CN114088852A

CN114088852A - Method and apparatus for analyzing non-denaturing mass spectrum

Info

Publication number: CN114088852A
Application number: CN202010859583.9A
Authority: CN
Inventors: 尹思成; 沈毅珺; 张一帆
Original assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Current assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2022-02-25

Abstract

The invention discloses a method and a device for analyzing a non-denaturing mass spectrum. The analysis method comprises the following steps: desalting and eluting a non-denatured protein sample to be detected, and directly performing mass spectrum sample loading analysis on the eluent on line, wherein the flow rates of the elution and the mass spectrum sample loading are both 0.019-0.021 ml/min; preferably 0.020 ml/min. Compared with a Native-MS result under a conventional ESI source, the Cys-ADC detection analysis of the full-coupling strategy has the advantages that the response value and the accuracy are greatly improved; in addition, the matched nano-ESI source and/or nano liquid phase do not need to be purchased, only a low-flow-rate spray needle needs to be configured, the cost of detection and analysis is greatly reduced, and the method is suitable for industrial large-scale detection; and can be used as a multi-attribute method.

Description

Method and apparatus for analyzing non-denaturing mass spectrum

Technical Field

The invention relates to the field of proteomics detection and analysis, in particular to an analysis method and device of non-denaturing mass spectrometry.

Background

The current conventional antibody drug coupling ratio (DAR) determination method is mainly liquid chromatography, and represents methods such as Hydrophobic Interaction Chromatography (HIC) and reverse phase chromatography (RPLC). Both methods require characterization of the peak components by Mass Spectrometry (MS). For mass spectrometry, lysine-conjugated antibody-conjugated drugs (Lys-ADCs) can be analyzed by conventional RPLC-MS platforms; whereas Cys-ADC is usually analyzed by non-denaturing mass spectrometry (Native-MS).

For cysteine conjugated antibody conjugated drugs (Cys-ADCs), the disulfide bond between the heavy and light chains, if a Drug (Drug), usually a cytotoxic Drug, is attached, maintains the spatial structure between the chains by non-covalent bonds. Conventional liquid chromatography-mass spectrometry conditions are generally reversed phase systems, and the mobile phase contains a high concentration of organic phase, which can disrupt the non-covalent bonds between the ADC subunits. Hydrophobic Interaction Chromatography (HIC) is effective in separating ADCs containing different numbers of drugs, but is incompatible with mass spectrometry due to the high concentration of non-volatile salts in the mobile phase; in addition, the hydrophobic interaction chromatography is possible to separate post-translational modification or degradation substances such as oxidative deamidation and the like, so that the difficulty of analysis is increased. For photometry, the content of antibody conjugated drugs cannot be accurately determined, especially for ADC with high coupling ratio, because some small molecules have strong interference.

Native-MS can not effectively separate ADC, but can effectively desalt ADC samples and maintain the integrity of ADC, and the DAR value closest to the samples in the natural state is obtained through mass spectrum detection. Native-MS can not only effectively measure DAR, but also can quantify each component of ADC containing different numbers of small molecules, so as to obtain the most real medicine distribution condition.

At present, a plurality of documents report that Native-MS is successfully applied to an analysis example of Cys-ADC at home and abroad. On the one hand, the successfully applied analytes are ADCs (DAR. ltoreq.4) coupled using a partial coupling strategy. Whereas Cys-ADC (DAR ≧ 7) of the full coupling strategy is generally more difficult to ionize in the Mass spectrum due to the higher number of small molecules and the greater hydrophobicity, the Mass spectrum response tends to be poor enough to guarantee the accuracy of the results (Chen, Jia, et. Development of a Natural nanoElectroluminescence Mass Spectrometry Method for Determination of the Drug-to-Antibody Ratio of Antibody-Drug Conjugates, "Analytical Chemistry, vol.85, No. 3,2013, pp.1699-1704; Pacholroz, Kamil J., and Perdita E.Barran." Use of a Charge recovery Antibody Analysis of Cyste-bound Antibody-amplification, Mass Spectrometry 23, 2016. The. A. B. A. B. A. B. A. B. A. a. A. a full coupling of a. A. a full coupling strategy of a coupling strategy. On the other hand, in Cys-ADC analysis, Native-MS is generally matched with a special nano-ESI source and a corresponding nano liquid phase system, and the cost is very high. For a biological macromolecule laboratory generally equipped with a conventional ESI source, the ionization efficiency of Native-MS analysis of Cys-ADC (DAR is more than or equal to 7) of a full coupling strategy by using the conventional ESI source is low, and the mass spectrum response is poor, so that effective analysis cannot be carried out; however, for the mode of performing offline desalting and then performing mass spectrum sampling by using a nano-ESI source, although a nano liquid phase system does not need to be matched, the sample preparation method is complex, poor in repeatability and low in flux, and is not suitable for being used as a conventional analysis method.

Disclosure of Invention

The invention aims to overcome the defects of low flux and high cost of the existing non-denaturing mass spectrometry (Native-MS) method, and provides a non-denaturing mass spectrometry analysis method and a non-denaturing mass spectrometry analysis device, which do not need to be additionally provided with a nano-ESI source and a nano liquid phase system, realize high flux and simultaneously reduce cost, can obtain structural information of a sample to be detected in a natural state, obtain a real drug distribution condition, and can be simultaneously used for ADC analysis, complete molecular weight measurement and the like.

The inventors found in practice that for Native-MS equipped with a conventional liquid phase system and ion source, in Cys-ADC analysis with Lys-ADC or partial conjugation strategy, the mass spectral response can meet the need for further deconvolution calculations even if the ionization efficiency is not good, due to the small number of drugs conjugated; however, in Cys-ADC analysis of the full coupling strategy, the ionization efficiency of the method is low, and the mass spectrum response is poor, so that effective analysis cannot be carried out. After analysis, the inventor believes that the Cys-ADC coupling drugs of the full coupling strategy are more in number and more hydrophobic, and are generally more difficult to ionize in mass spectrum, so that the mass spectrum response value is poorer, and the accuracy of the result is difficult to guarantee. Starting from improving the mass spectrum response value of the Cys-ADC of the full coupling strategy, the adjustment of a mass spectrum ion source and parameters thereof is firstly thought out, but the effect of the Cys-ADC analysis of the full coupling strategy is little; therefore, further trying to adjust other conditions or parameters of the Native-MS system, surprisingly finding that the flow rate of desalting elution and the flow rate of a sample to be detected entering mass spectrum sample loading analysis both obviously affect the mass spectrum response value; therefore, the flow rate is tried to be controlled within a certain range, mass spectrum parameters influencing mass spectrum response values within the flow rate range are screened by searching and designing experiments, desalting equipment adaptive to the flow rate is selected, a large number of online and offline tests are continuously carried out, and finally the Native-MS analysis method capable of carrying out online desalting-elution-mass spectrum sample loading analysis on the protein sample to be detected is obtained.

The invention is realized by the following technical scheme:

a first aspect of the invention provides a method of analysis of non-denaturing mass spectra comprising: desalting and eluting a non-denatured protein sample to be detected, and directly performing mass spectrum sample loading analysis on an eluent on line; the flow rates of the elution and the mass spectrum sample loading are both 0.019-0.021 ml/min; preferably 0.020 ml/min.

When the flow rates of the elution and the mass spectrum loading are both in the range, the response value of the mass spectrum is greatly improved.

In a preferred embodiment of the invention, the flow rates of elution and mass loading are both 0.020 ml/min.

The protein sample to be detected can be conventional in the field, and is preferably an antibody conjugated drug; more preferably a Cys-antibody conjugate drug.

In a preferred embodiment of the invention, the sample to be tested is a Cys-antibody conjugated drug with DAR not less than 5, 5.5, 6, 6.5 or 7.

According to the invention, said desalting is effected by ultra high liquid chromatography (UPLC); preferably, the ultra-high liquid chromatograph uses a size exclusion chromatographic column; more preferably, the size exclusion chromatographic column has a volume of 0.52 to 2.49 ml.

In a preferred embodiment of the invention, the size exclusion column is an ethylene bridge hybrid particle (BEH) column having a volume of 0.52ml and a particle pore size of

The particle size was 1.7. mu.m.

Conventionally, the mass spectrum sample loading analysis is realized by a mass spectrometer with an ion source; the ion source may be conventional in the art, preferably electrospray ionisation.

The parameters of the ion source of the mass spectrometer are selected as follows: the action voltage (CP) of the ion source can be 1-4 kV; preferably 2.5 kV.

The cone hole voltage (CV) of the ion source can be 40-150V; preferably 95V.

The Source Temperature (ST) of the ion source can be 50-150 ℃; preferably 100 deg.c.

The Desolvation Temperature (DT) of the ion source can be 200-450 ℃; preferably 450 deg.c.

The action voltage, the taper hole voltage, the source temperature and the desolvation temperature of the ion source can be independently adjusted, and two, three or four of the action voltage, the taper hole voltage, the source temperature and the desolvation temperature can be simultaneously adjusted to adapt to the flow velocity of elution and mass spectrum sampling.

In a preferred embodiment of the present invention, the ion source is electrospray ionization, the capillary voltage (i.e., the applied voltage) of the electrospray ionization is 2.5kV, and the desolvation temperature is 450 ℃.

In a more preferred embodiment of the present invention, the ion source is electrospray ionization, the capillary voltage of electrospray ionization is 2.5kV, the desolvation temperature is 450 ℃, the cone-hole voltage is 95V, and the source temperature is 100 ℃.

According to the invention, the non-denaturing treatment is carried out by methods conventional in the art, including: diluting a protein sample to be detected by using a mobile phase with the pH value of 6.0-7.0, performing enzyme digestion and desugarization, and replacing a protein system to be detected by using the mobile phase into a mobile phase system to be injected.

Preferably, the mobile phase has a pH of 6.5.

In a preferred embodiment of the present invention, the mobile phase is an ammonium acetate buffer solution with pH 6.5; preferably, the concentration of the ammonium acetate buffer solution is 50-150 mM.

In a more preferred embodiment of the invention, the mobile phase is 100mM ammonium acetate buffer pH 6.5.

The enzymatic desugarization may be conventional in the art, and preferably, means removal of sugar chains from glycoproteins by PNGaseF.

One purpose of the replacement is to completely replace a protein sample system to be detected into a mobile phase system; the second purpose is to replace the enzyme used for desugaring so as not to affect the ionization efficiency of mass spectrum.

A second aspect of the present invention provides an apparatus for the analysis method of the first aspect, comprising: the method comprises the following steps of desalting a protein sample to be detected after non-denaturing treatment, and mass spectrum equipment which is connected with the desalting equipment and is used for directly carrying out mass spectrum sample loading analysis on desalted eluent, wherein the mass spectrum equipment comprises a spray needle for receiving the eluent, and the spray needle is a spray needle for controlling the flow rate of the eluent and the mass spectrum sample loading to be 0.019-0.021 ml/min; preferably, the aperture of the spray needle is 27-45 μm; more preferably, the pore diameter is 30 to 36 μm.

The desalting apparatus may be conventional in the art and is preferably an ultra-high liquid chromatograph, such as an ACQUITY

BEH SEC(

1.7μm，2.1×150mm)。

The mass spectrometry apparatus may be conventional in the art, and is preferably a mass spectrometer, such as a Waters Vion IMS Qtof.

The spray needle can be selected from low-flow spray needles Waters, 186007529.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

(1) compared with a Native-MS result under a conventional ESI source, the Cys-ADC detection analysis of a full coupling strategy is carried out, so that the response value and the accuracy are greatly improved;

compared with Native-MS results of nano-ESI source configuration, the response value of the method is equivalent to the accuracy effect;

the invention has the detection effect equivalent to that of the prior art in Cys-ADC detection analysis of a partial coupling strategy.

(2) By using the method, a matched nano-ESI source and/or nano liquid phase does not need to be purchased, only a low-flow-rate spray needle needs to be configured, the cost of detection and analysis is greatly reduced, and the method is suitable for industrial large-scale detection.

(3) The method does not need a complicated pretreatment process, can perform whole-process online analysis only after simple conventional desugaring treatment, increases flux, and is convenient and fast to operate.

(4) The invention can be used as a multi-attribute method (MAM method) to simultaneously detect and measure a plurality of key quality attributes, such as DAR, integral-mass and ADC (analog-to-digital converter) polymer proportion, simplify the process and increase the flux.

Drawings

FIG. 1 is a schematic view of an apparatus according to example 1;

in the figure: the device comprises an ultra-high performance liquid chromatograph (UPLC) 1, a solvent manager 11, a sample manager 12, a size exclusion chromatographic column 13, a Mass Spectrometer (MS) 2, an ESI source 21, a low-flow-rate spray needle 211 and a mass spectrum detector 22.

FIG. 2 is a TIC chart (total ion current chromatogram) and TUV chart of an experiment under Original conditions in example 2;

wherein: (a) the TIC graph and (b) are TUV graphs.

FIG. 3 is the Original spectrum and deconvolution of the experiment under Original conditions in example 2;

wherein: (a) is the original spectrogram, and (b) is the deconvolution chart.

FIG. 4 shows the results of DT and CP fitting experiments in DOE2 in example 2;

wherein: (a) the effect p value and (b) the prediction curve.

FIG. 5 is a TIC and TUV plot for Axil 1 conditions in example 2;

wherein: (a) the TIC graph and (b) are TUV graphs.

FIG. 6 is the original spectrum and deconvolution under Axil 1 condition in example 2;

wherein: (a) is the original spectrogram, and (b) is the deconvolution chart.

FIG. 7 is the original spectrum of the experiment in example 2 under the optimum parameter conditions;

wherein: (a) the experimental parameter condition-1, (b) the experimental parameter condition-2, and (c) the experimental parameter condition-3.

FIG. 8 is a graph of deconvolution under the optimum parameters of the experiment in example 2;

FIG. 9 is the original spectrum under the condition of the theoretically optimal parameters in example 2;

wherein: (a) is theoretical parameter condition-1, (b) is theoretical parameter condition-2, and (c) is theoretical parameter condition-3.

FIG. 10 is a deconvolution graph under the theoretical optimum parameter conditions in example 2;

FIG. 11 is a raw spectrum of the multiple DAR sample test in example 2;

wherein: (a) is L-ADC, (b) is M-ADC, and (c) is H-ADC.

FIG. 12 is a deconvolution plot of the multiple DAR sample test in example 2;

wherein: (a) is L-ADC, (b) is M-ADC, and (c) is H-ADC.

FIG. 13 is a graph of drug profiles for the finished ADC test in example 2.

FIG. 14 is a graph of the drug profile of Adcetris in example 3.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

1. Instruments and equipment:

(1) liquid chromatography: ACQUITY

(2) Size exclusion chromatography column: BEH SEC (

1.7μm，4.6×150mm)、BEH SEC(

1.7μm，2.1×150mm)。

(3) Mass spectrometer (Waters Vion IMS Qtof): the ion source is ESI, the mass analyzer is TOF, and the detector is ultraviolet detector (TUV).

(4) And (3) needle spraying: low flow rate needles (Waters, 186007529), mass spectrometers are self-contained with conventional needles.

2. Preparation method of self-made ADC sample

To a 10mg/ml solution of mAb was added 10 equivalents of TCEP (purchased from Sigma-Aldrich), stirred at 37 ℃ for 2 hours to completely open the interchain disulfide bonds of the antibody, and then excess TCEP was removed via a G25 desalting column. Small molecule compounds of corresponding equivalents (L-ADC prepared using 4 equivalents, M-ADC prepared using 6 equivalents, H-ADC prepared using 8 equivalents, ADC-a prepared using 9 equivalents, and ADC-B prepared using 10 equivalents) were dissolved using DMSO, added dropwise to the reduced antibody solution, and DMSO was added thereto to a final concentration of 10% (v/v), and the reaction was stirred at 25 ℃ for 0.5 hour under a sealed condition, and after completion of the reaction, the sample was filtered using a 0.22 μ M membrane. The unconjugated small molecules were removed by purification using TFF (tangential flow ultrafiltration system), 50mM PB and 1.0mM EDTA solution (pH 6.0) in buffer, 15 volumes displaced, purified, added with sucrose to a final concentration of 6%, and stored in a refrigerator at-20 ℃ until use.

The purity of the ADC sample prepared was 99%.

The monoclonal antibody refers to a preparation method of an anti-Trop 2 antibody RS7 in the example 1 of Chinese patent application CN201910654574.3 (application date, 2019, 7, 19), and specifically comprises the following steps:

the nucleotide sequence of the light chain of the RS7 is shown as SEQ ID NO. 1, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 2; the light and heavy chain nucleotide sequence is obtained by whole gene synthesis (Jinzhi Suzhou), and is respectively and independently constructed into a pV81 vector by two enzyme cutting of EcoR I and Hind III (purchased from TAKARA), and is transformed into Trans 1-T1 competent cells (purchased from Beijing whole gold organism, CD501-03) by connection, clones are selected from the vector for PCR identification and are sent for detection and sequencing confirmation, the positive clones are cultured and amplified for plasmid extraction, so that an antibody light chain eukaryotic expression plasmid RS 7-L/pV 81 and an antibody heavy chain eukaryotic expression plasmid RS7-H/pV81 are obtained, the two plasmids are subjected to enzyme cutting linearization by Xba I (purchased from Takara, 1093S), the light and heavy chain eukaryotic expression plasmid proportion is 1.5/1, the light and heavy chain eukaryotic expression plasmid is transformed into CHO cells (purchased from ATCC-, after 3 weeks of culture, the expression level was measured by HTRF (homogeneous time-resolved fluorescence), and cell pool (cell pool) expressing Top10 was selected and amplified and frozen. Resuscitating a cell pool into a 125ml shake flask (culture volume 30ml), 37 ℃, 5.0% CO₂Shaking culture at 130rpm, 3 days later expanding to 1000ml shake flask (culture volume 300ml), 37 deg.C, 5.0% CO₂Shaking culture at 130rpm, feeding a supplemented medium with an initial culture volume of 5-8% every other day from the fourth day, culturing until 10-12 days, finishing the culture, centrifuging the harvest solution at 9500rpm for 15min, removing cell precipitates, collecting supernatant, filtering with a 0.22 μm filter membrane, and purifying the treated sample by using a MabSelect affinity chromatography column (purchased from GE company) to prepare the antibody RS 7.

The mAb was used as a reference mAb (mAb-Ref) in example 4.

The small molecule compound is prepared by a preparation method of CLB-SN38(n1 ═ 10) in example 2 of Chinese patent application CN201910654574.3 (

application date

2019, 7, 19 and 10), and specifically comprises the following steps:

step A:

SN38 (from carbofuran, Calif.) 10g (25.5mmol) was suspended in 200mL of anhydrous dichloromethane, and 5mL of pyridine and 11.3g (51mmol) of Boc-anhydride were added and reacted at room temperature overnight. After the reaction, the solvent was evaporated under reduced pressure to obtain a pale yellow solid, which was washed with petroleum ether to obtain 9.8g of a final product with a yield of 78%. MS [ M + H ]]⁺： 493.2。

And B:

the compound Boc-SN 385 g (10.1mmol) was dissolved in 100mL of anhydrous dichloromethane, 2mL of pyridine and 2.47g (20.2mmol) of DMAP were added, the temperature was reduced to 0 ℃, 1.5g (5.0 mmol) of triphosgene was slowly added in dichloromethane, the reaction was carried out at 0 ℃ for 1h, 11.3g (20.0mmol) of the starting material VII (purchased from Ningonions Suzhou) was added, and the mixture was allowed to warm to room temperature for 5 h. After the reaction, the solvent was evaporated to dryness under reduced pressure, and the compound VI was obtained by column chromatography separation in a yield of 65% as 7.1g of a pale yellow solid. MS [ M + H ]]⁺：1077.3。

And C:

compound VI was dissolved in 100mL of anhydrous dichloromethane (5 g, 4.64mmol), DBU (3.5 mL, 23.2mmol) was added, the reaction was carried out at room temperature for 30min, then SMCC (ex borrelium) 3.1g (9.28mmol) was added, and the reaction was continued at room temperature for 1 h. After the reaction, the solvent was evaporated to dryness under reduced pressure, and column chromatography was carried out to give compound V as a pale yellow solid (2.58 g) in 51% yield. MS [ M + H ]]⁺：1089.7。

Step D:

dissolving 3g (2.76mmol) of the compound V in 30mL of anhydrous dichloromethane, adding 10mL of trifluoroacetic acid, reacting at room temperature for 30 minutes, pouring the solution after the reaction into 100mL of anhydrous ether, separating out yellow solid, washing with ether to obtain 1.65g of a final product, namely a light yellow solid, and directly using the crude product for the next reaction.

Step E:

1g (1.0mmol) of the compound III was dissolved in 20mL of anhydrous dichloromethane, and 1.34g (1.2mmol) of polyethylene glycol carboxylic acid, 0.76g (2.0mmol) of HATU and 695. mu.L (4.0mmol) of DIEA were added thereto and reacted at room temperature for 2 hours. After the reaction, the solvent was evaporated to dryness under reduced pressure, and the crude product was subjected to column chromatography to give CLB-SN38 as a pale yellow oil 0.9 g. 1H-NMR (500MHz, CDCl3) delta 9.83(s,1H),8.45(s,1H), 8.05-8.03(M,1H),7.61-7.59(M,1H),7.44-7.38(M,5H),7.04(s,1H),6.72(s,2H), 6.35(s,1H),5.74-5.70(M,1H),5.43-5.31(M,5H),5.19(s,2H),4.92-4.89(M, 1H),4.31(s,1H),3.79-3.76(M,3H),3.75-3.58(M,51H),3.56-3.49(M,7H), 3.41-3.36 (M,6H),3.15-3.07(M, 3.07), 2.60-2H, 1H), 2H + 1H, 2H, 1H, 2H, 5, 7H, 2H, 7H, 2H, 7H, 2H, 2, 7, 2, 7H, 7, 2H, 2, 7H, 2, 7, 2H, 2H, 7, 2: 1459.1.

3. non-denaturing treatment agent

(1) Ammonium acetate aqueous solution: 100mM, pH 6.5.

(2) PNGaseF enzyme: reian organisms, Suzhou, QPF-001.

4. Non-denaturing treatment step

Diluting the sample to 2mg/ml by using a mobile phase, adding PNGaseF, carrying out enzyme digestion at 37 ℃ for 3h for desugarization, and then carrying out ultrafiltration substitution to obtain the ammonium acetate system to be injected, wherein the final concentration of the sample is 2 mg/ml.

The mobile phase is 100mM ammonium acetate water solution, and the pH value is adjusted to 6.5 by acetic acid.

The PNGaseF enzyme was added to the diluted sample at a ratio of 1:100 (v/v).

Example 1

As shown in fig. 1, the present embodiment is an analysis apparatus for non-denaturing mass spectrometry, including: an ultra-high performance liquid chromatograph 1 (desalting device) and a mass spectrometer 2 (mass spectrometry device); the ultra-high performance liquid chromatograph 1 comprises: a solvent manager 11, a sample manager 12 and a size exclusion chromatographic column 13 connected in sequence; the mass spectrometer 2 comprises: ESI source 21 and mass detector 22, wherein: the ESI source from a conventional needle with a low flow needle 211 of the present invention is replaced; a solvent manager 11 of the ultra-high performance liquid chromatograph 1 conveys a solvent mobile phase to a sample manager 12, and the solvent mobile phase is mixed with a protein sample to be detected in the sample manager 12 and then input into a size exclusion chromatographic column 13 for desalination; the desalted protein sample to be detected directly enters the ESI source 21 on line through a low-flow-rate spray needle 211 after eluting from the size exclusion chromatographic column 13, and the mass spectrometer 22 detects and analyzes gas-phase ions passing through the ESI source 21.

The low flow rate nozzle 211 used in this embodiment has a flow rate of 0.020ml/min for mass spectrum sampling and a pore size of about 30-36 μm.

Example 2 and comparative example

Through an earlier-stage exploration experiment, 4 key ion source parameters (called key source parameters for short) are screened out:

<1> capillary voltage (CP, kV);

<2> cone voltage (CV, V);

<3> source temperature (ST, ° C);

<4> desolvation temperature (DT,. degree.C.).

This was examined by designing a 4-factor 2 level DOE experiment.

The sample used in this example and comparative example was ADC-A in an amount of 100 mg.

The needle used in the comparative example was a conventional needle, and the conditions were the same as those of the low flow rate needle used in the examples except for the volume of the column, the elution rate, and the key source parameters.

1. Liquid phase and mass spectrum conditions:

(1) comparative example: ESI was derived from a conventional needle with:

a chromatographic column: ACQUITY

BEH SEC(

1.7μm，4.6×150mm)。

Column temperature of the chromatographic column: the sample was loaded at 25 ℃ in a volume of 5. mu.l, i.e.10. mu.g.

Liquid chromatography elution rate: isocratic elution was carried out at a flow rate of 0.065ml/min for 10 min.

Mass spectral parameters DOE1 are shown in table 1.

TABLE 1 DOE1 Mass Spectrometry Critical Source parameters

The "+" and "-" values in the table represent the maximum and minimum values of the corresponding parameters, respectively.

(2) Example 2: low flow rate needle injection:

a chromatographic column:

BEH SEC(

1.7μm，2.1×150mm)。

Liquid chromatography elution rate: isocratic elution was carried out at a flow rate of 0.020ml/min for 30 min.

Mass spectral parameters DOE2 are shown in table 2.

TABLE 2 DOE2 Mass Spectrometry Critical Source parameters

(3) Other mass spectral parameters are shown in table 3:

TABLE 3 Mass Spectrometry other parameters

Mass spectrometer	Q-TOF
		Ion source	ESI
Mass spectrometry data acquisition mode	MS
		Ion mode	Positive ion
Mass range	m/z 700–8000
		Resolution ratio	40000

2. And screening out optimal parameters of the experiment according to the results of the two DOE experiments, comparing the optimal parameters with optimal parameters of software fitting, and determining the final parameters to test the subsequent samples, wherein the repeatability, the specificity and the accuracy of the test method are improved. The method comprises the following steps:

2.1 arrangement of the Experimental data

2.1.1 comparative example 1: analysis of the results of the experiment with DOE1

As shown in FIGS. 2-3, before the DOE experiment, the best conditions (origin conditions in Table 1) obtained by the previous experiments were repeated, and the Original spectrogram response value is 1230. And (3) trying to fit the data of the DOE1 by taking the response value of the original spectrogram as Y, finding that no proper model matching exists, and preliminarily judging that no response surface exists between the selected parameters. As shown in table 4, in the 16 DOE conditions and 19 with three centroids, the condition of the first three response values is 3 centroids:

TABLE 4 center point parameters and response values

Name (R)	Condition	CP	CV	ST	DT	Response value
							Mid
	0	2.5	95	100	325	3680
							Mid(50Cg)	0	2.5	95	100	325	3540
Mid(0Cg)	0	2.5	95	100	325	2910
							Original	/	1.5	40	120	450	1230

As can be seen, the peak response value is improved by about 3 times compared with the response value under the Original condition (3680vs 1230).

2.1.2 example 2: analysis of the results of the experiment with DOE2

As shown in table 5, the condition of response top 5, except the Axial1 condition, has the maximum CP and DT:

table 5 experimental parameters of DOE2 before response 5

Name (R)	Condition	CP	CV	ST	DT	Response value
							Axial
1	000A	2.5	95	100	450	17300
							14	+--+	4	40	50	450	13000
10	++-+	4	150	50	450	12800
							6	+-++	4	40	150	450	11800
7	++++	4	150	150	450	10800

FIGS. 4 to 5 show the results of experiments under Axil 1.

As can be seen by comparison, the response value of the optimal condition of the low-flow-rate needle of the invention is improved by about 5 times compared with the response value of the optimal condition of the conventional needle of the comparative example (17300vs 3680).

As shown in fig. 4, the data of DOE2 were also fitted with the response value Y, and as a result, it was found that only CP (capillary voltage) and DT (desolvation temperature) are key factors of the four key source parameters, which corroborates the experimental results; at this point CP3.5, DT450, is the theoretical optimum parameter.

In summary, the parameter conditions (CP, CV, ST, DT) of Axil 1 were selected as the optimal parameters for the experiment.

2.2 comparison of the Experimental optimal parameters with the theoretical optimal parameters

In order to screen out the final conditions, sample introduction is carried out for three times respectively under the experimental optimal parameters and the theoretical optimal parameters, response values are compared, and the experimental results are shown in figures 5-10 and tables 6-7:

(1) experimental optimal parameters (CP2.5, CV95, ST100, DT450)

(2) Theoretical optimal parameters (CP3.5, CV95, ST100, DT450)

(3) Average response value comparison and DAR calculation

The DAR calculation formula is as follows:

wherein: DAR is the weighted average drug-antibody coupling ratio; the ionic strength (ion intensity) is the corresponding peak strength of the DARn, and n is the number of DARn coupled small molecules.

TABLE 6 average response value comparison

TABLE 7 DAR calculated comparison

From this, it is found that the average response value of the experimental optimum parameter is about 20% higher than that of the theoretical optimum parameter for the Cys-ADC sample, and the average DAR values are 7.37 and 7.44, respectively, which are closer to the DAR value measured by the orthogonal method (measured by the RPLC-MS method). Therefore, the optimum parameters for the experiment are selected as the optimum parameters for the method of this embodiment.

3. Method verification

3.1 multiple DAR sample testing

(1) For the specificity of the test method, testing ADC samples L-ADC, M-ADC and H-ADC with different DAR values by using the optimal experimental parameters; the DAR measured by the LC-MS method of the three samples is respectively as follows: 1.9, 3.5 and 5.6.

The experimental results are shown in FIGS. 11 to 12.

(2) The results are calculated as shown in tables 8 to 10.

TABLE 8 DAR test results (L-ADC)

TABLE 9 DAR test results (M-ADC)

TABLE 10 DAR test results (H-ADC)

The measured DAR values for the multiple DAR samples are shown in table 11 together with the statistical results of the DAR values measured by the remaining orthogonal methods:

TABLE 11 statistics of DAR value determination by orthogonal method

DAR，by UV	2.8	4.6	6.7
				DAR，by HIC	3.1	4.7	6.1
DAR，by RPLC-MS	1.9	3.5	5.6
				DAR, by the invention Native-MS	1.2	2.8	5.0

3.2 official sample testing

(1) The final test was performed with the ADC finished product to verify the accuracy of the complete molecular weight/DAR determination and the distribution of the drug.

(2) The results are shown in FIG. 13 and tables 12 to 13:

TABLE 12 detection results of naked antibody control (mAb reference, mAb-Ref)

TABLE 13 ADC finished product test results

In the table, L is the linker in the ADC, D is the cytotoxic drug conjugated in the ADC, and the numbers before L and D indicate the number of linker and drug, respectively.

The result shows that the error between the actually measured molecular weight and the theoretical molecular weight of the monoclonal antibody reference product is within 50ppm, which proves that the system runs normally. The error between the actual measured complete molecular weight and the theoretical complete molecular weight of the ADC is less than 50ppm, and the DAR is very close to the calculation result of the current batch release phase inversion method (the DAR is 7.6), thereby proving the orthogonality of the two methods and the accuracy of the current DAR value. Meanwhile, the method can be used as an MAM method, and the determination of the complete molecular weight and the drug distribution of the ADC is completed simultaneously in a single experiment.

Example 3

In this example, the ion source parameters were CP2.5 kV, CV 95V, ST100 ℃ and DT450 ℃.

The protein sample in this example was Adcetris (commercial Cys-ADC) and the results are shown in FIG. 14, where D0, D2, D4, D6 and D8 are the DAR values represented by the peaks. The results show that the DAR value measured in this example (4.0) is comparable to that of the literature (Debaene,

"Innovative Native MS methods for Antibody Drug conjugation Characterization" High Resolution Native MS and IM-MS for Average DAR and DAR Distribution assessment "Analytical Chemistry, vol.86, No.21,2014, pp.10674-10683.) the same DAR values were measured.

SEQUENCE LISTING

<110> Shanghai Compound Dangjiang biomedical corporation

<120> method and apparatus for analyzing non-denaturing mass spectrum

<130> P20012579C

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1353

<212> DNA

<213> Homo sapiens

<400> 1

caggtccaac tgcagcaatc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg cttctggata caccttcaca aactatggaa tgaactgggt gaagcaggcc 120

cctggacaag ggcttaaatg gatgggctgg ataaacacct acactggaga gccaacatat 180

actgatgact tcaagggacg gtttgccttc tccttggaca cctctgtcag cacggcatat 240

ctccagatca gcagcctaaa ggctgacgac actgccgtgt atttctgtgc aagagggggg 300

ttcggtagta gctactggta cttcgatgtc tggggccaag ggtccctggt caccgtctcc 360

tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1080

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccgggt aaa 1353

<210> 2

<211> 642

<212> DNA

<213> Homo sapiens

<400> 2

gacatccagc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcagc 60

atcacctgca aggccagtca ggatgtgagt attgctgtag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctactcg gcatcctacc ggtacactgg agtccctgat 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg cagtttatta ctgtcagcaa cattatatta ctccgctcac gttcggtgct 300

gggaccaagg tggagatcaa acgtactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

Claims

1. A method of non-denaturing mass spectrometry comprising: desalting a non-denatured protein sample to be detected, eluting, and directly performing mass spectrum sample loading analysis on an eluent on line, wherein the flow rates of elution and mass spectrum sample loading are both 0.019-0.021 ml/min; preferably 0.020 ml/min.

2. The assay of claim 1, wherein the protein sample is an antibody-conjugated drug; preferably a Cys-antibody conjugate drug; more preferably a Cys-antibody conjugate drug with a drug-antibody conjugate ratio (DAR) ≥ 5, 5.5, 6, 6.5 or 7.

3. The analytical method of claim 1, wherein said desalting is accomplished by ultra high liquid chromatography; preferably, the ultra-high liquid chromatograph uses a size exclusion chromatographic column; more preferably, the size exclusion chromatographic column has a volume of 0.52 to 2.49 ml; preferably an ethylene bridge hybrid particle chromatographic column having a particle pore size of

The particle size was 1.7. mu.m.

4. The method of claim 1, wherein the mass spectrometric sample loading analysis is performed by a mass spectrometer having an ion source; preferably, the ion source is electrospray ionization.

5. The analytical method of claim 4, wherein the ion source has an applied voltage of 1 to 4kV, preferably 2.5 kV;

and/or the cone hole voltage of the ion source is 40-150V, preferably 95V;

and/or the source temperature of the ion source is 50-150 ℃, and preferably 100 ℃;

and/or the desolvation temperature of the ion source is 200-450 ℃, preferably 450 ℃.

6. The analytical method of claim 5, wherein the ion source has an applied voltage of 2.5kV and the desolvation temperature is 450 ℃.

7. The analytical method of claim 5, wherein the ion source has an applied voltage of 2.5kV, a desolvation temperature of 450 ℃, a cone-hole voltage of 95V, and a source temperature of 100 ℃.

8. The assay of claim 1, wherein said non-denaturing treatment comprises: diluting a protein sample to be detected by using a mobile phase with the pH value of 6.0-7.0, performing enzyme digestion and desugarization, and replacing a protein system to be detected by using the mobile phase into a mobile phase system to be injected; preferably, the mobile phase has a pH of 6.5.

9. The assay of claim 8, wherein the mobile phase is an ammonium acetate buffer at pH 6.5; preferably, the concentration of the ammonium acetate buffer solution is 50-150 mM; preferably 100 mM;

and/or the enzyme is used for removing sugar chains from the glycoprotein through PNGaseF.

10. An apparatus for use in an assay method according to any one of claims 1 to 9, comprising: the method comprises the following steps of desalting a protein sample to be detected after non-denaturing treatment, and mass spectrum equipment which is connected with the desalting equipment and is used for directly carrying out mass spectrum sample loading analysis on desalted eluent, wherein the mass spectrum equipment comprises a spray needle for receiving the eluent, and the spray needle is a spray needle which is suitable for the eluent and the mass spectrum sample loading and has the flow velocity of 0.019-0.021 ml/min; preferably, the aperture of the spray needle is 27-45 μm; more preferably, the pore diameter is 30 to 36 μm.