为自由巯基肽段(2C)碎片离子图；
图29为自由巯基肽段(5C)碎片离子图；
图30为自由巯基肽段(6C)碎片离子图；
图31为自由巯基肽段(7C)碎片离子图；
图32为自由巯基肽段(8C)碎片离子图；
图33为自由巯基肽段(9C)碎片离子图；
图34为自由巯基肽段(11C+12C)碎片离子图；
图35～40为本发明实施例3中西妥昔单抗(Cetuximab，
Figure PCTCN2016077810-appb-000003
)的二硫键配对分析结果；
图35为抗体蛋白二硫键肽段(1C/2C)碎片离子图；
图36为抗体蛋白二硫键肽段(6C/7C)碎片离子图；
图37为自由巯基肽段(1C)碎片离子图；
图38为自由巯基肽段(2C)碎片离子图；
图39为自由巯基肽段(6C)碎片离子图；
图40为自由巯基肽段(7C)碎片离子图；
图41～52为本发明实施例4中融合蛋白rhCTLA4-Ig的二硫键配对分析结果；
图41为融合蛋白二硫键肽段(1C/4C)碎片离子图；
图42为融合蛋白二硫键肽段(2C/3C)碎片离子图；
图43为自由巯基肽段(1C)碎片离子图；
图44为自由巯基肽段(4C)碎片离子图；
图45为自由巯基肽段(2C)碎片离子图；
图46为自由巯基肽段(3C)碎片离子图；
图47为融合蛋白二硫键肽段(5C/5C)碎片离子图；
图48为融合蛋白二硫键肽段(2C/3C)碎片离子图；
图49为融合蛋白二硫键肽段(6C/7C)碎片离子图；
图50为自由巯基肽段(5C)碎片离子图；
图51为自由巯基肽段(2C)碎片离子图；
图52为自由巯基肽段(3C)碎片离子图；
图53～63为本发明实施例5中TNFR2 Fc融合蛋白依那西普(Etanercept)二硫键配对分析结果；
图53A为本发明实施例抗体蛋白二硫键肽段(1C/2C+3C/5C)CID二级碎 片离子图，图53B为二硫键肽段(1C/2C+3C/5C)ETD二级碎片离子图，C为部分还原的α+β肽段CID三级碎片离子图；
图54A为本发明实施例抗体蛋白二硫键肽段(7C/8C+9C/11C)CID二级碎片离子图，图54B为二硫键肽段(7C/8C+9C/11C)ETD二级碎片离子图，图54C为部分还原的α+β肽段CID三级碎片离子图；
图55A为本发明实施例抗体蛋白二硫键肽段(10C/12C+13C/16C)CID二级碎片离子图，图55B为二硫键肽段(10C/12C+13C/16C)ETD二级碎片离子图，图55C为部分还原的α+β肽段CID三级碎片离子图；
图56A为本发明实施例抗体蛋白二硫键肽段(17C/18C+19C+20C+21C/22C)一级质谱图，图56B为二硫键肽段(17C/18C+19C+20C+21C/22C)ETD二级碎片离子图，图56C为部分还原的β+γ肽段CID三级碎片离子图；
图57A为本发明实施例抗体蛋白二硫键肽段(4C/6C)CID二级碎片离子图，图57B为二硫键肽段(4C/6C)ETD二级碎片离子图；
图58A为本发明实施例抗体蛋白二硫键肽段(14C/15C)CID二级碎片离子图，图58B为二硫键肽段(14C/15C)ETD二级碎片离子图；
图59A为本发明实施例抗体蛋白二硫键肽段(17C/18C)CID二级碎片离子图，图59B为二硫键肽段(17C/18C)ETD二级碎片离子图；
图60A为本发明实施例抗体蛋白二硫键肽段(23C/23C)CID二级碎片离子图，图60B为二硫键肽段(23C/23C)ETD二级碎片离子图；
图61A为本发明实施例抗体蛋白二硫键肽段(24C+25C/24C+25C)CID二级碎片离子图，图61B为二硫键肽段(26C/27C)ETD二级碎片离子图；
图62A为本发明实施例抗体蛋白二硫键肽段(26C/27C)CID二级碎片离子图，图62B为二硫键肽段(26C/27C)ETD二级碎片离子图；
图63A为本发明实施例抗体蛋白二硫键肽段(28C/29C)CID二级碎片离子图，图63B为二硫键肽段(28C/29C)ETD二级碎片离子图；
附图1～52中肽段中氨基酸之间的每一条虚线表示检测到的该肽段的一个碎片离子(b离子或y离子)，y离子是从肽段羧基端开始，b离子是从肽段氨基端开始。附图53～56依照肽段的氨基酸序列的先后顺序将三条肽段分别命名为α、β、γ。一般碎片离子越多越丰富，即表明该肽段与理论肽段一致，而碎片离子较少，则不一定与理论肽段一致，可能是具有相同分子量的其他肽段，碎片离子的多少与肽段的长度及碎裂模式有关，太短或太长的肽段在诱导碰撞电离碎裂模式下一般碎片离子都较少。
具体实施方式
以下结合具体实施例对本发明进一步阐述，不应理解为是对本发明的限制。下列实施例中未注明具体条件的实验方法，均是按照常规条件或制造厂商所建议的条件进行实验。实施例中使用的胞内蛋白酶Lys-C酶购买自罗氏公司(Roche)，其余的蛋白酶若无特别说明，都购买自普洛麦格公司(Promega)。
以下实施例中，变性的定义是指在高浓度盐等作用下，蛋白质分子间氢键等次级键被破坏，蛋白质分子就从原来有序的卷曲的紧密结构变为无序的松散的伸展状结构，但其一级结构并未改变。
以下实施例中，还原的定义是指蛋白质二硫键在还原试剂作用下断开。
下列实施例中未注明具体条件的实验方法，均是按照常规条件或制造厂商所建议的条件进行实验。
实施例1狄迪诺塞麦(Denosumab)二硫键配对分析
所用抗体蛋白为Denosumab(上海抗体药物国家工程研究中心有限公司提供)。
抗体蛋白狄迪诺塞麦(Denosumab)轻链含有5个半胱氨酸Cys，分别为Cys23、Cys89、Cys135、Cys195、Cys215，重链含13个半胱氨酸Cys，分别为Cys22、Cys96、Cys136、Cys 149、Cys205、Cys224、Cys225、Cys228、Cys231、Cys262、Cys322、Cys368、Cys426，从轻链N-末端开始到C-末端分别标示为1C～5C，从重链N-末端开始到C-末端分别标示为6C～18C，理论配对链内二硫键为1C/2C、3C/4C、6C/7C、9C/10C、15C/16C、17C/18C，链间二硫键为5C/8C，铰链区二硫键为11C+12C+13C+14C/11C+12C+13C+14C。
实验结果表明，胰蛋白酶联合胞内蛋白酶Lys-C酶切，胰蛋白酶联合弹性蛋白酶酶切，胰蛋白酶联合胰凝乳蛋白酶酶切，单一酶胰蛋白酶(1：25)酶切等方式均不能确认6C/7C二硫键肽段。胰蛋白酶加入量为1：15及以上时，二硫键肽段6C/7C的丰度相对较低，其b、y离子个数较少，优选地胰蛋白酶加入量为1：10二硫键肽段6C/7C碎片离子信息较为丰富，这比通过几种特异性酶进行组合酶切确认所有二硫键配对的方式更简单更方便。
1.1抗体蛋白二硫键配对分析(胰蛋白酶酶切)
抗体蛋白二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(1)中蛋白100μg，以1:10(wt/wt，酶/蛋白)加入胰蛋白酶酶解抗体蛋白， 37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至体积终浓度0.1％；另一半体积样品加入1μL0.1M DTT(二硫苏糖醇)，于37℃水浴30min后，加入甲酸FA至体积终浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件如表1所示：
表1 ESI-Q-TOF MS分析质谱参数和液相条件
毛细管电压(V) 3000 采集时间(min) 0-96
一级锥孔电压(V) 35 质量校准范围(m/z) 100-3000
二级锥孔电压(V) 4 采集质量范围(m/z) 100-3000
脱溶剂气温度(℃) 250 采样时间(sec) 0.500
脱溶剂气流速(L/H) 500 LockSpray扫描时间(sec) 0.500
锥孔气流速(L/H) 50 LockSpray扫描间隔(sec) 60
ESI源温(℃) 130 Ramp collision Energy(V) 15-30
碰撞能量(eV) 6 -- --
流动相A：0.1％FA-H2O，流动相B：0.1％FA-ACN，质谱清洗液：50％CAN，质谱IntelliStart阀清洗液：50％MeOH，柱温：45℃，检测波长：214nm，进样体积：5μL或10μL，样品室温度：10℃，液相梯度洗脱条件：流动相B在80分钟内从1％到36％。
实验结果：实验结果见下表2～3，其中表2为本发明实施例非还原条件抗体蛋白二硫键肽段分子量与理论分子量对比表；表3为本发明实施例1非还原条件抗体蛋白自由巯基肽段分子量与理论分子量对比表：
表2非还原条件抗体蛋白二硫键肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000004
Figure PCTCN2016077810-appb-000005
经ESI-Q-TOF质量肽谱图分析，二硫键肽段(1C/2C)碎片离子图见图1， 二硫键肽段(3C/4C)碎片离子图见附图2，二硫键肽段(5C/8C)碎片离子图见附图3，二硫键肽段(6C/7C)碎片离子图见附图4，二硫键肽段(9C/10C)碎片离子图见附图5，二硫键肽段(11C+12C+13C+14C/11C+12C+13C+14C)碎片离子图见附图6，二硫键肽段(15C/16C)碎片离子图见附图7，二硫键肽段(17C/18C)碎片离子图见附图8，自由巯基肽段(1C)碎片离子图见附图9，自由巯基肽段(2C)碎片离子图见附图10，自由巯基肽段(3C)碎片离子图见附图11，自由巯基肽段(4C)碎片离子图见附图12，自由巯基肽段(6C)碎片离子图见附图13，自由巯基肽段(7C)碎片离子图见附图14，自由巯基肽段(8C)碎片离子图见附图15，自由巯基肽段(9C)碎片离子图见附图16，自由巯基肽段(10C)碎片离子图见附图17，自由巯基肽段(11C+12C+13C+14C)碎片离子图见附图18，自由巯基肽段(15C)碎片离子图见附图19，自由巯基肽段(17C)碎片离子图见附图20，自由巯基肽段(18C)碎片离子图见附图21。
1.2：抗体蛋白二硫键配对分析(胰蛋白酶1：25酶切)
抗体蛋白二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(1)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶酶解抗体蛋白，37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：能确认1C/2C、3C/4C、5C/8C、9C/10C、15C/16C和17C/18C共6对二硫键，含6C/7C二硫键可以找到，但丰度较低，碎片离子信息不丰富未能确认，同时铰链区11C+12C+13C+14C/11C+12C+13C+14C二硫键也未能确 认。
1.3：抗体蛋白二硫键配对分析(胰蛋白酶联合胰凝乳蛋白酶酶切)
抗体蛋白二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(2)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，以1：50(wt/wt)加入胰凝乳蛋白酶联合酶解抗体蛋白，37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：能确认3C/4C、5C/8C、9C/10C、11C+12C+13C+14C/11C+12C+13C+14C和15C/16C等二硫键配对，无法确认6C/7C二硫键肽段。
1.4：抗体蛋白二硫键配对分析(胰蛋白酶联合胰凝乳蛋白酶酶切)
抗体蛋白二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(1)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，37℃孵育3小时后，再以1:50(wt/wt)加入胰凝乳蛋白酶，继续37℃孵育1小时。
平行取(1)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，37℃孵育3小时40分钟后，再以1:50(wt/wt)加入胰凝乳蛋白酶，继续37``℃孵育20分钟。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：未能确认6C/7C二硫键肽段。
1.5：抗体蛋白二硫键配对分析(胰蛋白酶联合弹性蛋白酶酶切)
抗体蛋白二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(1)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，以1:50(wt/wt)加入弹性蛋白酶，37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：由于弹性蛋白酶酶切位点较大，肽段较短，碎片离子信息也不丰富，未能确认6C/7C二硫键肽段。
1.6：抗体蛋白二硫键配对分析(胰蛋白酶联合Lys-C酶切)
抗体蛋白二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM  EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(2)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，以1：200(wt/wt)加入Lys-C联合酶解抗体蛋白，37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：能确认1C/2C、3C/4C、15C/16C和17C/18C等二硫键肽段，但无法确认6C/7C二硫键肽段。
综上，与现有技术相比，本发明通过单一酶胰蛋白酶在非还原条件下，能直接确认抗体蛋白Denosumab所有二硫键配对方式，可以排除抗体蛋白特殊氨基酸序列对酶解蛋白时的空间位阻作用，全部二硫键配对只通过单一酶酶切方式即能确认，方法简单方便、有效可靠。同时，该方法可以检测到部分错配的二硫键肽段。
实施例2戈利木单抗(Golimumab)二硫键配对分析
本发明单克隆抗体戈利木单抗轻链含有5个半胱氨酸Cys，分别为Cys23、Cys88、Cys135、Cys 195、Cys215，重链含11个半胱氨酸Cys，分别为Cys22、Cys96、Cys153、Cys 209、Cys229、Cys235、Cys238、Cys270、Cys330、Cys376、Cys434，从轻链N-末端开始到C-末端分别标示为1C～5C，从重链N-末端开始到C-末端分别标示为6C～16C，理论配对链内二硫键为1C/2C、3C/4C、6C/7C、8C/9C、13C/14C、15C/16C，链间二硫键为5C/10C，铰链区二硫键为11C+12C/11C+12C。
实验结果表明，单一酶胰蛋白酶酶切时不能确认5C/10C和11C+12C/11C+12C，单一酶Lys-C酶切时不能确认1C/2C和6C/7C，而将两种酶联合起来可以确认各自不能确认的二硫键肽段，避免Lys-C酶切时造成的肽段较长碎片离子不丰富的缺点，也避免了胰蛋白酶酶切时专一性不够易产生部分漏切导致肽段丰度较低的缺点。另外实验结果表明，戈利木单抗(Golimumab) 铰链区二硫键11C+12C/11C+12C由于氨基酸序列的特异性无法确认具体配对方式。
以下实施例中使用的单克隆抗体是戈利木单抗(上海抗体药物国家工程研究中心有限公司提供)。
2.1：抗体二硫键配对分析(胰蛋白酶联合Lys-C酶切)
单克隆抗体二硫键配对分析步骤：
(1)蛋白变性处理：同1.1分析步骤(1)
(2)酶解
取(1)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，以1﹕200加入Lys-C，联合酶解单克隆抗体，37℃孵育4小时。
(3)还原和终止酶解，同1.1分析步骤(3)
(4)ESI-Q-TOF质量肽谱图分析，同1.1分析步骤(4)
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果见下表，其中表4是非还原条件单克隆抗体二硫键肽段分子量与理论分子量对比表，表5是单克隆抗体自由巯基肽段分子量与理论分子量对比表：
表4非还原条件单克隆抗体二硫键肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000006
Figure PCTCN2016077810-appb-000007
表5单克隆抗体自由巯基肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000008
经ESI-Q-TOF质量肽谱图分析，二硫键肽段(1C/2C)碎片离子图见附图22，二硫键肽段(5C/8C)碎片离子图见附图23，二硫键肽段(6C/7C)碎片离子图见附图24，二硫键肽段(9C/10C)碎片离子图见附图25，二硫键肽段(11C+12C/11C+12C)碎片离子图见附图26，自由巯基肽段(1C)碎片离子图见附图27，自由巯基肽段(2C)碎片离子图见附图28，自由巯基肽段(5C)碎片离子图见附图29，自由巯基肽段(6C)碎片离子图见附图30，自由巯基肽段(7C)碎片离子图见附图31，自由巯基肽段(8C)碎片离子图见附图32，自由巯基肽段(9C)碎片离子图见附图33，自由巯基肽段(11C+12C)碎片离子图见附图34。
2.2：单克隆抗体二硫键配对分析(胰蛋白酶酶切)
单克隆抗体二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM  EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(2)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶酶解单克隆抗体，37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终体积浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终体积浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与2.1中相同。
实验结果：能确认除5C/10C和11C+12C/11C+12C以外的二硫键配对。
2.3：单克隆抗体二硫键配对分析(Lys-C酶切)
单克隆抗体二硫键配对分析步骤：
(1)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(2)酶解
取(1)中蛋白100μg，以1:200(wt/wt)加入Lys-C酶解单克隆抗体，37℃孵育4小时。
(3)还原和终止酶解
待(2)中样品酶解完成后，取出一半体积样品加入甲酸FA至终体积浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终体积浓度0.1％，终止酶解反应，同时酸化肽段。
(4)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与2.1中相同。
实验结果：能确认除1C/2C和6C/7C以外的二硫键肽段。
综上，与现有技术相比，本发明通过胰蛋白酶联合胞内蛋白酶Lys-C酶切方式，能直接确认戈利木单抗(Golimumab)所有二硫键配对方式，可以排除单克隆抗体特殊氨基酸序列对酶解蛋白时的空间位阻作用，全部二硫键配对只通一种酶切方式即能确认，方法简单方便、有效可靠。
实施例3西妥昔单抗(Cetuximab，
Figure PCTCN2016077810-appb-000009
)二硫键配对分析
西妥昔单抗(Cetuximab)轻链含有5个半胱氨酸Cys，分别为Cys23、Cys88、Cys134、Cys194、Cys214，重链含11个半胱氨酸Cys，分别为Cys22、Cys95、Cys146、Cys 202、Cys222、Cys228、Cys231、Cys263、Cys323、Cys369、Cys427，从轻链N-末端开始到C-末端分别标示为1C～5C，从重链N-末端开始到C-末端分别标示为6C～16C，理论配对链内二硫键为1C/2C、3C/4C、6C/7C、8C/9C、13C/14C、15C/16C，链间二硫键为5C/10C，铰链区二硫键为11C+12C/11C+12C。
实验结果表明，通过利用特异性脱糖酶去除抗体蛋白N-糖后再进行蛋白变性，然后酶解，可以排除抗体蛋白N-糖对特异性酶在酶解解蛋白时的空间位阻作用，降低N-糖对含N-糖位点肽段离子化效率的抑制，从而克服现有技术对含有N-糖位点二硫键肽段需要采用酶切位点较多特异性酶联合胰蛋白酶才能确认的缺点；且实验结果表明，单一酶胰蛋白酶酶切时不能确认西妥昔单抗的5C/10C和11C+12C/11C+12C两条二硫键肽段，单一酶胞内蛋白酶Lys-C酶切时不能确认1C/2C和6C/7C两条二硫键肽段，而胰蛋白酶联合胞内蛋白酶Lys-C酶切方式可以确认西妥昔单抗的全部二硫键配对，避免胞内蛋白酶Lys-C酶切时造成的肽段较长碎片离子不丰富的缺点，也避免了胰蛋白酶酶切时专一性不够易产生部分漏切导致肽段丰度较低的缺点。
以下实施例中所用抗体蛋白为西妥昔单抗(上海抗体药物国家工程研究中心有限公司提供)。
3.1：抗体蛋白二硫键配对分析(胰蛋白酶联合Lys-C酶切)
抗体蛋白二硫键配对分析步骤：
(1)N-糖(N-Glycosylations)去除
取1mg抗体蛋白样品，加入50mM NH4HCO3(pH8.0)溶液至体积20～50μL，加入1μL肽N-糖苷酶PNGase F(NEB，500000u/mL)，混匀后，37℃孵育24小时。
(2)蛋白变性处理，同1.1分析步骤(1)
(3)酶解
取(2)中蛋白100μg，以1:20(wt/wt)加入Trypsin，以1：200加入Lys-C，联合酶解抗体蛋白，37℃孵育4小时。
(4)还原和终止酶解，同1.1分析步骤(3)
(5)ESI-Q-TOF质量肽谱图分析，同1.1分析步骤(4)
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与实施例1.1中相同。实验结果：实验结果见下表，其中表6为非还原条件抗体蛋白二硫键肽段分子量与理论分子量对比表；表7为非还原条件抗体蛋白自由巯基肽段分子量与理论分子量对比表。
表6非还原条件抗体蛋白二硫键肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000010
表7非还原条件抗体蛋白自由巯基肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000011
Figure PCTCN2016077810-appb-000012
经ESI-Q-TOF质量肽谱图分析，抗体蛋白二硫键肽段(1C/2C)碎片离子图见附图35，二硫键肽段(6C/7C)碎片离子图见附图36，自由巯基肽段(1C)碎片离子图见附图37，自由巯基肽段(2C)碎片离子图见附图38，自由巯基肽段(6C)碎片离子图见附图39，自由巯基肽段(7C)碎片离子图见附图40。
3.2：抗体蛋白完整分子量分析(胰蛋白酶酶切)
抗体蛋白二硫键配对分析步骤：
(1)N-糖(N-Glycosylations)去除
取1mg抗体蛋白样品，加入50mM NH4HCO3(pH8.0)溶液至体积20～50μL，加入1μL肽N-糖苷酶PNGase F(NEB，500000u/mL)，混匀后，37℃孵育24小时。
(2)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(3)酶解
取(2)中蛋白100μg，以1:20(wt/wt)加入Trypsin酶解抗体蛋白，37℃孵育4小时。
(4)还原和终止酶解
待(3)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(5)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与3.1中相同。
实验结果：能确认除5C/10C和11C+12C/11C+12C以外的二硫键配对。
3.3：抗体蛋白完整分子量分析(Lys-C酶切)
抗体蛋白二硫键配对分析步骤：
(1)N-糖(N-Glycosylations)去除
取1mg抗体蛋白样品，加入50mM NH4HCO3(pH8.0)溶液至体积20～50μL，加入1μL肽N-糖苷酶PNGase F(NEB，500000u/mL)，混匀后，37℃孵育24小时。
(2)蛋白变性处理
取蛋白样品0.5mg加入到380μl变性缓冲液中(8M盐酸胍+5mM EDTA+0.5M Tris，pH8.3)混匀，37℃孵育60分钟。超滤离心或经脱盐柱脱盐至50mM的NH4HCO3(pH8.0)缓冲液中。
(3)酶解
取(2)中蛋白100μg，以1:200加入Lys-C酶解抗体蛋白，37℃孵育4小时。
(4)还原和终止酶解
待(3)中样品酶解完成后，取出一半体积样品加入甲酸FA至终浓度0.1％；另一半体积样品加入1μL0.1M DTT，于37℃水浴30min后，加入甲酸FA至终浓度0.1％，终止酶解反应，同时酸化肽段。
(5)ESI-Q-TOF质量肽谱图分析
混匀后离心10min(转速≥12000rpm)，ACQUITY UPLC BEH300 C18色谱柱(1.7μm，2.1×150mm，Waters公司)分离后，ESI-Q-TOF质谱分析，BiopharmaLynx软件分析。
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与3.1中相同。
实验结果：能确认除1C/2C和6C/7C以外的二硫键配对。
综上，与现有技术相比，本发明通过特异性脱糖酶去除N-糖，可以排除融合蛋白N-糖对特异性酶在酶解解蛋白时的空间位阻作用，降低N-糖对肽段离子化效率的抑制，通过胰蛋白酶联合胞内蛋白酶Lys-C酶切一种方式即能确认抗体蛋白(如西妥昔单抗)所有二硫键配对方式，方法简单方便、有效可靠。
实施例4融合蛋白(rhCTLA4-Ig)二硫键配对分析
融合蛋白rhCTLA4-Ig单链共有9个半胱氨酸Cys，分别为Cys21、Cys48、Cys 66、Cys 92、Cys120、Cys171、Cys231、Cys277、Cys335，从N-末端开始到C-末端分别标示为1C～9C，理论配对为单链链内二硫键：1C/4C、2C/3C、 6C/7C、8C/9C，链间二硫键5C/5C。
实验结果表明，不去除N-糖，只采用胰蛋白酶酶切只能确认Fc段6C/7C、8C/9C两对二硫键，2C/3C这对含有N-糖位点二硫键肽段需要采用酶切位点较多特异性酶联合胰蛋白酶以使得含2C和3C的二硫键肽段不含N-糖，从而排除N-糖对二硫键肽段离子化效率及酶切位阻的影响，最后才能确认2C/3C二硫键。利用特异性脱糖酶去除融合蛋白N-糖后再进行蛋白变性，通过胰蛋白酶和/或胰凝乳蛋白酶等酶切方式可确认2C/3C这对含有N-糖位点二硫键肽段。实验结果还表明，融合蛋白N-糖去除后2C/3C、6C/7C这两对二硫键配对方式在胰蛋白酶和/或胰凝乳蛋白酶的酶切条件下均能确认。
以下实施例中所用融合蛋白为rhCTLA4-Ig(上海抗体药物国家工程研究中心有限公司提供)。
4.1：融合蛋白二硫键配对分析(胰蛋白酶酶切)
融合蛋白二硫键配对分析步骤：
(1)N-糖(N-Glycosylations)去除
取1mgrhCTLA4-Ig融合蛋白样品，加入50mM NH4HCO3(pH8.0)溶液至体积20～50μL，加入1μL肽N-糖苷酶PNGase F(NEB，500000u/mL)，混匀后，37℃孵育24小时。
(2)蛋白变性处理，同1.1分析步骤(1)
(3)酶解
取(2)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶酶解抗体蛋白，37℃孵育4小时。
(4)还原和终止酶解，同1.1分析步骤(3)
(5)ESI-Q-TOF质量肽谱图分析，同1.1分析步骤(4)
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：实验结果见下表，其中表8为本发明实施例1非还原条件融合蛋白二硫键肽段分子量与理论分子量对比表；表9为本发明实施例1非还原条件融合蛋白自由巯基肽段分子量与理论分子量对比表。
表8非还原条件融合蛋白二硫键肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000013
Figure PCTCN2016077810-appb-000014
表9非还原条件融合蛋白自由巯基肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000015
经ESI-Q-TOF质量肽谱图分析，二硫键肽段(1C/4C)碎片离子图见附图41，二硫键肽段(2C/3C)碎片离子图见附图42，自由巯基肽段(1C)碎片离子图见附图43，自由巯基肽段(4C)碎片离子图见附图44，自由巯基肽段(2C+3C)碎片离子图见附图45，自由巯基肽段(6C)碎片离子图见附图46。
4.2：融合蛋白完整分子量分析(胰蛋白酶联合胰凝乳蛋白酶酶切)
融合蛋白二硫键配对分析步骤：
(1)N-糖(N-Glycosylations)去除，同4.1分析步骤(1)
(2)蛋白变性处理，同4.1分析步骤(2)
(3)酶解
取(2)中蛋白100μg，以1:25(wt/wt)加入胰蛋白酶，以1:50(wt/wt)加入胰凝乳蛋白酶酶解蛋白，37℃孵育4小时。
(4)还原和终止酶解，同4.1分析步骤(4)
(5)ESI-Q-TOF质量肽谱图分析，同4.1分析步骤(5)
所述ESI-Q-TOF MS分析典型参数包括质谱参数和液相条件优选与1.1中相同。
实验结果：实验结果见下表，其中：表10为本发明实施例2非还原条件融合蛋白二硫键肽段分子量与理论分子量对比本发明实施例表；表11为本发明实施例2非还原条件融合蛋白自由巯基肽段分子量与理论分子量对比表。
表10非还原条件融合蛋白二硫键肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000016
Figure PCTCN2016077810-appb-000017
表11非还原条件融合蛋白自由巯基肽段分子量与理论分子量对比表
肽段 理论分子量(Da) 测试分子量(Da) Mass error(ppm)
2C:QADSQVTEVCAATY 1484.65 1484.64 -1.0
3C:LDDSICTGTSSGNQVNL 1723.76 1723.75 -1.9
5C:VIDPEPCPDSDQEPK 1667.73 1667.73 0
6C:TPEVTCVVVDVSHEDPEVK 2081.00 2081.00 0
经ESI-Q-TOF质量肽谱图分析，二硫键肽段(5C/5C)碎片离子图见附图47，二硫键肽段(2C/3C)碎片离子图见附图48，二硫键肽段(6C/7C)碎片离子图见附图49，自由巯基肽段(5C)碎片离子图见附图50，自由巯基肽段(2C)碎片离子图见附图51，自由巯基肽段(3C)碎片离子图见附图52。
综上，与现有技术相比，本发明通过特异性脱糖酶去除N-糖，可以排除融合蛋白N-糖对特异性酶在酶解解蛋白时的空间位阻作用，降低N-糖对肽段离子化效率的抑制，仅通过胰蛋白酶和/或胰凝乳蛋白酶酶切方式均能确认含N-糖位点二硫键肽段，并且碎片离子信息丰富，全部二硫键配对也仅通过2种酶切方式即能确认。
实施例5 TNFR2 Fc融合蛋白依那西普(Etanercept)二硫键配对分析
TNFR2 Fc融合蛋白依那西普(Etanercept)单链含有29个半胱氨酸Cys，分别为Cys18、Cys31、Cys32、Cys35、Cys45、Cys53、Cys56、Cys71、Cys 74、Cys78、Cys88、Cys96、Cys98、Cys104、Cys112、Cys115、Cys121、Cys139、Cys142、Cys157、Cys163、Cys178、Cys240、Cys246、Cys249、Cys281、Cys341、Cys387、Cys445，从N-末端开始到C-末端分别标示为1C～29C，链内Fab段理论二硫键配对应为：1C/2C，3C/5C，4C/6C，7C/8C，9C/11C，10C/12C，13C/16C，14C/15C，17C/18C，19C/20C，21C/22C；铰链区二硫键为23C/23C，24C/24C，25C/25C；Fc段二硫键为：26C/27C，28C/29C。
本发明根据TNFR2 Fc融合蛋白的氨基酸序列，优选了Trypsin、Lys-C和Glu-C组合酶解的一种方式，通过将反应体系pH值降低为6.5，同时在反应体系中加入烷基化试剂N-乙基马来酰亚胺(NEM)，屏蔽自由巯基，降低二硫键错配的几率，准确确认了该融合蛋白所有的二硫键配对。
以下实施例中，采用的样品为：TNFR2 Fc融合蛋白依那西普(Etanercept)采用的液质联用系统(LC-MS/MS)为：Thermo Fisher Scientific公司Thermo Orbitrap Elite质谱仪，配有：ESI源，Orbitrap质量分析器，以及ETD源。液相 系统为Dionex公司的Ultimate 3000并配有：LPG-3400(RS)泵，WPS-3000(RS)进样器，TCC-3000(RS)柱温箱，VWD-3000(RS)检测器。数据采集使用软件ThermoXcalibur，数据处理分析软件：Pepfinder，Protome Discoverer，pLabel。
步骤一，脱N-糖处理：
将1mg抗体蛋白样品溶于50mM Tris-HCl(pH 6.5)体系中，加入PNGase F酶1μL，并加入NEM使其终浓度为2mM，混匀，37℃孵育18小时。
步骤二，蛋白变性处理：
将1mg脱N-糖抗体蛋白加入到盐酸胍蛋白变性液(8M盐酸胍、5mM EDTA、0.5M Tris,pH8.3)中，补加水使盐酸胍浓度为6M，并加入N-乙基马来酰亚胺(NEM)，终浓度为2mM，混匀，37℃孵育60分钟。变性后的样品超滤脱盐至100mM Tris-HCl(pH6.5)溶液中。
步骤三，酶解：
取100μg置换到100mM Tris-HCl(pH6.5)溶液中的抗体蛋白，分别按酶：样品＝1：25(wt/wt)的比例加入Trypsin，1：100(wt/wt)的比例加入Lys-C，1：25(wt/wt)的比例加入Glu-C，酶解抗体蛋白，加入100mM Tris-HCl(pH6.5)溶液使样品浓度为1.0mg/mL，37℃孵育18小时，FA淬灭酶切反应，FA终浓度为1％，样品13000rpm离心10min后取上清进行分析。
步骤四，LTQ-Orbitrap Elite质谱分析：
酶切后的样品经Acquity UPLC BEH300 C18(1.7μm 2.1×150mm，Waters公司)色谱柱分离后，再进入Thermo LTQ-Orbitrap Elite质谱，采用多种碎裂模式进行分析，首先获得同一前体离子的CID二级谱和ETD二级谱，然后再对ETD谱中的前三个最大丰度的离子进行CID三级碎裂。
表12 LTQ-Orbitrap Elite分析典型质谱参数优选
Figure PCTCN2016077810-appb-000018
Figure PCTCN2016077810-appb-000019
液相条件优选如下：
流动相A：0.1％FA-H2O；流动相B：0.1％FA-ACN；柱温：45℃；检测波长：214nm；进样体积：10μg；样品室温度：10℃；流速：0.15mL/min；液相梯度洗脱条件：流动相B在60分钟内从3％到20％,40分钟后到35％。
步骤五，数据分析：
数据使用Pepfinder和pLabel分析软件分析，通过CID二级谱可以初步得到抗体蛋白二硫键的基本配对方式，再人工结合ETD二级质谱可以进一步确证肽段之间的连接方式，最后通过ETD二级谱中部分离子的CID三级谱则可最终确定两个半胱氨酸之间的连接方式。
实验结果：
本实施例找到了TNFR2 Fc融合蛋白29对二硫键，其配对方式与理论一致，确认的二硫键肽段如表13所示。
表13非还原条件融合蛋白二硫键肽段分子量与理论分子量对比表
Figure PCTCN2016077810-appb-000020
Figure PCTCN2016077810-appb-000021
本实施例发明确认的二硫键肽段根据其连接特征可分为三类。第一类为含链间二硫键的三条肽段的连接，包括：1C/2C+3C/5C，7C/8C+9C/11C，10C/12C+13C/16C，参见图53-55。对于这类离子，Pepfinder软件在进行CID二级谱图匹配时只能将两条肽段离子作为另一条肽段的修饰进行匹配(参见图53A，图54A和图55A)，尽管其匹配状况良好，但因另外两条肽段被视为一体的修饰，因此另外两条肽段之间的连接顺序，以及当同一条肽段含多个C时具体哪两个C连接的信息则无法获得。因三条肽段连接的二硫键在ETD模式下并不能完全被还原，往往会产生大量的部分还原离子，如α+β或β+γ两条肽段连接的离子(依照肽段的氨基酸序列的先后顺序将三条肽段分别命名为α、β、γ)，参见图53B，图54B和图55B。基于此，可对这些部分还原离子的CID三级碎片离子图进行匹配，进而判断出具体哪两个半胱氨酸连接(参见图53C，图54C和图55C)。
第二类离子是既含有链间又含有链内二硫键(inter-/intra-peptide bonds)的复杂型肽段离子:17C/18C+19C+20C+21C/22C。对这类离子，受限于目前Pepfinder软件无法进行CID二级谱图匹配，需要人工解谱。首先根据酶解位点和母离子进行粗略计算，推测可能的肽段组合，然后根据ETD二级碎片离子谱进行单条肽段和部分还原肽段的匹配，最后根据部分还原肽段β+γ三级谱图判断出半胱氨酸的连接状况，参见图56。其中图56A为本发明实施例抗体蛋白二硫键肽段(17C/18C+19C+20C+21C/22C)一级质谱图，图56B为二硫键肽段(17C/18C+19C+20C+21C/22C)ETD二级碎片离子图，图56C为部分还原的β+γ肽段CID三级碎片离子图。
第三类为含链间二硫键(inter-peptide bonds)的两条肽段的连接，这类肽段包括：4C/6C，14C/15C，17C/18C，23C/23C，24C+25C/24C+25C，26C/27C，28C/29C本实施例发明通其CID二级碎片离子图中的b/y碎片离子和ETD二级碎片离子图中二硫键断裂后形成的单条肽段确认了其二硫键连接状况，参见图57-63。其中图57A为本发明实施例抗体蛋白二硫键肽段(4C/6C)CID二级碎片离子图，图57B为二硫键肽段(4C/6C)ETD二级碎片离子图；图58A为本发明实施例抗体蛋白二硫键肽段(14C/15C)CID二级碎片离子图，图58B为二硫键肽段(14C/15C)ETD二级碎片离子图；图59A为本发明实施例抗体蛋白二硫键肽段(17C/18C)CID二级碎片离子图，图59B为二硫键肽段(17C/18C)ETD二级碎片离子图；图60A为本发明实施例抗体蛋白二硫键肽段(23C/23C) CID二级碎片离子图，图60B为二硫键肽段(23C/23C)ETD二级碎片离子图；图61A为本发明实施例抗体蛋白二硫键肽段(24C+25C/24C+25C)CID二级碎片离子图，图61B为二硫键肽段(26C/27C)ETD二级碎片离子图；图62A为本发明实施例抗体蛋白二硫键肽段(26C/27C)CID二级碎片离子图，图62B为二硫键肽段(26C/27C)ETD二级碎片离子图；图63A为本发明实施例抗体蛋白二硫键肽段(28C/29C)CID二级碎片离子图，图63B为二硫键肽段(28C/29C)ETD二级碎片离子图。
工业应用性
结合以上实验结果可以充分说明，本发明的蛋白质二硫键配对分析方法可简单、高效、准确地分析蛋白分子中的二硫键连接，具有重复性好，谱图解析简单的优点，使用本发明方法可准确全面地确证蛋白质分子中的二硫键连接的肽段。本发明方法适于工业生产中较大批次的质量监控。

Claims (17)

  1. 一种蛋白质二硫键配对分析方法，包括制备用于分析的蛋白质样品和将所述样品进行质量肽谱图分析，得到所述蛋白质的二硫键连接表征，包括下述步骤：
    1)对蛋白质进行变性处理；
    2)根据待分析蛋白质的分子结构确定特异性蛋白酶类型，在非还原条件下用所确定的特异性蛋白酶酶解步骤1)获得的变性的蛋白质；
    3)终止酶解反应得到用于分析的蛋白质样品；
    4)分析步骤3)的蛋白质样品获得所述蛋白的二硫键连接表征。
  2. 如权利要求1所述的蛋白质二硫键配对分析方法，其中步骤2)所述的特异性蛋白酶选自胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、胞内蛋白酶Lys-C和蛋白内切酶Glu-C的一种或几种组合。
  3. 如权利要求1所述的方法，其中根据待分析蛋白质的分子结构，若蛋白质结构中含有N-糖链则进一步在步骤1)之前使用肽N-糖苷酶F去除所述蛋白质的N-糖。
  4. 如权利要求1所述的方法，其中步骤1)中使用盐酸胍进行蛋白变性，盐酸胍浓度为5.0～7.6M。
  5. 如权利要求1所述的方法，其中所述的蛋白质为IgG2型单克隆抗体单克隆抗体，所述方法包括：
    a)用盐酸胍对蛋白进行变性处理；
    b)在非还原条件下用胰蛋白酶酶解步骤a)获得的变性蛋白；
    c)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
    d)将步骤c)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
  6. 如权利要求5所述的方法，其中所述胰蛋白酶的用量为1:5～1﹕12(wt/wt，酶/蛋白)；所述ESI-Q-TOF MS方法包括：
    将步骤c)获得的样品用50mM NH4HCO3，pH8.0溶液稀释至1.5-2mg/mL，然后用色谱柱分离，进行Q-TOF质谱分析。
  7. 如权利要求6所述的方法，其中所述IgG2型单抗为Denosumab。
  8. 如权利要求1所述的方法，其中所述的蛋白质为戈利木单抗，所述方法包括：
    i)用盐酸胍对蛋白进行变性处理；
    ii)在非还原条件下用胰蛋白酶和胞内蛋白酶Lys-C联合酶解步骤i)获得的变性蛋白；
    iii)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
    iv)将步骤iii)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
  9. 如权利要求8所述的方法，其中蛋白酶用量为：胰蛋白酶﹕蛋白质为1﹕25(wt/wt)；胞内蛋白酶Lys-C﹕蛋白质为1﹕200(wt/wt)。
  10. 如权利要求1所述的方法，其中所述的蛋白质为西妥昔单抗，所述方法包括：
    I)用肽N-糖苷酶F去除蛋白质的N-糖；
    II)用盐酸胍对蛋白进行变性处理；
    III)在非还原条件下用胰蛋白酶和胞内蛋白酶Lys-C联合酶解步骤II)获得的变性蛋白；
    IV)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
    V)将步骤IV)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
  11. 如权利要求10所述的方法，其中步骤III)中胰蛋白酶﹕蛋白质为1﹕20(wt/wt)，胞内蛋白酶Lys-C﹕蛋白质为1﹕200(wt/wt)。
  12. 如权利要求1所述的方法，其中所述蛋白质为融合蛋白rhCTLA4-Ig，所述方法包括下述步骤：
    A)用肽N-糖苷酶F去除蛋白质的N-糖；
    B)用盐酸胍对蛋白进行变性处理；
    C)在非还原条件下用胰蛋白酶和/或胰凝乳蛋白酶酶解步骤B)获得的变性蛋白；
    D)将酶解后的蛋白分为两份样品，其中一份样品加入DTT进行还原，另一份样品不还原；
    E)将步骤D)获得的两份样品终止酶解反应，使用ESI-Q-TOF MS方法进行质量肽谱图分析。
  13. 如权利要求12所述的方法，其中步骤C)所述蛋白酶用量为：胰蛋白酶﹕融合蛋白为1﹕25(wt/wt)；胰凝乳蛋白酶﹕融合蛋白为1﹕50(wt/wt)。
  14. 如权利要求1所述的方法，其中所述蛋白质为TNFR2Fc融合蛋白依那西普，所述方法包括：
    ①在微酸性条件下对蛋白质进行脱N-糖处理；
    ②用盐酸胍对步骤①处理后的蛋白质进行变性处理；
    ③在非还原条件下使用胰蛋白酶、胞内蛋白酶Lys-C和蛋白内切酶Glu-C组 合酶解步骤②变性处理后的蛋白质；
    ④终止酶解后获得的肽段样品进行LTQ-Orbitrap Elite质谱分析。
  15. 如权利要求14所述的方法，其中步骤①和步骤③的反应体系pH为6.5。
  16. 如权利要求14所述的方法，其中步骤③的反应体系中含有烷基化试剂N-乙基马来酰亚胺。
  17. 如权利要求14所述的方法，其中步骤③中特异性蛋白酶用量为胰蛋白酶：融合蛋白为1：25(wt/wt)，胞内蛋白酶Lys-C：融合蛋白为1：100(wt/wt)，蛋白内切酶Glu-C：融合蛋白为1：25(wt/wt)。
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