CN112285238B - Liquid chromatography for detecting content of free micromolecules of ADC (azodicarbonamide) medicine - Google Patents

Abstract

The invention provides a liquid chromatography for detecting the content of free micromolecules of ADC (azodicarbonamide) medicines, which comprises the following steps of: adding a salt solution and an organic solvent into an ADC sample, centrifuging, concentrating the supernatant, and detecting by adopting a reverse phase chromatography to obtain the content of free micromolecules. In the ADC sample treatment process, the protein can be completely precipitated under the combined action of the salt and the organic solvent, the interference of a protein residual peak on detection is avoided, and the method has high sensitivity.

Description

Liquid chromatography for detecting content of free micromolecules of ADC (azodicarbonamide) medicine

Technical Field

The invention relates to the technical field of chemical analysis and detection, in particular to a liquid chromatography for detecting the content of free small molecules of ADC (azodicarbonamide) medicines.

Background

The antibody-conjugated drug (ADC) is prepared by coupling a monoclonal antibody drug and a small-molecule cytotoxic drug, targeting a monoclonal antibody to tumor cells, decomposing and releasing small-molecule toxin to kill the tumor cells after endocytosis, and has the advantages of improving targeting property and drug effect and reducing toxic and side effects of small molecules. In ADC drug design, the commonly used linker (linker) is maleimide-vc dipeptide, and the commonly used small molecule toxin (drug) is MMAE (Monomethylauristatin E). The synthesis process comprises the following steps: the maleimide and MMAE were linked to form maleimide-vc-MMAE (linker-drug, LD). The antibody is added with a reducing agent to open the interchain disulfide bond, and LD is coupled to the sulfhydryl group. The remaining LD will be inactivated by addition of excess cysteine to form Cys-maleimide-vc-MMAE (CLD), terminating the coupling reaction. By this step, LD is theoretically all converted to CLD. Eventually by a purification process, CLD is removed, but there may still be a small amount of free CLD remaining in the product. In addition, the conjugated small molecules (LD and MMAE) in the product may be detached from the ADC during storage of the drug. Free small molecular toxin has no targeting property and high toxicity, and can influence the safety of ADC medicines. It is important to monitor the amount of free small molecules in the ADC.

The conventional procedure for detecting free small molecules by reverse phase chromatography is to add 3 times volume of organic solvent into ADC drug to denature and precipitate ADC, and then remove protein precipitate by centrifugation to obtain supernatant of small molecule toxin. The supernatant was subjected to reverse phase chromatography using a C18 column and the small molecules were quantified by external standard methods. The disadvantages are that: (1) 3 times volume of organic solvent is added in the pretreatment of the sample, so that the concentration of free micromolecules is diluted to be lower, and the sensitivity is not enough for the product with trace free micromolecules. (2) Protein residues are still detected after a part of ADC products are pretreated by an organic solvent, which is mainly related to the protection of a formula of a preparation buffer solution on the proteins, and a large amount of protein residues can interfere with the detection of free small molecules and can shorten the service life of a chromatographic column.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide a liquid chromatography for detecting the content of free small molecules in ADC drugs, which has high sensitivity and no protein interference.

In order to achieve the purpose, the invention provides a liquid chromatography for detecting the content of free micromolecules of ADC drugs, which comprises the following steps:

adding a salt solution and an organic solvent into an ADC sample to obtain a mixture, centrifuging, concentrating the supernatant, and detecting by adopting a reverse phase chromatography to obtain the content of the free micromolecules.

In the invention, the ADC refers to an antibody conjugate drug, or is called an antibody drug conjugate. The ADC is not particularly limited in kind, and any ADC that is conventional in the art may be used, such as microtubule inhibitor conjugated antibody drugs (MMAE conjugated antibody drugs, DM1 conjugated antibody drugs, etc.), DNA damaging agent conjugated antibody drugs (calicheamicin conjugated antibody drugs, etc.). In some embodiments of the invention, the ADC is an anti-HER 2 monoclonal antibody-vc-MMAE conjugate drug.

Preferably, in the mixture, the concentration of the salt solution is more than or equal to 5mmol/L; more preferably 5 to 5mol/L.

In the present invention, the volume content of the organic solvent in the mixture is preferably 60% or more, and more preferably 60% to 75%.

Preferably, the volume ratio of the ADC sample, the salt solution and the organic solvent is 100;

the concentration of the salt solution is preferably 2mol/L.

Preferably, the salt solution is a NaCl solution or a KCl solution.

The solvent of the salt solution is preferably ultrapure water.

In the present invention, preferably, the organic solvent is selected from one or more of methanol, ethanol, acetonitrile, isopropanol and acetone.

The buffer for the ADC sample is not particularly limited in the present invention, and may be any suitable buffer known to those skilled in the art.

In the present invention, the buffer of the ADC sample is preferably a combination of one or more of histidine, succinic acid, sucrose, trehalose, polysorbate 20 and polysorbate 80.

Preferably, according to the invention, a linear standard curve is drawn by preparing a standard solution from the MMAE standard and the CLD standard before detection.

The method for drawing the linear standard curve is not particularly limited, and may be a method known to those skilled in the art, and the following steps are preferably adopted in the present invention:

MMAE standards were diluted to 50. Mu.g/mL with acetonitrile and used as MMAE stock solutions.

CLD standards were diluted to 100 μ g/mL with acetonitrile and used as CLD stock solutions.

MMAE mother liquor and CLD mother liquor were formulated as a series of linear standard solutions, the concentrations of which in some embodiments of the invention are shown in table 1:

TABLE 1 Linear Standard solution concentration

Preferably, the liquid chromatography is specifically as follows:

adding a salt solution and an organic solvent into an ADC sample, centrifuging, concentrating, drying and redissolving supernatant, and detecting by adopting a reverse phase chromatography to obtain the content of free micromolecules.

In the invention, the preferable solvent for re-dissolving is acetonitrile.

The concentration of acetonitrile is preferably 10% to 50%, and more preferably 30%.

According to the invention, after the sample is centrifugally concentrated and dried, a proper amount of acetonitrile is added for redissolving, so that the volume of the sample solution can be reduced, the concentration of small molecules in the sample is improved, and the accurate detection is more convenient.

In some embodiments of the invention, 100. Mu.L of the solution to be tested is added, 4. Mu.L of the salt solution is added, and 296. Mu.L of the organic solvent is added.

Then, reversed phase chromatography was used for detection.

In the preferred method, the mobile phase A of the reverse phase chromatography is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution.

The reversed phase chromatography preferably adopts a C18 reversed phase chromatographic column with octadecylsilane as a filling agent, the wavelength of an ultraviolet detector is 214nm, the column temperature is preferably 45 ℃, the flow rate is preferably 0.3mL/min, and the injection volume is preferably 10 muL.

Preferably, the elution gradient of the reverse phase chromatography is as follows:

0-20 min, the volume ratio of the mobile phase A to the mobile phase B is 7:3;

20-40 min, the volume ratio of the mobile phase A to the mobile phase B is 3:7;

40-60 min, the volume ratio of the mobile phase A to the mobile phase B is 7:3;

60-90 min, and the volume ratio of the mobile phase A to the mobile phase B is 7:3.

See table 2 for details:

table 2: elution gradient

Time (min)	A％	B％
			0	70	30
20	30	70
			20	70	30
30	70	30

In the invention, the free small molecules are MMAE and CLD.

The MMAE refers to monomethyl auristatin E.

The CLD refers to Cys-maleimide-vc-MMAE.

Cys means cysteine.

Compared with the prior art, the invention provides a liquid chromatography for detecting the content of free micromolecules of ADC (azodicarbonamide) medicaments, which comprises the following steps of: and adding a salt solution and an organic solvent into the ADC sample, centrifuging, concentrating the supernatant, and detecting by using a reverse phase chromatography to obtain the content of the free micromolecules. In the ADC sample treatment process, the protein can be completely precipitated under the combined action of the salt and the organic solvent, the interference of a protein residual peak on detection is avoided, and the method has high sensitivity.

Drawings

FIG. 1 is a standard curve overlay map;

FIG. 2 is a chromatogram of sample 1, sample 2, and a blank solution of example 1;

FIG. 3 is a chromatogram of sample 1 of example 2;

FIG. 4 is a chromatogram of comparative example 1.

Detailed Description

In order to further illustrate the present invention, the following examples are provided to describe the liquid chromatography method for detecting the content of free small molecules in ADC drugs.

Example 1

1. Preparation of small molecule standard substance stock solution

The MMAE standard was precisely weighed at 5.038mg, diluted to 50. Mu.g/mL MMAE stock solution with an appropriate amount of 30% ACN (acetonitrile).

CLD standard 5.104mg was accurately weighed and diluted to 100. Mu.g/mL CLD stock solution with an appropriate amount of 30% ACN.

2. Preparation of small molecule standard curve

MMAE and CLD stocks were aspirated and diluted with 30% acn to make a series of linear standard solutions, at concentrations as given in table 1:

table 1: concentration of Linear Standard solution

3. Sample preparation

The following formulation buffer components were as follows:

contains 15mM histidine, 6% sucrose and 0.02% polysorbate 20 solution.

3.1 blank solution: taking 100 mu L of ADC sample preparation buffer solution, adding 4 mu L of 2M NaCl solution and 296 mu L of isopropanol, centrifuging at 13000rpm for 30min, sucking 200 mu L of supernatant into a new 1.5mL centrifuge tube, centrifuging, concentrating, drying, adding 50 mu L of 30% ACN for redissolving, and fully mixing.

3.2 ADC sample 1 solution: 100 μ L of LADC sample 1 (20 mg/mL of anti-HER 2 monoclonal antibody-vc-MMAE conjugate drug dissolved in formulation buffer) was added with 4 μ L of 2M NaCl and 296 μ L of isopropanol, centrifuged at 13000rpm at room temperature for 30min, 200 μ L of the supernatant was aspirated into a new 1.5mL centrifuge tube, centrifuged, concentrated and dried, reconstituted with 50 μ L of 30% ACN, and mixed well.

3.3 ADC sample 2 solution: 100 μ L of LADC sample 2 (20 mg/mL of anti-HER 2 monoclonal antibody-vc-MMAE conjugate drug was dissolved in the formulation buffer), 4 μ L of 2M NaCl and 296 μ L of isopropanol were added, the mixture was centrifuged at 13000rpm at room temperature for 30min, 200 μ L of the supernatant was aspirated into a new 1.5mL centrifuge tube, the mixture was centrifuged, concentrated and dried, 50 μ L of 30% ACN was added to redissolve the mixture, and the mixture was thoroughly mixed.

4. Chromatographic conditions

Adopting a chromatographic column Poroshell 120 SB-C18.1 x 150mm 2.7 μm, an ultraviolet detector wavelength of 214nm, a column temperature of 45 ℃, a flow rate of 0.3mL/min and a sample injection volume of 10 μ L; wherein the mobile phase A was 0.1% aqueous TFA solution, the mobile phase B was 0.1% TFA acetonitrile solution, the gradient elution procedure is shown in Table 2.

Table 2: elution gradient

Time (min)	A％	B％
			0	70	30
20	30	70
			20	70	30
30	70	30

5. Calculation of results

The blank solution was not perturbed at MMAE and CLD peak positions.

The concentration of MMAE and CLD standard curve solutions is taken as the horizontal axis, and the chromatographic peak area is taken as the vertical axis to prepare a linear standard curve.

The MMAE standard curve is y =25665.02x-1784.16 ² ＝0.999。

The CLD standard curve is y =21909.62x-4099.45 ² ＝0.999。

The result is shown in figure 1, and figure 1 is a standard curve overlapping map which is sequentially SD1-SD7 from bottom to top.

FIG. 2 is a chromatogram of sample 1, sample 2, and a blank solution.

The MMAE, CLD content in ADC samples was calculated as shown in table 3:

TABLE 3 Small molecule content results

Note: ADC sample 1 and ADC sample 2 product batches were different.

Example 2

1. Preparation of small molecule standard substance stock solution

The MMAE standard was precisely weighed at 5.038mg, diluted to 50. Mu.g/mL MMAE stock solution with an appropriate amount of 30% ACN.

CLD standard 5.104mg was accurately weighed and diluted to 100. Mu.g/mL CLD stock solution with an appropriate amount of 30% ACN.

2. Preparation of small molecule standard curve

MMAE and CLD stocks were aspirated and diluted with 30% acn to make a series of linear standard solutions, at concentrations as given in table 1:

table 1: concentration of Linear Standard solution

3. ADC sample 1 solution: 100 μ L of ADC sample 1 (same as ADC sample 1 in example 1) was added with 4 μ L of 2M KCl and 296mL of acetone, centrifuged at 13000rpm for 30min at room temperature, 200 μ L of the supernatant was taken out and put in a new 1.5mL centrifuge tube, centrifuged, concentrated and dried, added with 50 μ L of 30% ACN, redissolved, and mixed well.

4. Chromatographic conditions

Adopting a chromatographic column Poroshell 120 SB-C18.1 x 150mm 2.7 μm, an ultraviolet detector wavelength of 214nm, a column temperature of 45 ℃, a flow rate of 0.3mL/min and a sample injection volume of 10 μ L; wherein mobile phase A was 0.1% aqueous TFA, mobile phase B was 0.1% TFA in acetonitrile, and the gradient elution procedure is shown in Table 2.

Table 2: elution gradient

Time (min)	A％	B％
			0	70	30
20	30	70
			20	70	30
30	70	30

5. Calculation of results

The concentration of MMAE and CLD standard curve solutions is taken as the horizontal axis, and the chromatographic peak area is taken as the vertical axis to prepare a linear standard curve.

The MMAE standard curve is y =22882.89x-992.78 ² ＝0.999。

The CLD standard curve is y =22109.10x-5136.75 ² ＝0.999。

MMAE was not detected in ADC sample 1. The CLD peak area was 4738, and the concentration was calculated by substituting it into a standard curve to obtain 0.45. Mu.g/mL.

FIG. 3 is a chromatogram of sample 1.

Comparative example 1

1. Sample preparation

ADC sample 1 solution: 100mL of ADC sample 1 (same as ADC sample 1 in example 1) was added with 300. Mu.L of methanol, centrifuged at 13000rpm for 30min at room temperature, 200. Mu.L of the supernatant was taken out and put in a new 1.5mL centrifuge tube, centrifuged, concentrated and dried, and 50. Mu.L of 30% ACN was added for redissolution and mixed well.

2. Chromatographic conditions

And (3) chromatographic column: poroshell 120 SB-C18.1 x 150mm 2.7 μm, ultraviolet detector wavelength 214nm, column temperature 45 deg.C, flow rate 0.3mL/min, sample volume 10 μ L; wherein the mobile phase A was 0.1% aqueous TFA solution, the mobile phase B was 0.1% TFA acetonitrile solution, the gradient elution procedure is shown in Table 2.

Table 2: elution gradient

Time (min)	A％	B％
			0	70	30
20	30	70
			20	70	30
30	70	30

3. As a result, the

The residual protein peak was significant, the retention time was 7.8min, and there was a tailing phenomenon, covering the CLD-out peak position (8.2 min), resulting in undetected CLD. The retention time of MMAE was 7.2min, so the protein residual peak was not interfering with the detection of MMAE. The chromatogram is shown in FIG. 4.

As can be seen from the above examples, the detection method provided by the invention avoids protein interference and has higher accuracy and sensitivity.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (3)

1. A liquid chromatography for detecting the content of free small molecules of ADC drugs comprises the following steps:

adding a salt solution and an organic solvent into an ADC sample to obtain a mixture, centrifuging, concentrating supernate, and detecting by adopting a reverse phase chromatography to obtain the content of free micromolecules;

the volume ratio of the ADC sample, the salt solution and the organic solvent is 100;

the concentration of the salt solution is 2mol/L;

the mobile phase A of the reverse phase chromatography is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution;

the elution gradient of the reverse phase chromatography is:

0 to 20min, mobile phase A:70% → 30%, mobile phase B:30% → 70%;

20 to 30min, mobile phase A:70 percent; mobile phase B:30 percent;

the free small molecules are MMAE and CLD;

the CLD is Cys-maleimide-vc-MMAE;

the salt solution is a NaCl solution or a KCl solution;

the solvent of the salt solution is ultrapure water;

the organic solvent is selected from one or more of methanol, ethanol, acetonitrile, isopropanol and acetone;

the ADC drug is an anti-HER 2 monoclonal antibody-vc-MMAE conjugate drug;

the column is a C18 reverse phase column packed with octadecylsilane.

2. The liquid chromatography method of claim 1, further comprising:

and preparing a standard solution by using the MMAE standard substance and the CLD standard substance, and drawing a linear standard curve.

3. Liquid chromatography according to claim 1, characterized in that it is in particular:

adding a salt solution and an organic solvent into an ADC sample, centrifuging, concentrating, drying and redissolving supernatant, and detecting by adopting a reverse phase chromatography to obtain the content of free micromolecules.

Images