WO2014110874A1 - 一种测定蛋白纯化工艺过程中样品的糖基化和末端修饰情况的方法 - Google Patents

一种测定蛋白纯化工艺过程中样品的糖基化和末端修饰情况的方法 Download PDF

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WO2014110874A1
WO2014110874A1 PCT/CN2013/074596 CN2013074596W WO2014110874A1 WO 2014110874 A1 WO2014110874 A1 WO 2014110874A1 CN 2013074596 W CN2013074596 W CN 2013074596W WO 2014110874 A1 WO2014110874 A1 WO 2014110874A1
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immunoglobulin
terminal
glycosylation
antibody
heavy chain
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PCT/CN2013/074596
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French (fr)
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朱保国
彭育才
杨嘉明
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珠海市丽珠单抗生物技术有限公司
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Priority to JP2015544313A priority Critical patent/JP6027258B2/ja
Priority to US14/760,313 priority patent/US9645156B2/en
Priority to EP13871613.9A priority patent/EP2947454A4/en
Publication of WO2014110874A1 publication Critical patent/WO2014110874A1/zh
Priority to HK16105427.4A priority patent/HK1217536A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • the present invention relates to the field of biotechnology.
  • the present invention provides a method of determining glycosylation and terminal modification of a sample during an immunoglobulin purification process.
  • the present invention also relates to a kit for determining protein glycosylation and terminal modification in immunoglobulin purification.
  • monoclonal antibodies have achieved great success and tremendous development in the biomedical industry and the entire pharmaceutical industry. Compared with traditional small molecule drugs, monoclonal antibodies have the advantages of high specificity, remarkable curative effect, small side effects and low dosage. In terms of drug molecular properties, antibodies have greater heterogeneity. This property of antibodies is caused by a variety of factors, and post-translational modification is one of the most important intrinsic factors. Common post-translational modifications of antibodies include glycosylation, N-terminal pyroglutamate, C-terminal delysine, deamidation, oxidation, isomerization, and the like.
  • Antibody IgG glycosylation occurs in the heavy chain Fc region of asparagine, which is N-glycosylated and is an important structural component of antibodies.
  • the core unit of IgG sugar chain is composed of two N-acetylglucosamine and two mannose double-stranded structures. According to the difference of terminal galactose, core fucose, terminal sialic acid, etc., a variety of different sugars can be formed. Chain structure.
  • the glycosylation of IgG is heterogeneous and manifests itself in different glycoforms and levels.
  • Differences in glycosylation can affect the biological activity and pharmacokinetic profile of the antibody, such as CDC, ADCC, in vivo clearance half-life, and the like.
  • the N-terminal amino acid of the antibody IgG is glutamine
  • the cyclization reaction is likely to occur to form pyroglutamic acid (pyroglutamic acid, pyroE.
  • pyroglutamic acid pyroE
  • This reaction can be carried out spontaneously or under enzymatic conditions.
  • de-lysine (-K) is prone to occur. In most cases, the two have no effect on the biological activity of the antibody, but it has also been reported that some antibodies may affect the antigen and glutamate.
  • an object of the present invention is to provide a method for simultaneously measuring glycosylation and terminal modification of a sample during purification of an immunoglobulin (i.e., antibody).
  • the invention also relates to a kit for determining protein glycosylation and terminal modification in immunoglobulin purification.
  • the present invention provides a method for simultaneously determining glycosylation and terminal modification of a sample during an immunoglobulin purification process, the method comprising the steps of:
  • step 2) after the immunoglobulin component in step 1) is denatured by a denaturant, reduced by a reducing agent, thereby splitting the light chain and the heavy chain;
  • step 2) separating the light and heavy chains of immunoglobulin in step 2) using reversed-phase ultra-high pressure liquid chromatography;
  • step 3 using the mass spectrometry to determine the molecular weight of the light and heavy chains obtained in step 3);
  • the chromatographic data in step 3) and the mass spectrometry data in step 4) are analyzed to determine the glycosylation and terminal modification of the immunoglobulin.
  • the immunoglobulin is preferably a human immunoglobulin, preferably a human immunoglobulin IgG, and more preferably a human immunoglobulin IgG1 and IgG2 subtype.
  • the glycosylation and terminal modification of the immunoglobulin preferably include light chain N-terminal pyroglutamation of immunoglobulin, aspartic acid glycosylation and N-terminal pyroglutamination of the heavy chain, C-terminal De-lysine.
  • the step 1) comprises: using a conventional strong cation exchange chromatography column, the loading buffer salt is 20 mM sodium phosphate buffer salt, the elution buffer salt is 20 mM sodium phosphate and 1 M chlorination.
  • Sodium buffered saline (pH 6.0), UV absorbance at 280 nm to monitor the effluent fraction.
  • the step 2) comprises: adding 10-30 ⁇ of a 1-6 M aqueous solution of guanidine hydrochloride to a certain amount of immunoglobulin, mixing uniformly, and adding an aqueous solution of 1-4 dithiothreitol (DTT) to make The immunoglobulin is denatured and reduced, wherein the final concentration of DTT in the reaction solution is 25-100 mM, and the final concentration of immunoglobulin is 0.2-3 ⁇ ⁇ / ⁇ .
  • the final concentration of DTT in said step 2) is 50 mM.
  • the step 2) the immunoglobulin is denatured, the temperature of the reduction reaction is 50-65 ° C, and the reaction time is 45 min-120 min.
  • the step 2) the immunoglobulin is denatured, the reduction reaction temperature is 65 ° C, and the reaction time is 45 min.
  • the step 3) specifically comprises: separating the light chain and the heavy chain of the immunoglobulin in the step 2) by reverse ultrahigh pressure liquid chromatography to achieve baseline separation of the light chain and the heavy chain, according to the present invention
  • the liquid phase system used may be UPLC (Waters, ACQUITY column: Waters, ACQUITY UPLC column, BEH C4, 1.7 ⁇ (particle size), 300 ⁇ (pore size), 2.1 x 50 mm. Mobile phase elution conditions The separation of the light and heavy chains is greatly affected.
  • the chromatographic conditions are set to: the column temperature is set to 55-65 ° C, the injection volume is 0.2-3 ⁇ ⁇ ; the mobile phase X is 0.1% formic acid, The mobile phase was 0.1% formic acid acetonitrile at a flow rate of 0.4 mL/min; the gradient elution conditions were: flow rate
  • the step 4) specifically comprises: determining the molecular weights of the light chain and the heavy chain obtained in the step 3) by electrospray ionization mass spectrometry, wherein at 0-5 min, the flow path leads to the waste liquid, 5-16 min, the flow path Passing to the mass spectrometer, and then collecting the mass spectrometry data in positive ion mode; preferably, the mass spectrometry conditions are set as follows: the cone gas flow is 50.0 L/Hr, the desolvation gas is 500-800.0 L/Hr, and the desolvation temperature is 350-500 °. C, the scanning range is 400-2500Da, and the scanning time is 0.5-2s.
  • the sampling cone voltage has a large influence on the mass spectrometry signal, and is set to 20-40 V, preferably 20-30V.
  • the step 5) specifically includes: calculating an N-terminal pyroglutamate ratio of the light chain of the immunoglobulin by the chromatographic peak area obtained in the step 3), and passing the mass spectrometry data obtained in the step 4) Deconvolution calculation yields the relative glycoform content of the heavy chain of the immunoglobulin and the N-terminal pyroglutamic acid and C-terminal delysine ratio.
  • the method determines the use in a kit for protein glycosylation and terminal modification in immunoglobulin purification.
  • the ratio of various heavy chains is proportional to the intensity of the molecular weight peak, so the relative content of glycoforms and the ratio of the N-terminal pyroglutamic acid and the C-terminal delysine of the heavy chain can be calculated from the data of the heavy chain mass spectrometry.
  • the N-terminal pyroglutamate ratio of the light chain was calculated from the peak area of the chromatogram.
  • mass spectrometers such as MALDI-MS
  • MALDI-MS mass spectrometers
  • the mass spectrometry method has good compatibility and convenient operation, the resolution of the measured molecular weight is low.
  • the enzymatic hydrolysis fluorescent labeling method uses N-glycolylase PNGaseF to digest IgG to obtain sugar.
  • the chain is purified by fluorescence labeling, high performance liquid chromatography or capillary electrophoresis.
  • the method is highly selective and accurate, but the sample processing process is complicated, takes a long time (usually takes 2 days), and requires a large sample size.
  • ESI-MS is also used to determine IgG enzymatic fragment for glycosylation analysis, but the selectivity of papain cleavage site is low, which will increase by-products and affect data analysis; immunoglobulin G-degrading enzyme S ( IdeS ) is highly selective, but the cost of IdeS is high, and it is not suitable for batch detection of routine or process development samples.
  • IdeS immunoglobulin G-degrading enzyme S
  • the use of LC-MS for tryptic peptide mapping can theoretically detect the glycosylation and terminal modification of antibodies.
  • there are many technical difficulties in the separation, determination and data analysis of sugar-containing peptides, and their detection sensitivity is low, which is not suitable for the detection of low-content glycosyl groups.
  • the sample processing process is complex and time consuming, and the enzymatic digestion process may have an effect on the original end modification of the antibody.
  • the method uses ESI-MS to determine the multi-charged ions of the antibody, and then performs deconvolution calculations, which greatly improves the resolution and accuracy of the detection results ( ⁇ 30 ppm).
  • the sample of the invention is simple to process, only requires a reducing agent to carry out the reaction (45 min), the sample amount is small (5 g), and the UPLC-MS detection of the sample is completed in only 16 minutes, and the antibody glycosylation can be simultaneously obtained. Data for N-terminal pyroglutamate and C-terminal delysine.
  • the invention is particularly applicable to experiments with low sample volumes, such as clonal screening, and rapid batch detection of process development processes; at the same time, the invention is also applicable to sample testing at conventional dosages (100 ⁇ ⁇ ).
  • the method of the present invention glycosylation and terminal modification of different antibodies such as IgG1, IgG2 can be detected, and the method can be used for the detection of samples during antibody process development.
  • carboxypeptidase B and cation exchange chromatography (CEX-HPLC) the structure of the antibody charge isomer can be characterized and identified by this method.
  • Immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE.
  • IgG can be divided into subtypes such as IgG1, IgG2, IgG3, and IgG4.
  • IgGl Currently, 70%-80% of monoclonal antibody drugs sold on the market belong to IgGl. Protein-like.
  • the invention provides a method for determining glycosylation and terminal modification of a sample during an immunoglobulin purification process, in particular to provide glycosylation and terminal modification of an immunoglobulin IgGl, IgG2. The method of the situation.
  • the method can simultaneously and rapidly determine the glycosylation, N-terminal pyroglutamate and C-terminal delysine of immunoglobulin during protein purification.
  • the present invention can correctly separate the light chain and the heavy chain by reducing a small amount of protein during protein purification, and does not affect the original glycosylation and terminal modification of the immunoglobulin.
  • Ultra-high pressure liquid chromatography-mass spectrometry analysis of reduced human immunoglobulin (ie, antibody) enables simultaneous and rapid glycosylation of a small amount of immunoglobulin (especially human immunoglobulin), N-terminal focal valley The cyclization of the amino acid and the C-terminal delysine were carried out.
  • Figures 1A-1D show the results of comparison of the different DTT amounts in Example 1 for the separation of the light and heavy chains of antibody A.
  • 2A-2C show the effect of different DTT reduction reaction temperatures and times in Example 1 on antibody light light heavy chain separation and terminal modification.
  • Figures 3A-3D show the effect of elution gradients 1, 2, 3, and 4 on the separation of light and heavy chain chromatography in Example 1, respectively.
  • Figures 4A-4D show the effect of different cone voltages (20V, 25V, 30V, 40V) in Example 1 on the peak intensity of heavy chain deconvolution molecular weight mass spectrometry.
  • Figure 5A shows the chromatogram of the antibody A after reduction in Example 2
  • Figure 5B-1 to Figure 5B-3 show the light chain, pyroglutamic acid light chain and heavy chain mass spectrometry determined after reduction of Antibody A, respectively.
  • Figure. Fig. 5C shows a chromatogram of the antibody B after reduction in Example 2
  • Fig. 5D-1 to Fig. 5D-2 show the light chain and heavy chain mass spectra of the antibody B after reduction, respectively.
  • pyroE is N-terminal pyroglutamic acid
  • -K is C-terminal delysine
  • -H 2 0 is dehydration.
  • Figure 6A shows a chromatogram of the antibody A cation exchange resin component 1 and component 5 after reduction in Example 3;
  • Figure 6B shows the heavy chain mass spectrum of the antibody A component 1 and component 5 after reduction.
  • pyroE is N-terminal pyroglutamic acid, -K is C-terminal delysine, and -3 ⁇ 40 is dehydration.
  • Figure 7 shows a chromatogram of the antibody C (IgG2) measured in Example 4 after reduction.
  • pyroE is N-terminal pyroglutamic acid
  • -K is C-terminal delysine
  • -3 ⁇ 40 is dehydration.
  • the antibody A in the following examples is a chimeric antibody IgG1 (specific preparation method is as shown in Example 1-6 of Chinese Patent No.: CN 101177453B, pages 10-13 of the specification, wherein C2-selected in Example 6 on page 13 of the specification 11-12
  • the chimeric antibody is the antibody A) of the invention
  • the antibody B is the humanized antibody IgG1, (produced by Zhuhai Livumab Biotechnology Co., Ltd., the specific preparation method such as Chinese patent: CN 102675460A specification 12-18 pages
  • the AT-132 antibody screened in Example 7 on pages 17-18 of the specification is the antibody B) of the present invention
  • the antibody C is a fully human antibody IgG2, produced by Amgen Canada Inc.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the medicinal materials, reagent materials and the like used in the following examples are commercially
  • the light and heavy chains obtained by the reaction were separated by C4 reverse ultra-high pressure liquid chromatography using a liquid phase system of UPLC (Waters, ACQUITY column: Waters, ACQUITY UPLC column, BEH C4, 1.7 ⁇ (particle size), 300 ⁇ (Aperture), 2.1x50 mm.
  • the chromatographic conditions were set to: column temperature set to 60 ° C, injection volume 1 ⁇ ⁇ ; mobile phase X was 0.1% formic acid water, mobile phase ⁇ was 0.1% formic acid acetonitrile, flow rate was 0.4mL/min ; gradient elution conditions
  • the molecular weights of the light and heavy chains obtained by chromatographic separation were determined by electrospray ionization mass spectrometry. At 0-5 min, the flow path leads to waste liquid, 5-16 min, the flow path leads to mass spectrometry, and then the positive ion is used.
  • the mass spectrometry data was set in the mode; the mass spectrometry conditions were set as follows: the cone gas flow was 50.0 L/Hr, the desolvation gas was 800.0 L/Hr, the desolvation temperature was 500 ° C, the scan range was 400-2500 Da, and the scan time was ls, sampling. The cone voltage was set to 25V. The results are shown in Figures 1A-1D.
  • the method parameters are as follows: Lock Mass (Da): 556.2771; TIC Threshold: 300-500; Deconvolution m/z Range: light chain 850-2000, heavy chain It is 950-1500; Protein MW Range: light chain is 20000-30000 Da, heavy chain is 42000-60000 Da.
  • the ratio of each glycoform of the antibody IgG was calculated by normalization based on the intensity of each glycoform molecular weight peak in the heavy chain mass spectrum.
  • the light chain containing the N-terminal pyroglutamic acid and the light chain containing no pyroglutamic acid can achieve baseline separation, so the chromatographic peak area is integrated to calculate the proportion of light chain N-terminal pyroglutamate.
  • the heavy chain N-terminal pyroglutamate and the C-terminal delysine are obtained by molecular weight analysis of the G0F heavy chain.
  • the specific results are shown in Figure 2 and Table 1. It can be seen from Fig. 2A-1 to Fig. 2A-3 that when the reduction temperature is 37 ° C, the antibody light and heavy chains are not completely separated, indicating that the reduction reaction is not complete at this temperature. 2B-1 to 2B-3 and 2C-1 to 2C-3, when the reduction temperature is increased to 50 ° C and 65 ° C (reaction time ⁇ 45 min), the antibody is light The separation of heavy chains is ideal, indicating that DTT reduction is more thorough at high temperatures above 50 °C.
  • reaction conditions were determined as follows: Reaction at 50-65 ° C, reaction time: 451 ⁇ ! 1 ⁇ reaction time ⁇ 120 min.
  • the method is excellent in precision and reproducibility.
  • the mobile phase X was 0.1% formic acid water
  • the mobile phase Y was 0.1% formic acid acetonitrile
  • the flow rate was 0.4 mL/min.
  • Gradient 1 0-5 min, 10% Y; 5-5.1 min, 10%-18% Y; 5.1-15 min, 18%-28% Y; 15-15. lmin, 28%-90% Y; 15.1-19.0 Min, 90% Y; 19.0- 19. lmin, 90%-10% Y, 19.1-22.0 min, 10% Y.
  • Gradient 2 0-5min, 10% Y; 5-5. lmin, 10%-25% ⁇ ; 5.1-8min, 25%-27% ⁇ ; 8-18min, 27%-30% ⁇ ; 18-18.
  • the optimal mobile phase elution gradient for light and heavy chain baseline separation is gradient 4: 0-5min, 10% Y; 5-5. lmin, 10%-25%Y; 5.1-6min, 25%-26% 6-10min, 26%-27% ⁇ ; 10-15min, 27%-32% ⁇ ; 15-15. lmin, 32%-90% ⁇ ; 15.1-18.0min, 90% ⁇ ; 18.0-18. lmin, 90%-10% ⁇ , 18.1-21.0min, 10% ⁇ .
  • the chromatographic separation results of the light and heavy chains are shown in Figure 3.
  • the optimal mobile phase elution gradient for light and heavy chain baseline separation is gradient 4: 0-5min, 10% Y; 5-5. lmin, 10%-25%Y; 5.1-6min, 25%-26% 6-10min, 26%-27% ⁇ ; 10-15min, 27%-32% ⁇ ; 15-15. lmin, 32%-90% ⁇ ; 15.1-18.0min, 90% ⁇ ;
  • Mass spectrometry conditions are optimized based on the optimized mobile phase elution gradient (0-5 min, 10% Y; 5-5. lmin, 10%-25% Y; 5.1-6 min, 25%-26% ⁇ ; 6-10 min, 26%-27% ⁇ ; 10-15min, 27%-32% ⁇ ; 15-15. lmin, 32%-90% ⁇ ;
  • the reaction product was separated by the chromatographic conditions in Example 1.1, and the mass spectrometric sampling cone voltages were set to 20 V, 25 V, 30 V and 40 V, respectively, and other mass spectrometry conditions were identical to those in Example 1.1.
  • the total ion current (peak area) of the light and heavy chains increases; as shown in Figures 4A to 4D, the deconvoluted molecular weight mass spectrometry signal of the heavy chain is between 20-25V.
  • the increase was significantly increased, there was no significant difference between 25-30V, and there was a significant decrease at 40V.
  • the mass spectrometry sampling cone voltage of the method is determined to be 25-30V.
  • Example 2 Determination of glycosylation and end of antibody A and antibody B (IgGl) using the UPLC-MS method of the present invention
  • the end modification was carried out under optimized reducing conditions (5 ⁇ ⁇ Antibody A was added to 10 ⁇ 6 M guanidine hydrochloride solution, then 0.5 L DTT solution was added 2 L, and finally an appropriate amount of 6 M guanidine hydrochloride solution was added to make the final concentration of DTT 50 mM, 65 °C reaction for 45 min), UPLC separation (consistent with Example 1.1), ESI-MS detection (consistent with Example 1.1) and normalized data processing (compared with Example 1.2) for analysis of the antibodies A and B Base and end modification.
  • the first amino acid at the N-terminus of the light chain and heavy chain of antibody A is glutamine (Gln), which is prone to cyclinization of pyroglutamate; the first amino acid at the N-terminus of the light chain of antibody B is glutamic acid (Glu), which is not easy to occur.
  • the pyroglutamic acid is cyclized, and the heavy chain is glutamine which is susceptible to cyclization.
  • 5A is a chromatogram obtained by the UPLC-MS method of the present invention after reduction of the antibody A
  • FIG. 5B is a mass spectrum measured by the antibody, wherein FIG. 5B-1 is a chromatographic peak having a retention time of 8.19 min in FIG. 5A.
  • FIG. 5B-2 A deconvoluted mass spectrum of an unmodified light chain (LC) with a molecular weight of 23056 Da.
  • Figure 5B-2 is a deconvoluted mass spectrum of the N-terminal pyroglutamate light chain with a retention time of 9.67 min in Figure 5A with a molecular weight of 23039 Da.
  • Figure 5B-3 is a deconvoluted mass spectrum of the heavy chain (HC) with a retention time of 11.27 min in Figure 5A.
  • the mass spectral peaks of different masses in Figure 5B-3 represent different glycoforms and terminal modifications, respectively.
  • the molecular weight of IgG1, its molecular weight and theoretical molecular weight are shown in Table 4.
  • the method measures that the light and heavy chain molecular weight of antibody A is very consistent with its theoretical value and has high accuracy; and it can distinguish mass spectral peaks with a mass difference of 17 Da, such as 50542Da (GOF, pyroglutamic acid, delysine) and 50559Da (GOF). , de-lysine), indicating high resolution.
  • 5C is a chromatogram of the antibody B measured by the UPLC-MS method of the present invention
  • FIG. 5D is a mass spectrum of the antibody, wherein FIG. 5D-1 is the peak of the retention time of 11.57 min in FIG. 5C.
  • the deconvoluted mass spectrum of the light chain (LC) has a non-pyroglutamic acid molecular weight of 23,056 Da.
  • Figure 5D-2 is a deconvoluted mass spectrum of the heavy chain (HC) with a retention time of 13.26 min in Figure 5C.
  • the mass spectral peaks of different mass numbers in Figure 5D-2 represent different glycoforms and terminal modifications, respectively.
  • the measured value of the molecular weight of the light heavy chain of antibody B is in good agreement with the theoretical value.
  • Example 3 The UPLC-MS method of the present invention was used to determine the glycosylation and terminal modification of each component of the purified antibody A.
  • a conventional strong cation exchange chromatography column was used, and the loading buffer salt was 20 mM.
  • the elution rate is 200-400 cm/h
  • the effluent component is monitored by UV absorption at 280 nm
  • the antibody is collected according to the retention time.
  • Component A Component 1 (4000-4300 minutes), Component 2 (4300-4500 minutes), Component 3 (4500-4650 minutes), Component 4 (4650-4800 minutes), Component 5 (4800- 5100 minutes).
  • the optimized reducing conditions (5 ⁇ ⁇ Antibody A was added to 10 ⁇ 6 ⁇ guanidine hydrochloride solution, then 0.5 ⁇ DTT solution 2 L was added, and finally an appropriate amount of 6 M guanidine hydrochloride solution was added to make DTT final concentration 50 mM, 65 ° C reaction for 45 min. ), UPLC separation (consistent with Example 1.1), ESI-MS detection (consistent with Example 1.1) and normalized data processing (compared with Example 1.2) Analysis of glycosylation and terminal modification of the IgG1 components .
  • Figure 6A-1 to Figure 6A-2 are chromatograms obtained after reduction of component 1 and component 5, and Figure 6B-1 to Figure 6B-2 are weights of component 1 and component 5.
  • the mass spectrum of the chain The results showed that from component 1 to component 5, the light chain N-terminal pyroglutamate (from 95.36% to 48.16%) and heavy chain pyroglutamination (from 86.49% to 65.53%) decreased in turn; glycosylation In part, GIF decreased from 14.39% (component 1) to 8.27% (component 5), while Man5 and G0F-GN increased from 5.53% and 6.72% to 9.16% and 14.11, respectively.
  • Example 4 Determination of glycosylation and terminal modification of antibody C (IgG2) by the UPLC-MS method of the present invention
  • the optimized reducing conditions in the present invention 5 ⁇ ⁇ Antibody A was added to 10 ⁇ M ⁇ 6 guanidine hydrochloride solution, and then Add 2 L of 0.5M DTT solution, finally add appropriate amount of 6 M guanidine hydrochloride solution to make DTT final concentration 50 mM, react at 65 ° C for 45 min), separate by UPLC (consistent with Example 1.1), ESI-MS detection (with Example 1.1) Consistent) and normalized data processing (compared to Example 1.2) Analysis of glycosylation and terminal modification of Antibody C.
  • Figure ⁇ is a chromatogram of the antibody C after reduction using the UPLC-MS method of the present invention, the light chain (LC) retention time is 6.6 min, and the heavy chain (HC) retention time is 13.7 min.
  • the first amino acid at the N-terminus of the light and heavy chains of antibody C is glutamic acid (Glu), which is not easily cyclized to form pyroglutamic acid, so no pyroglutamic acid light chain or heavy chain is detected; most of the heavy chain The C-terminal delysine occurred.
  • Glu glutamic acid
  • the glycoform of antibody C mainly includes G0F, GIF, Man5, GO and G2F, and the corresponding molecular weights of the delysine-containing heavy chain are 50206Da, 50367Da, 49978Da, 50059Da and 50531Da, which are consistent with the theoretical values; the content is 58.0%, respectively. , 19.5%, 13.7%, 6.6%, 2.2%. Therefore, the method of the invention is equally applicable to the detection of immunoglobulin IgG2 glycosylation and terminal modification.
  • Example 5 Kit Method for Determining Glycosylation and Terminal Modification of Antibody A Using the Present Invention
  • the kit consists of Reagent A and Reagent B, wherein Reagent A is a 6 M guanidine hydrochloride solution; Reagent B is a 0.5 M DTT solution.
  • the method for detecting the glycosylation and terminal modification of antibody A using the kit is as follows: Take 20 ⁇ ⁇ Antibody A (protein concentration should be greater than ⁇ / ⁇ if less than 1 ⁇ ⁇ / ⁇ , use ultrafiltration centrifuge tube with molecular weight cutoff lOkDa Concentration), add a certain amount of reagent A to the total volume of the solution is 36 L, then add 4 reagent B, and react at 65 ° C for 45 min. The reaction product was isolated by UPLC (consistent with Example 1.1), ESI-MS assay (consistent with Example 1.1) and normalized data processing (consistent with Example 1.2) for analysis of glycosylation and terminal modification of Antibody A.

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Abstract

本发明提供一种在免疫球蛋白纯化过程中测定免疫球蛋白的糖基化和末端修饰情况的方法，该方法能够同时、快速地对免疫球蛋白糖基化、N端焦谷氨酸化和C端脱赖氨酸进行测定，所述方法包括以下步骤：1）使用阳离子交换树脂分离免疫球蛋白，按保留时间收集不同组分；2）使步骤1）中免疫球蛋白组分经变性剂变性后，使用还原剂还原，从而拆分轻链和重链；3）使用反相超高压液相色谱分离步骤2）中免疫球蛋白的轻链和重链；4）使用质谱测定步骤3）中获得的轻链和重链的分子量；5）分析步骤3）中的色谱数据和步骤4）中的质谱数据，从而测定所述免疫球蛋白的糖基化和末端修饰情况。

Description

一种测定蛋白纯化工艺过程中样品的糖基化和末端修饰情况的方法
技术领域 本发明涉及生物技术领域。 具体而言， 本发明提供了一种测定免疫球蛋白纯化工 艺过程中样品的糖基化和末端修饰情况的方法。 同时本发明还涉及一种免疫球蛋白纯 化中测定蛋白糖基化和末端修饰情况的试剂盒。
近十年来， 单克隆抗体在生物医药界、 乃至整个医药行业里取得重大的成功和巨大 的发展。 与传统小分子药物相比， 单克隆抗体具有特异性强， 疗效显著， 副作用小， 用 量少等优点。 就药物分子特性而言， 抗体具有更大的不均一性。 抗体的这一特性是由多 种因素引起的， 翻译后修饰是其中最重要的内在因素。 常见的抗体翻译后修饰包括糖基 化， N端焦谷氨酸化， C端脱赖氨酸， 脱酰胺， 氧化， 异构化等。 在抗体药物研发的多 个环节， 如分子鉴定、 工艺开发、 质量监控等， 均需要对翻译后修饰进行检测分析。 抗体 IgG糖基化发生在重链 Fc区的天冬酰胺， 属 N-糖基化， 是抗体的重要结构组 成。 IgG糖链的核心单元由两个 N-乙酰葡萄糖胺和三个甘露糖的双叉结构连接组成， 根 据末端半乳糖、 核心岩藻糖、 末端唾液酸等的差异， 可以组成多种不同的糖链结构。 IgG 的糖基化是不均一的， 表现为不同的糖型和含量。 糖基化差异可以影响抗体的生物学活 性和药代特征， 如 CDC、 ADCC、 体内清除半衰期等。 抗体 IgG 的 N 端氨基酸为谷氨酰胺时， 容易发生环化反应而生成焦谷氨酸 (pyroglutamic acid, pyroE 此反应可以自发进行， 也可以在酶催化条件下进行。在抗体 IgG分子的 C端， 则容易发生脱赖氨酸（-K)。 在大部分情况下， 两者对抗体的生物活性 没有影响， 但亦有报道指某些抗体的 Ν端焦谷氨酸化可能会影响其与抗原的结合力。 此 外， Ν端的谷氨酰胺焦谷氨酸化和 C端脱赖氨酸均会影响抗体的电荷分布， 而电荷特性 是抗体质量监控的重要指标之一。 因此， 建立快速检测抗体 IgG糖基化和末端修饰的分析方法在抗体研发中具有重要 意义。 目前， 本领域一般对糖基化和末端修饰单独进行检测。 酶解荧光标记法是 IgGl糖 基化测定的经典定量方法， 但样品处理过程相当复杂， 耗时长， 而且需要样品量大； 也 有使用质谱测定 IgG酶解片段进行糖基化分析， 如 papain酶和 IdeS酶等， 但这些方法都 存在一些不足， 如酶切位点选择性不强， 或成本过高， 或样品处理复杂等， 不适宜常规 或工艺开发样品的批量检测。 应用 LC-MS进行肽图分析理论上可以同时检测抗体的糖 基化和末端修饰， 但含糖多肽的分离、 测定和数据分析存在诸多技术难点， 不适用于糖 基化定量分析， 而且样品处理过程复杂， 耗时长， 酶切过程也可能对抗体原有末端修饰 产生影响。 在免疫球蛋白纯化过程中， 需要对蛋白的纯化情况进行随时检测， 检测需用 样品量大， 检测耗时长仍是目前最大的问题。 因此， 目前仍未有适用于抗体研发的对少 量 IgG的糖基化和末端修饰同时进行快速测定的报道。 发明内容 为了解决上述技术问题， 本发明的目的是提供一种同时测定免疫球蛋白（即抗体） 纯化工艺过程中样品的糖基化和末端修饰情况的方法。 同时本发明还涉及一种免疫球 蛋白纯化中测定蛋白糖基化和末端修饰情况的试剂盒。 本发明提供一种同时测定免疫球蛋白纯化工艺过程中样品的糖基化和末端修饰情 况的方法， 所述方法包括以下步骤：
1 ) 使用阳离子交换层析法分离免疫球蛋白， 按保留时间收集不同组分；
2) 使步骤 1 ) 中免疫球蛋白组分经变性剂变性后， 使用还原剂还原， 从而拆分轻链 和重链；
3 ) 使用反相超高压液相色谱分离步骤 2) 中免疫球蛋白的轻链和重链；
4) 使用质谱测定步骤 3 ) 中获得的轻链和重链的分子量；
5 ) 分析步骤 3 ) 中的色谱数据和步骤 4) 中的质谱数据， 从而测定所述免疫球蛋白 的糖基化和末端修饰情况。 其中， 所述免疫球蛋白优选为人免疫球蛋白， 优选为人免疫球蛋白 IgG， 进一步优 选为人免疫球蛋白 IgGl和 IgG2亚型。 并且， 所述免疫球蛋白的糖基化和末端修饰情况优选包括免疫球蛋白的轻链 N端焦 谷氨酸化， 以及重链的天冬酰胺糖基化和 N端焦谷氨酸化、 C端脱赖氨酸。 在本发明提供的测定方法中， 所述步骤 1 ) 包括： 采用常规强阳离子交换层析柱的上样缓冲盐为 20 mM磷酸钠缓冲盐，洗脱缓冲盐为 20 mM磷酸钠和 1M氯化钠缓冲盐 （pH=6.0)， 紫外吸收 280nm监测流出组分。 具体地， 所述步骤 2) 包括： 向一定量的免疫球蛋白中加入 10-30 μ 的 1-6 M盐酸 胍水溶液， 混合均匀后加入 1-4 二硫苏糖醇 （DTT) 水溶液， 使免疫球蛋白发生变性、 还原反应，其中，反应溶液中 DTT终浓度为 25-100mM，免疫球蛋白终浓度为 0.2-3μ§/μΙ^。 优选地， 所述步骤 2) 中的 DTT终浓度为 50 mM。 优选地， 所述步骤 2) 免疫球蛋白发生变性、 还原反应温度为 50-65 °C， 反应时间为 45 min-120min。 更优选地， 所述步骤 2) 免疫球蛋白发生变性、 还原反应温度为 65 °C， 反应时间为 45 min。 具体地， 所述步骤 3 ) 具体包括： 采用反向超高压液相色谱分离步骤 2)中免疫球蛋白的轻链和重链， 以实现所述轻链 和重链的基线分离， 据本发明的具体实施方案， 采用的液相系统可以为 UPLC ( Waters, ACQUITY 色谱柱： Waters，ACQUITY UPLC column，BEH C4， 1.7μηι (粒径）， 300Α (孔 径）， 2.1 x50 mm。 流动相洗脱条件对轻链和重链的分离影响较大， 优选地， 色谱条件设定为： 色谱柱温度设定为 55-65 °C， 进样量 0.2-3μ§; 流动相 X为 0.1 %甲酸水， 流动相 Υ为 0.1 %甲酸乙腈， 流速为 0.4mL/min; 梯度洗脱条件为: 流速
时间 （min) 流动相 X 流动相 Y
(mL/min)
0 90 10 0.4
5 90 10 0.4 5.1 75 25 0.4
15 65 35 0.4
15.1 10 90 0.4
18 10 90 0.4
18.1 90 10 0.4
21 90 10 0.4
具体地， 所述步骤 4) 具体包括： 采用电喷雾电离质谱测定步骤 3 ) 中获得的轻链和重链的分子量， 其中在 0-5min， 流路通往废液， 5-16min， 流路通往质谱， 然后采用正离子模式采集质谱数据； 优选地， 质谱条件设定为： 锥孔气流为 50.0L/Hr， 脱溶剂气体为 500-800.0L/Hr， 脱溶剂温度为 350-500 °C， 扫 描范围为 400-2500Da， 扫描时间为 0.5-2s。 采样锥孔电压对质谱信号影响较大， 设定为 20-40 V, 优选地， 设定为 20-30V。 具体地， 所述步骤 5 ) 具体包括： 由步骤 3 ) 中获得的色谱峰面积计算得到所述免疫球蛋白的轻链的 N端焦谷氨酸化 比例， 由步骤 4)中获得的质谱数据通过去卷积化计算， 得到所述免疫球蛋白的重链的糖 型相对含量以及 N端焦谷氨酸和 C端脱赖氨酸比例。 具体地， 所述方法在免疫球蛋白纯化中测定蛋白糖基化和末端修饰情况试剂盒中的 应用。 在免疫球蛋白还原反应中， 蛋白的二硫键在 DTT的作用下发生断裂， 生成两条一 样的轻链和两条一样的重链。 使用 C4反相 -超高压液相色谱分离轻重链混合物， 含或 不含 N端焦谷氨酸的轻链与重链 （含不同糖型和末端修饰） 能实现基线分离， 然后采 用 ESI-MS在线测定其分子量。 由于含不同糖型或末端修饰的重链有不同的分子量， 而且各种重链的比例与其分子量峰的强度成正比， 因此糖型相对含量和重链 N端焦谷 氨酸化、 C端脱赖氨酸比例均可以由重链质谱数据计算获得。 此外， 轻链 N端焦谷氨 酸化比例由色谱峰面积计算获得。 目前本领域已有一些技术来测定蛋白质的分子量或其修饰情况， 但与本发明的方 法相比， 都存在缺陷或不足之处。 例如质谱仪 (MS)， 如 MALDI-MS, 常用于测定抗体 完整蛋白水平的分子量。 质谱方法虽然对样品兼容性好， 操作方便， 但测得分子量结 果分辨率较低； 作为抗体 IgG糖基化的经典定量方法一一酶解荧光标记法采用 N糖酐 酶 PNGaseF酶切 IgG获得糖链， 经纯化后再进行荧光标记、 高效液相色谱或毛细管电 泳分析； 该方法选择性强， 准确度高， 但样品处理过程复杂， 耗时长（通常需要 2天）， 而且需要样品量较大 （一般至少为 100μ§); 也有使用 ESI-MS测定 IgG酶解片段进行 糖基化分析， 但 papain酶切位点的选择性低， 会增加副产物而影响数据分析； 免疫球 蛋白 G降解酶 S ( IdeS ) 选择性很强， 但 IdeS的成本高， 不适宜常规或工艺开发样品 的批量检测； 应用 LC-MS 进行胰酶肽图分析理论上可以同时检测抗体的糖基化和末 端修饰， 但含糖多肽的分离、 测定和数据分析存在诸多技术难点， 而且其检测灵敏度低， 不适于低含量糖基的检测， 此外样品处理过程复杂， 耗时长， 酶切过程可能对抗体原有 末端修饰产生影响。 相比之下， 本方法采用 ESI-MS测定抗体多电荷离子， 然后进行去卷积化计算， 大大提高了分辨率和检测结果的准确度 (<30ppm)。 此外， 本发明样品处理简单， 只需 还原剂进行反应（45min)，样品用量少（5 g)，而且样品的 UPLC-MS检测只需在 16min 内完成， 便可同时得到抗体糖基化、 N端焦谷氨酸化和 C端脱赖氨酸的数据。 本发明 尤其适用于样品量少的实验， 如克隆筛选， 以及工艺开发过程的批量快速检测； 同时， 本发明也适用于常规用量 （100μ§) 的样品检测。 采用本发明的方法，可以检测不同抗 体如 IgGl、 IgG2 的糖基化和末端修饰， 而且该方法可用于抗体工艺开发过程中样品 的检测。 此外， 结合羧肽酶 B和阳离子交换色谱（CEX-HPLC ), 应用本方法还可以对 抗体电荷异构体的结构进行表征和鉴定。 免疫球蛋白可以分为 IgG、 IgA、 IgM、 IgD、 IgE五类，其中 IgG可分为 IgGl、 IgG2、 IgG3、 IgG4等亚型， 目前市场上销售的单克隆抗体药物 70%-80%属于 IgGl类蛋白。 根据抗体 IgGl 中人源氨基酸序列的组成， 又可划分为嵌合型抗体 IgGl、 人源化抗体 IgGl 等多种蛋白。 本发明提供了一种测定免疫球蛋白纯化工艺过程中样品的糖基化和 末端修饰情况的方法， 尤其提供了一种免疫球蛋白 IgGl、 IgG2的糖基化和末端修饰情 况的方法。 该方法能够同时、 快速地对蛋白纯化过程中免疫球蛋白的糖基化、 N端焦 谷氨酸化和 C端脱赖氨酸进行测定。 具体而言， 本发明通过在蛋白纯化过程中对少量 蛋白进行还原， 可以使其轻链和重链正确拆分开， 且不影响该免疫球蛋白原有的糖基化 和末端修饰情况。 经还原的人免疫球蛋白 （即抗体） 进行超高压液相色谱 -质谱联用分 析， 可以同时、 快速地对少量的免疫球蛋白 （特别是人免疫球蛋白） 糖基化、 N端焦 谷氨酸环化和 C端脱赖氨酸进行测定。 附图说明 以下， 结合附图来详细说明本发明的实施方案， 其中： 图 1A-1D显示了实施例 1中不同 DTT用量对抗体 A轻重链分离情况的比较结果。 图 2A-2C显示了实施例 1中不同 DTT还原反应温度和时间对抗体 A轻重链分离和 末端修饰情况的影响。 图 3A-3D分别显示了实施例 1 中洗脱梯度 1、 2、 3、 4对轻重链色谱分离情况的影 响比较。 图 4A-4D显示了实施例 1中不同锥孔电压 （20V， 25V, 30V, 40V) 对重链去卷积 化分子量质谱峰强度的影响。 图 5A显示了实施例 2中抗体 A经还原后测定的色谱图； 图 5B-1至图 5B-3分别显 示了抗体 A经还原后测定的轻链，焦谷氨酸化轻链和重链质谱图。图 5C显示了实施例 2 中抗体 B经还原后测定的色谱图；图 5D-1至图 5D-2分别显示了抗体 B经还原后测定的 轻链和重链质谱图。 pyroE为 N端焦谷氨酸， -K为 C端脱赖氨酸， -H20为脱水。 图 6A显示了实施例 3中抗体 A阳离子交换树脂组分 1和组分 5经还原后测定的色 谱图； 图 6B显示了抗体 A组分 1和组分 5经还原后测定的重链质谱图。 pyroE为 N端 焦谷氨酸， -K为 C端脱赖氨酸， -¾0为脱水。 图 7显示了实施例 4中抗体 C (IgG2) 经还原后测定的色谱图。 pyroE为 N端焦谷 氨酸， -K为 C端脱赖氨酸， -¾0为脱水。 具体实施方式 以下参照具体的实施例来说明本发明。 本领域技术人员能够理解， 这些实施例仅用 于说明本发明， 其不以任何方式限制本发明的范围。 下述实施例中的抗体 A 为嵌合型抗体 IgGl， （具体制备方法如中国专利： CN 101177453B说明书 10-13页实施例 1-6所示，其中说明书 13页实施例 6筛选出的 C2-11-12 嵌合抗体即为本发明的抗体 A); 抗体 B为人源化抗体 IgGl， （由珠海丽珠单抗生物技术 有限公司生产， 具体制备方法如中国专利： CN 102675460A说明书 12-18页实施例 1-7 所示， 其中说明书 17-18页实施例 7筛选出的 AT-132抗体即为本发明的抗体 B); 抗体 C 为全人源抗体 IgG2， 由 Amgen Canada Inc.生产。 下述实施例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的 药材原料、 试剂材料等， 如无特殊说明， 均为市售购买产品。 实施例 1 免疫球蛋白还原方法的条件筛选
1.1 考察还原剂 DTT的用量 考察了 4个不同用量的 DTT对轻、重链分离的影响。取 5μ§抗体 A蛋白 4份， 分别 加入到 ΙΟμΙ^ 6 Μ盐酸胍溶液中，再分别加入 0.1M DTT溶液 2 L禾卩 5 L，以及 0.5M DTT 溶液 2 L和 4 L，最后加适量 6 M盐酸胍溶液使 DTT终浓度分别为 10mM，25mM，50mM 和 lOOmM, 使其与所述 IgGl蛋白在 65°C下反应 45 min。 采用 C4反向超高压液相色谱分离反应所得的轻链和重链，使用的液相系统为 UPLC ( Waters, ACQUITY 色谱柱： Waters, ACQUITY UPLC column, BEH C4， 1.7μηι (粒径）， 300Α (孔径）， 2.1x50 mm。 色谱条件设定为： 色谱柱温度设定为 60°C， 进样量 1μ§; 流 动相 X为 0.1%甲酸水， 流动相 Υ为 0.1%甲酸乙腈， 流速为 0.4mL/min; 梯度洗脱条件
流速
时间 （min) 流动相 X 流动相 Y
(mL/min)
0 90 10 0.4
5 90 10 0.4
5.1 75 25 0.4
15 65 35 0.4
15.1 10 90 0.4 18 10 90 0.4
18.1 90 10 0.4
21 90 10 0.4 采用电喷雾电离质谱测定经色谱分离获得的轻链和重链的分子量， 其中在 0-5min， 流路通往废液， 5-16min， 流路通往质谱， 然后采用正离子模式采集质谱数据； 质谱条件 设定为： 锥孔气流为 50.0L/Hr， 脱溶剂气体为 800.0L/Hr， 脱溶剂温度为 500°C， 扫描范 围为 400-2500Da， 扫描时间为 ls， 采样锥孔电压设定为 25V。 结果如图 1A-1D所示， DTT终浓度为 10mM时， 抗体 A轻重链未能完全拆分； 当 DTT浓度为 25M时， 轻重链大部分情况下可以拆分完全， 但有极个别样品色谱分离不理 想； 当 DTT浓度为 50-100mM时， 所有抗体样品轻重链完全拆分。 为保证抗体轻重链拆 分和色谱分离， 确定使用 25-100mM DTT作为 0.2-3μ§/μΙ^抗体蛋白的合适还原剂用量， 50mM为优选用量。
1.2 考察还原反应的温度和时间 取 5 g抗体 A若干份分别加入到 ΙΟμΙ^ 6Μ盐酸胍溶液中，再加入 0.5Μ DTT溶液 2μ∑， 最后加适量 6 Μ盐酸胍溶液使 DTT终浓度为 50mM， 考察了 37°C、 50°C、 65 °C这三个反 应温度以及 20 min到 120 min的反应时间对抗体 IgGl轻、重链分离和末端修饰结果的影 响。 色谱和质谱条件与实施例 1.1一致。 实验数据使用 Waters公司的 BiopharmaLynx 1.3软件进行处理。 选择" Intact Protein" 模式进行去卷积化 (Deconvolution)处理， 方法参数如下： Lock Mass (Da): 556.2771; TIC Threshold: 300-500； Deconvolution m/z Range:轻链为 850-2000， 重链为 950-1500; Protein MW Range: 轻链为 20000-30000 Da， 重链为 42000-60000 Da。 抗体 IgG各糖型的比例根 据重链质谱图中各糖型分子量峰的强度进行归一化计算得到。 含 N端焦谷氨酸的轻链与 不含焦谷氨酸的轻链可实现基线分离， 因此对色谱峰面积进行积分， 可计算出轻链 N端 焦谷氨酸化的比例。重链 N端焦谷氨酸化和 C端脱赖氨酸是通过对 G0F重链的分子量分 析获得的。 具体结果如图 2和表 1所示。 其中， 由图 2A-1至图 2A-3可见， 当还原温度为 37°C 时， 抗体轻重链未完全分开， 说明该温度下还原反应不彻底。 由图 2B-1至图 2B-3以及 图 2C-1至图 2C-3可见， 当还原温度升高为 50°C及 65 °C (反应时间≥45min)时， 抗体轻 重链分离情况比较理想， 说明 DTT还原在 50°C以上的高温下反应比较彻底。此外， 从下 表 1 中可以看出， 在相同的还原温度下， 随着反应时间的延长， 样品轻重链末端的修饰 比例均呈上升趋势； 同样， 在相同的反应时间里， 随着反应温度的升高， 样品轻重链末 端修饰比例也均逐渐增加。 并且， 考虑到高温条件下可能促进 N端焦谷氨酸化和 C端脱 赖氨酸反应， 须考察样品制备过程对末端修饰的影响。 如表 1所示， 当 45!^!1≤反应时间
<120min, 末端修饰数据的变化幅度均不大， 因此， 所用样品制备条件对抗体末端修饰的 影响较小。
表 1 不同 DTT还原温度和时间条件下抗体 IgGl末端修饰情况比较
DTT还原条件 修饰比例 （％) 时间 焦谷氨酸化 焦谷氨酸化 脱赖氨酸 温度
(min) (轻链） (重链） (重链）
30 一 81.22 85.42
45 70.47 80.59 85.04
37 °C 60 70.55 81.68 84.32
90 75.53 81.89 85.12
120 77.66 80.33 86.71
30 71.38 81.87 84.39
45 77.13 83.10 86.60
50 °C 60 78.75 82.31 87.13
90 80.62 81.39 87.45
120 80.67 79.78 88.45
65 °C 20 79.51 83.96 87.17 30 80.56 81.20 87.17
45 81.12 82.62 87.41
60 81.11 82.13 88.45
90 81.58 81.81 88.05
120 81.12 81.24 89.30 综上， 确定还原反应条件为： 在 50-65°C下反应， 反应时间为： 451^!1≤反应时间 ≤120min。
1.3 方法精密度和重现性 在优化后的实验条件下 （取 5μ§抗体 A若干份分别加入到 10μΙ^ 6Μ盐酸胍溶液中， 再加入 0.5Μ DTT溶液 2 L， 最后加适量 6 M盐酸胍溶液使 DTT终浓度为 50mM， 65 °C 反应 45min)，评价本方法测定抗体 A的糖基化、 N端焦谷氨酸环化和 C端脱赖氨酸的精 密度和重现性。 色谱和质谱条件与实施例 1.1一致。 数据处理方法与实施例 1.2—致。 连续测定五次， 结果如表 2所示。 IgGl各糖型比例、 N端焦谷氨酸环化和 C端脱赖 氨酸修饰比例的测定结果 RSD%均小于 0.5%， 糖型含量测定结果的 RSD%小于 7%。 测定五个平行处理的样品， 结果如表 3所示。 N端焦谷氨酸环化和 C端脱赖氨酸修 饰比例的测定结果 RSD%均小于 1%， 糖型含量测定结果的 RSD%小于 6%。 表 2 方法精密度测定 末端修饰 百分比 (％) RSD
平均值 或糖型 1 2 3 4 5 (%) 轻链焦谷氨酸 83.51 83.55 83.49 83.59 83.58 83.54 0.05
G0F重链焦谷氨酸 90.10 89.58 90.04 90.04 89.81 89.91 0.24
G0F重链脱赖氨酸 87.95 88.17 88.15 88.43 87.99 88.14 0.22
G0F 74.40 74.03 73.22 73.80 74.25 73.94 0.62 GIF 12.64 12.79 12.76 13.01 13.13 12.87 1.55
Man5 4.61 4.75 5.35 5.10 4.79 4.92 6.09
GOF-GN 4.10 4.15 4.40 3.98 3.85 4.10 5.02
GO 4.24 4.28 4.27 4.12 3.98 4.18 3.06
表 3 方法重现性测定
Figure imgf000012_0001
综上， 该方法的精密度和重现性良好。
1.4 流动相优化 基于反相超高压液相色谱， 考察了流动相洗脱梯度对轻重链色谱分离的影响。 样品 使用 5 g抗体 A加入到 ΙΟμΙ^ 6Μ盐酸胍溶液中， 再加入 0.5Μ DTT溶液 2μ 最后加适 量 6 Μ盐酸胍溶液使 DTT终浓度为 50mM， 65°C反应 45min。 反应产品采用不同流动相 梯度进行分离， 其他色谱条件和质谱条件与实施例 1.1一致。 流动相 X为 0.1%甲酸水， 流动相 Y为 0.1%甲酸乙腈， 流速为 0.4mL/min， 考察了四种洗脱梯度， 具体如下： 梯度 1 : 0-5min， 10%Y; 5-5.1min, 10%-18%Y; 5.1-15min, 18%-28%Y; 15-15. lmin, 28%-90%Y; 15.1-19.0min, 90%Y; 19.0- 19. lmin, 90%-10%Y， 19.1-22.0min, 10%Y。 梯度 2: 0-5min, 10%Y; 5-5. lmin, 10%-25%Υ; 5.1-8min, 25%-27%Υ; 8-18min, 27%-30%Υ; 18-18. lmin, 30%-90%Υ; 18.1-21.0min, 90%Υ; 21.0-21. lmin, 90%-10%Υ， 21.1-24.0min, 10%Υ。 梯度 3: 0-5min, 10%Y; 5-5. lmin, 10%-25%Y; 5.1-8min, 25%-26%Y; 8-18min, 26%-28%Y; 18-18. lmin, 28%-90%Y; 18.1-21.0min, 90%Y; 21.0-21. lmin, 90%-10%Y， 21.1-24.0min, 10%Y。 梯度 4: 0-5min, 10%Y; 5-5. lmin, 10%-25%Υ; 5.1-6min, 25%-26%Υ; 6-10min, 26%-27%Υ; 10-15min, 27%-32%Υ; 15-15. lmin, 32%-90%Υ; 15.1-18.0min, 90%Υ; 18.0-18. lmin, 90%-10%Υ, 18.1-21.0min, 10%Υ。 轻链和重链的色谱分离结果如图 3所示。 综上， 实现轻重链基线分离的最佳流动相 洗脱梯度为梯度 4: 0-5min, 10%Y; 5-5. lmin, 10%-25%Y; 5.1-6min, 25%-26%Υ; 6-10min, 26%-27%Υ; 10-15min, 27%-32%Υ; 15-15. lmin, 32%-90%Υ; 15.1-18.0min, 90%Υ;
18.0- 18. lmin, 90%-10%Υ, 18.1-21.0min, 10%Υ。
1.5 质谱条件优化 基于优化后的流动相洗脱梯度 ( 0-5min, 10%Y; 5-5. lmin, 10%-25%Y; 5.1-6min, 25%-26%Υ; 6-10min, 26%-27%Υ; 10-15min, 27%-32%Υ; 15-15. lmin, 32%-90%Υ;
15.1- 18.0min, 90%Υ; 18.0-18. lmin, 90%-10%Υ， 18.l-21.0min, 10%Υ)， 考察了质谱参数对 基线分离后的轻链和重链质谱信号的影响。 锥孔气流， 脱溶剂气体和脱溶剂温度等对质 谱信号影响不大， 基本采用仪器供应商建议参数。 本发明方法对采样锥孔电压进行了优 化。 样品使用 5 g抗体 A加入到 ΙΟμΙ^ 6Μ盐酸胍溶液中， 再加入 0.5Μ DTT溶液 2 L， 最后加适量 6 M盐酸胍溶液使 DTT终浓度为 50mM， 65 °C反应 45min。 反应产品采用实 施例 1.1中的色谱条件进行分离，质谱采样锥孔电压分别设定为 20V， 25V, 30V和 40V， 其他质谱条件与实施例 1.1一致。 随着电压的升高， 轻重链的总离子流（峰面积）都有所 增加； 而如图 4A至图 4D所示， 重链的去卷积化分子量质谱信号在 20-25V之间时有较 明显增加， 在 25-30V之间没有明显差别， 在 40V时则有显著降低。 综上， 确定该方法 的质谱采样锥孔电压为 25-30V。 实施例 2 采用本发明的 UPLC-MS方法测定抗体 A和抗体 B ( IgGl )的糖基化和末 端修饰情况 采用优化后的还原条件（5 μ§抗体 A加入到 10 μ 6M盐酸胍溶液中，再加入 0.5 M DTT溶液 2 L， 最后加适量 6 M盐酸胍溶液使 DTT终浓度为 50mM， 65 °C反应 45 min), UPLC分离 （与实施例 1.1一致）， ESI-MS检测 （与实施例 1.1一致） 和归一化数据处理 (与实施例 1.2—致）分析抗体 A和抗体 B的糖基化和末端修饰。 抗体 A轻链和重链的 N端首个氨基酸均为谷氨酰胺 （Gln)， 易发生焦谷氨酸环化； 抗体 B轻链 N端首个氨基 酸为谷氨酸（Glu)， 不易发生焦谷氨酸环化， 而重链为易发生环化的谷氨酰胺。 图 5A为 抗体 A经过还原后采用本发明的 UPLC-MS方法测得的色谱图，图 5B为该抗体测得的质 谱图， 其中图 5B-1是图 5A中保留时间为 8.19min色谱峰即无修饰的轻链 (LC)的去卷 积化质谱图， 分子量为 23056 Da。 图 5B-2是图 5A中保留时间为 9.67min色谱峰即 N 端焦谷氨酸化的轻链的去卷积化质谱图， 分子量为 23039 Da。 图 5B-3是图 5A中保留 时间为 11.27min色谱峰即重链 (HC)的去卷积化质谱图， 图 5B-3中不同质量数的质谱 峰分别代表含不同糖型和末端修饰的 IgGl分子量，其测定分子量和理论分子量如表 4 所示。 该方法测得抗体 A的轻重链分子量与其理论值非常一致， 准确度高； 而且能区分 质量差为 17Da的质谱峰， 如 50542Da ( GOF, 焦谷氨酸，脱赖氨酸）和 50559Da ( GOF, 脱 赖氨酸）， 表明分辨率高。 图 5C为抗体 B经过还原后采用本发明的 UPLC-MS方法测得 的色谱图， 图 5D为该抗体测得的质谱图， 其中图 5D-1是图 5C中保留时间为 11.57min 色谱峰即轻链 (LC)的去卷积化质谱图， 无焦谷氨酸化分子量为 23056 Da。 图 5D-2是 图 5C中保留时间为 13.26min色谱峰即重链 (HC)的去卷积化质谱图， 图 5D-2中不同 质量数的质谱峰分别代表含不同糖型和末端修饰的 IgGl分子量。与抗体 A—样，抗体 B轻重链分子量的测定值与理论值十分一致。 通过归一化计算， 抗体 B重链 N端焦谷氨 酸化和 C 端脱赖氨酸分别为 70.6%和 97.8% ; G0F,G1F,G2F 和 GO 的含量分别为 65.7%，26.5%，4.6%禾卩 3.2%。 表 4抗体 A含不同糖型和末端修饰的重链分子量理论值和测定值 糖基化和末端修饰 理论分子量 （Da) 实测分子量 （Da)
G0F, 焦谷氨酸，脱赖氨酸 50542 50542
G0F, 焦谷氨酸，脱赖氨酸， 脱水 50524 50523
G0F, 脱赖氨酸 50559 50559 GOF, 焦谷氨酸 50670 50670
GIF, 焦谷氨酸，脱赖氨酸 50704 50704
Man5, 焦谷氨酸，脱赖氨酸 50314 50315
G0F-GN, 焦谷氨酸，脱赖氨酸 50338 50339
GO, 焦谷氨酸，脱赖氨酸 50395 50395
实施例 3 采用本发明的 UPLC-MS方法测定抗体 A经纯化后各组分的糖基化和末 端修饰情况 在抗体 A纯化工艺中， 采用常规强阳离子交换层析柱， 上样缓冲盐为 20mM 磷酸 钠缓冲盐，洗脱缓冲盐为 20mM磷酸钠和 1M氯化钠缓冲盐（pH=6.0)，流速 200-400cm/h 进行洗脱， 紫外吸收 280nm监测流出组分， 按保留时间先后收集抗体 A组分： 组分 1 (4000-4300分钟），组分 2(4300-4500分钟），组分 3 (4500-4650分钟），组分 4(4650-4800 分钟）， 组分 5 (4800-5100分钟）。采用优化后的还原条件（5μ§抗体 A加入到 10μΙ^ 6Μ 盐酸胍溶液中， 再加入 0.5Μ DTT溶液 2 L， 最后加适量 6 M盐酸胍溶液使 DTT终浓度 为 50mM， 65 °C反应 45min)， UPLC分离 （与实施例 1.1一致）， ESI-MS检测 （与实施 例 1.1一致） 和归一化数据处理 （与实施例 1.2—致） 分析该 IgGl各组分的糖基化和末 端修饰。 结果如表 5以及图 6A-1至图 6A-2和图 6B-1至图 6B-2所示。 表 5 阳离子交换树脂收集的 IgGl各纯化组分的糖基化和末端修饰检测结果 末端修饰 百分比 (％) 或糖型 组分 1 组分 2 组分 3 组分 4 组分 5 轻链焦谷氨酸 95.36 75.88 57.89 59.14 48.16
G0F重链焦谷氨酸 86.49 88.05 86.31 75.48 65.53
G0F重链脱赖氨酸 95.63 93.85 90.88 91.81 92.30
G0F 66.45 66.95 59.98 59.14 62.33 GIF 14.39 12.82 11.83 10.47 8.27
Man5 5.53 7.24 10.55 11.61 9.16
GOF-GN 6.90 6.88 10.09 11.49 14.11
GO 6.72 6.11 7.55 7.28 6.12 图 6A-1至图 6A-2为组分 1和组分 5经还原后测得的色谱图， 图 6B-1至图 6B-2为 组分 1和组分 5重链的质谱图。 测定结果显示， 由组分 1到组分 5， 轻链 N端焦谷氨酸化 （由 95.36%到 48.16%) 和重链焦谷氨酸化 （由 86.49%到 65.53%) 依次减少； 糖基化部分， GIF由 14.39% (组 分 1 ) 减少至 8.27% (组分 5)， Man5和 G0F-GN则分别由 5.53%和 6.72%增加至 9.16% 和 14.11。 因此， 本方明方法可用于监测抗体纯化工艺过程中样品的糖基化和末端修饰的 差异。 实施例 4 采用本发明的 UPLC-MS方法测定抗体 C (IgG2)的糖基化和末端修饰情 况 采用本发明中优化后的还原条件 （5μ§抗体 A加入到 10μΙ^ 6Μ盐酸胍溶液中， 再加 入 0.5M DTT溶液 2 L， 最后加适量 6 M盐酸胍溶液使 DTT终浓度为 50mM， 65°C反应 45min)， 经 UPLC分离（与实施例 1.1一致）， ESI-MS检测 （与实施例 1.1一致）和归一 化数据处理 （与实施例 1.2—致） 分析抗体 C的糖基化和末端修饰。 图 Ί为抗体 C经过 还原后采用本发明的 UPLC-MS方法测得的色谱图， 轻链 （LC) 的保留时间为 6.6min， 重链 (HC)的保留时间为 13.7min。抗体 C轻链和重链的 N端首个氨基酸均为谷氨酸 ( Glu)， 不易环化形成焦谷氨酸， 因此未检测到焦谷氨酸化的轻链或重链； 重链大部分发生了 C 端脱赖氨酸。 抗体 C的糖型主要包括 G0F， GIF, Man5， GO和 G2F， 对应的含脱赖氨 酸重链的分子量为 50206Da， 50367Da, 49978Da， 50059Da和 50531Da， 与理论值一致； 其含量分别为 58.0%， 19.5%, 13.7%, 6.6%, 2.2%。 因此， 本发明方法同样适用于免疫 球蛋白 IgG2糖基化和末端修饰的检测。 实施例 5 采用本发明测定抗体 A的糖基化和末端修饰情况的试剂盒方法 试剂盒由试剂 A和试剂 B构成， 其中试剂 A为 6M盐酸胍溶液； 试剂 B为 0.5 M DTT溶液。 使用试剂盒检测抗体 A的糖基化和末端修饰情况的方法具体如下： 取 20μ§抗体 A (蛋白浓度应大于 ΙμΒ/μΙ^若小于 1μ§/μί，可用截留分子量为 lOkDa 的超滤离心管进行浓缩）， 加入一定量试剂 A至溶液总体积为 36 L, 再加入 4 试剂 B， 在 65°C下反应 45 min。 反应产品采用 UPLC分离 （与实施例 1.1一致）， ESI-MS检 测 （与实施例 1.1一致）和归一化数据处理（与实施例 1.2—致）分析抗体 A的糖基化和 末端修饰。 连续 5天进行重复实验 （重新配置样品和测定）。 结果显示， 抗体 A的轻链和重链有效拆分， 并在色谱上实现基线分离。如表 6所示， 连续测定 5天， 轻链 N端焦谷氨酸化， 重链 N端焦谷氨酸化和 C端脱赖氨酸测定值的相 对标准偏差 RSD%小于 2%;糖链 G0F，G1F和 GO相对含量测定值的相对标准偏差 RSD% 小于 5%， Man5和 G0F-GN小于 10%。 综上， 本方法可实现标准化操作， 且重现性良好， 可用于建立测定免疫球蛋白糖基化和末端修饰的试剂盒方法。 表 6 应用本方法测定抗体 A末端修饰情况的重现性
Figure imgf000017_0001
以上对本发明具体实施方式的描述并不限制本发明， 本领域技术人员可以根据本发 明作出各种改变或变形， 只要不脱离本发明的精神， 均应属于本发明所附权利要求的范 围。

Claims

权 利 要 求 书
1、一种测定免疫球蛋白纯化工艺过程中样品的糖基化和末端修饰情况的方法，所述 方法包括以下步骤：
1 ) 使用阳离子交换层析法分离免疫球蛋白， 按保留时间收集不同组分；
2) 使步骤 1 ) 中免疫球蛋白组分经变性剂变性后， 使用还原剂还原， 从而拆分轻链 和重链；
3 ) 使用反相超高压液相色谱分离步骤 2) 中免疫球蛋白的轻链和重链；
4) 使用质谱测定步骤 3 ) 中获得的轻链和重链的分子量；
5 ) 分析步骤 3 ) 中的色谱数据和步骤 4) 中的质谱数据， 从而测定所述免疫球蛋白 的糖基化和末端修饰情况。
2、 根据权利要求 1所述的方法， 其特征在于， 所述步骤 1 ) 采用常规强阳离子交换 层析柱的上样缓冲盐为 20 mM 磷酸钠缓冲盐，洗脱缓冲盐为 20mM磷酸钠和 1M氯化钠 缓冲盐 （pH=6.0)， 紫外吸收 280nm监测流出组分。
3、 根据权利要求 1所述的方法， 其特征在于， 所述步骤 2) 包括： 向一定量的免疫 球蛋白中加入 10-30 μ 的 1-6 M盐酸胍水溶液，混合均匀后加入 1-4 μ 二硫苏糖醇 (DTT) 水溶液， 使免疫球蛋白发生变性、还原反应，其中，反应溶液中 DTT终浓度为 25-100mM， 免疫球蛋白终浓度为 0.2-3μ§/μΙ^。
4、根据权利要求 3所述的方法，其特征在于，所述步骤 2)中 DTT终浓度为 50 mM。
5、 根据权利要求 3所述的方法， 其特征在于， 所述步骤 2) 中免疫球蛋白发生变性、 还原反应温度为 50-65 °C， 反应时间为 45 min-120min。
6、 根据权利要求 5所述的方法， 其特征在于， 所述步骤 2) 中免疫球蛋白发生变性、 还原反应温度为 65 °C， 反应时间为 45 min。
7、 根据权利要求 1所述的方法， 其特征在于， 所述步骤 3 ) 包括： 采用 C4反向超 高压液相色谱分离步骤 2)中免疫球蛋白的轻链和重链， 以实现所述轻链和重链的基线分 离。
8、 根据权利要求 1所述的方法， 其特征在于， 所述步骤 4) 包括： 采用电喷雾电离质谱测定步骤 3 ) 中获得的轻链和重链的分子量， 其中在 0-5min， 流路通往废液， 5-16min， 流路通往质谱， 然后采用正离子模式采集质谱数据。 质谱条件设定为： 锥孔气流为 50.0L/Hr， 脱溶剂气体为 800.0L/Hr， 脱溶剂温度为 500 °C， 锥孔电压为 20-40 V, 扫描范围为 400-2500Da， 扫描时间为 ls。
9、 根据权利要求 8所述的方法， 其特征在于， 所述锥孔电压为 25-30V。
10、 根据权利要求 1所述的方法， 其特征在于， 所述步骤 5 ) 包括： 由步骤 3 )中获得的色谱峰面积计算得到所述免疫球蛋白的轻链的 N端焦谷氨酸化比例， 由步骤 4) 中获得的质谱数据计算得到所述免疫球蛋白的重链的糖型相对含量以及 N端 焦谷氨酸化和 C端脱赖氨酸比例。
11、 根据权利要求 1所述的方法， 其特征在于， 所述免疫球蛋白为人免疫球蛋白。
12、根据权利要求 11所述的方法，其特征在于，所述免疫球蛋白为人免疫球蛋白 IgGl 和 IgG2亚型。
13、 根据权利要求 1所述的方法， 其特征在于， 所述免疫球蛋白的糖基化和末端修 饰情况包括免疫球蛋白的轻链 N端焦谷氨酸化， 以及重链的天冬酰胺糖基化和 N端焦谷 氨酸化、 C端脱赖氨酸。
14、 根据权利要求 1-13任一项所述的方法， 其特征在于， 所述方法在免疫球蛋白纯 化中测定蛋白糖基化和末端修饰情况试剂盒中的应用。
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