LC-MS / MS method for the determination of palbociclib monoclonal antibody drug in blood samples

Abstract

The application discloses an LC-MS / MS detection method for a palbociclib monoclonal antibody drug in a blood sample, enriches and purifies the palbociclib monoclonal antibody drug in serum and plasma through coupling of a reagent I and magnetic beads, separates the enriched blood sample through magnetic attraction, discards supernatant, adds buffer I for elution, adds buffer II for dilution after elution, adds an internal standard and a protease for enzyme hydrolysis after high-temperature denaturation of the diluted blood sample, and finally, the high-sensitivity and rapid detection of the palbociclib monoclonal antibody drug concentration in the blood sample is realized through high performance liquid chromatography-tandem mass spectrometry. The detection method optimizes the operation steps of traditional operation, removes the protein reduction and alkylation steps in the pretreatment process, shortens the protease hydrolysis time, and is simple, rapid and convenient in the overall process, and can simultaneously meet multiple requirements of time efficiency, accuracy and low detection cost in clinical detection.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to an LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples. Background Technology

[0002] Therapeutic monoclonal antibodies (mAbs) are a class of antibody drugs that are highly homogeneous and have pharmacological effects against a single antigenic epitope, obtained through molecular biology techniques. They have been widely used in the targeted therapy of diseases such as tumors, autoimmune diseases, and infections.

[0003] As monoclonal antibody drugs are further developed and used clinically, clinical problems are gradually emerging. Significant differences exist in the degree of individual response to these drugs, with initial treatment response rates ranging from 40% to 90%. However, some patients who initially respond to treatment experience loss of response during treatment, leading to disease progression. Individual differences in treatment response and loss of response are believed to be related to factors such as the concentration of monoclonal antibody drugs in the blood and immunogenicity after drug use. Therefore, dosage optimization based on therapeutic drug monitoring can provide an effective means for precision treatment with monoclonal antibody drugs.

[0004] Currently, there are two main methods for detecting monoclonal antibody drug concentration: liquid chromatography-mass spectrometry (LC-MS) and immunoassay.

[0005] Immunoassay is a commonly used clinical method, but it has many drawbacks in monitoring monoclonal antibody therapy: narrow quantitative range (about two orders of magnitude); long development time and difficulty in obtaining highly specific antibodies; cross-immunization; non-specific binding; poor specificity; and the ability to measure only the free form of the antibody.

[0006] In recent years, liquid chromatography-mass spectrometry (LC-MS / MS) has gradually become an indispensable tool in monoclonal antibody drug research due to its advantages of high separation, high specificity, and accurate quantification. The "Pharmaceutical Expert Consensus on Therapeutic Drug Monitoring of Antitumor Biosimilars," initiated by the Chinese Pharmacological Society and the China-Japan Friendship Hospital in May 2020, strongly recommends the use of LC-MS / MS for therapeutic drug monitoring. Currently reported LC-MS / MS methods for monoclonal antibody drug concentration determination either have complex pretreatment procedures, time-consuming enzymatic digestion, or are too expensive to meet clinical testing needs.

[0007] Pembrolizumab is a commonly used anti-tumor monoclonal antibody drug in clinical practice. Due to its broad-spectrum anti-tumor efficacy (targeting dozens of different types of cancer, including melanoma and non-small cell lung cancer), it is increasingly being used in clinical cancer treatment. Currently, there is a lack of efficient, simple, and rapid methods for detecting its blood drug concentration using liquid chromatography-tandem mass spectrometry.

[0008] Chinese patent document CN 113804804 A discloses a "Rapid determination method for monoclonal antibody drugs in plasma by ultra-high performance liquid chromatography-tandem mass spectrometry". This method can simultaneously detect three monoclonal antibody drugs, rituximab, trastuzumab and cetuximab, in plasma. However, its pretreatment process (which takes about 6 hours) and chromatographic analysis process (11 minutes per sample) are both time-consuming, and it requires a high-sensitivity mass spectrometer (Waters-TQS) for detection, which limits its clinical application. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples, which provides a technical means for the detection of pembrolizumab monoclonal antibody concentration with high accuracy, good stability, high sensitivity, simple and rapid pretreatment process and low detection cost.

[0010] To solve the above-mentioned technical problems, the present invention provides an LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples, which includes the following steps:

[0011] S1. Take a blood sample, add enrichment reagent I for enrichment, and then use pre-washed magnetic beads to capture the target monoclonal antibody drug;

[0012] S2. After the enriched blood sample is separated by magnetic attraction, the supernatant is discarded, buffer I is added for rinsing, and then buffer II is added for dilution.

[0013] S3. After denaturing the diluted blood sample from step S2 at high temperature, internal standard solution and protease were added sequentially for enzymatic digestion.

[0014] S4. After the enzymatic hydrolysis is completed, add the enzymatic hydrolysis termination solution to stop the enzymatic hydrolysis reaction and obtain the peptide solution to be tested;

[0015] S5. Perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis on the peptide solution to be tested, and obtain the content of pembrolizumab in the blood sample by detecting the characteristic peptides of pembrolizumab separately.

[0016] Preferably, the blood sample is plasma or serum.

[0017] Preferably, in step S1, the enrichment reagent is PD-1 protein with a concentration of 5–30 μg / ml.

[0018] Preferably, in step S1, the magnetic beads used are one of Protein A, Protein G or streptavidin, the prewash is performed using one of phosphate buffer solution or Tris buffer solution, pH 6-9; the enrichment conditions are: temperature 8℃-40℃, time 10min-1.5hr, and oscillation speed 500rpm-2000rpm.

[0019] Preferably, in step S2, buffer I is either a phosphate buffer or a Tris buffer solution with a pH of 6–9.

[0020] Preferably, in step S2, the magnetic separation is performed by using a magnetic rack to separate the magnetic beads from the supernatant and then discarding the supernatant.

[0021] In step S2, buffer II is either ammonium bicarbonate solution or Tris buffer solution, with a concentration of 20 mM to 200 mM and a pH of 6 to 8.

[0022] In step S3, the high-temperature denaturation conditions are: temperature 60℃~100℃; time 5min~20min; oscillation speed 500rpm~2000rpm;

[0023] In step S3, the internal standard solution used contains pembrolizumab isotope peptide fragments at a concentration of 0.1–20 μg / mL;

[0024] In step S3, the protease is either trypsin or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are as follows: hydrolysis ratio: 1:10 to 1:200; hydrolysis time: 20 min to 16 hr; hydrolysis temperature: 15℃ to 60℃; shaking speed: 500 rpm to 2000 rpm.

[0025] In step S4, the enzymatic hydrolysis termination solution is either an aqueous solution of formic acid or a solution of formic acid and acetonitrile, with a volume percentage of formic acid of 5% to 25%.

[0026] Preferably, in step S5, the liquid chromatography conditions are as follows:

[0027] Column type: One of C4, C8, C12 and C18 column types;

[0028] Mobile phase A: an aqueous solution containing formic acid (0.01%–0.5%); Mobile phase B: an acetonitrile or methanol solution containing formic acid (0.01%–0.5%); Column temperature: 30℃–60℃;

[0029] The mobile phase employs gradient elution, with the following elution program: mobile phase A + mobile phase B = 100%; 0–0.5 min, mobile phase B volume remains at 2–20%; 3.0–3.5 min, mobile phase B volume increases to 20–95%; 3.5–4.5 min, mobile phase B volume gradually increases to 70–95%; 4.5–5.0 min, mobile phase B volume decreases to 2–20%.

[0030] Flow rate: 0.3–1.0 mL / min;

[0031] Injection volume: 2–50 μL.

[0032] Preferably, in step S5, the mass spectrometry conditions are as follows:

[0033] Ion source: Electrospray ion source; Ion mode: Positive ion mode;

[0034] Monitoring mode: Multiple response monitoring;

[0035] The ion source temperature is 500℃~750℃;

[0036] The spray voltage for positive ions is 4500V~5500V;

[0037] The air curtain gas pressure is 25–40 psi;

[0038] The pressure of the atomizing gas Gas1 and the auxiliary heating gas Gas2 is 30-60 psi.

[0039] This invention provides an LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples. The method involves enriching and purifying pembrolizumab monoclonal antibody in serum and plasma using magnetic beads coupled with enrichment reagent I. After magnetic separation, the supernatant is discarded, and the sample is washed with buffer I. Following washing, buffer II is added for dilution. The diluted blood sample is then denatured at high temperature, followed by the addition of an internal standard and enzymatic digestion. This method optimizes the traditional procedure (eliminating protein reduction and alkylation steps in the pretreatment process and shortening the enzymatic digestion time to 1 hour, resulting in a simple and rapid overall process that can complete all pretreatment operations in just 3 hours). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is then used to achieve highly sensitive and rapid detection of pembrolizumab monoclonal antibody concentration in blood samples. This provides a highly accurate, stable, sensitive, simple, rapid, and cost-effective technique for detecting pembrolizumab monoclonal antibody concentration, simultaneously meeting the multiple requirements of clinical testing for timeliness, accuracy, and low cost. Attached Figure Description

[0040] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is an embodiment of the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples of the present invention, showing the total chromatogram of pembrolizumab and internal standard;

[0042] Figure 2 This is a chromatogram of pembrolizumab from an embodiment of the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to the present invention;

[0043] Figure 3 This is a qualitative chromatogram of pembrolizumab from an embodiment of the LC-MS / MS detection method for pembrolizumab in blood samples according to the present invention;

[0044] Figure 4 This is a chromatogram of pembrolizumab internal standard from an embodiment of the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to the present invention;

[0045] Figure 5 This is a pembrolizumab standard curve of an embodiment of the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples of the present invention;

[0046] Figure 6 This is a flowchart of an embodiment of the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to the present invention. Detailed Implementation

[0047] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0048] Example 1

[0049] An LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples, such as Figure 6 As shown, it includes the following steps:

[0050] S1. Take a blood sample, add enrichment reagent I for enrichment, and then use pre-washed magnetic beads to capture the target monoclonal antibody drug.

[0051] S2. After the enriched blood sample is separated by magnetic attraction, the supernatant is discarded, buffer I is added for rinsing, and then buffer II is added for dilution.

[0052] S3. After denaturing the diluted blood sample from step S2 at high temperature, internal standard solution and protease were added sequentially for enzymatic digestion.

[0053] S4. After the enzymatic hydrolysis is completed, add the enzymatic hydrolysis termination solution to stop the enzymatic hydrolysis reaction and obtain the peptide solution to be tested;

[0054] S5. Perform liquid chromatography-mass spectrometry (LC-MS / MS) analysis on the peptide solution to be tested. The content of pembrolizumab in the blood sample is obtained by detecting the characteristic peptides of pembrolizumab separately.

[0055] Preferably, the blood sample is plasma or serum.

[0056] The LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples in Example 1 involves enriching and purifying pembrolizumab monoclonal antibody in serum and plasma using magnetic beads coupled with enrichment reagent I. After magnetic separation, the supernatant is discarded, and the sample is washed with buffer I, followed by dilution with buffer II. The diluted blood sample is then denatured at high temperature, followed by the addition of an internal standard and enzymatic digestion. This method optimizes the traditional procedure (eliminating protein reduction and alkylation steps in the pretreatment process and shortening the enzymatic digestion time to 1 hour, resulting in a simple and rapid overall process that can complete all pretreatment operations in just 3 hours). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is then used to achieve highly sensitive and rapid detection of pembrolizumab monoclonal antibody concentration in blood samples. This provides a highly accurate, stable, sensitive, simple, rapid, and cost-effective technique for detecting pembrolizumab monoclonal antibody concentration, simultaneously meeting the multiple requirements of clinical testing for timeliness, accuracy, and low cost.

[0057] Example 2

[0058] Based on the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to Example 1, in step S1, the enrichment reagent is PD-1 protein with a concentration of 5-30 μg / ml.

[0059] Preferably, in step S1, the magnetic beads used are one of Protein A, Protein G, or streptavidin; the prewashing is performed using one of phosphate buffer solution or Tris buffer solution, with a pH of 6–9; the enrichment conditions are: temperature 8℃–40℃, time 10 min–1.5 hr, and oscillation speed 500 rpm–2000 rpm.

[0060] Preferably, in step S2, buffer I is either a phosphate buffer or a Tris buffer solution with a pH of 6–9.

[0061] Preferably, in step S2, magnetic separation is performed by using a magnetic rack to separate the magnetic beads from the supernatant and then discarding the supernatant.

[0062] Preferably, in step S2, buffer II is either ammonium bicarbonate solution or Tris buffer solution, with a concentration of 20mM to 200mM and a pH of 6 to 8.

[0063] Preferably, in step S3, the high-temperature denaturation conditions are: temperature 60℃~100℃; time 5min~20min; oscillation speed 500rpm~2000rpm.

[0064] Preferably, in step S3, the internal standard solution used contains pembrolizumab isotope peptides at a concentration of 0.1–20 μg / mL.

[0065] Preferably, in step S3, the protease is either trypsin or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are as follows: hydrolysis ratio: 1:10 to 1:200; hydrolysis time: 20 min to 16 hr; hydrolysis temperature: 15℃ to 60℃; shaking speed: 500 rpm to 2000 rpm.

[0066] Preferably, in step S4, the enzymatic hydrolysis termination solution is either an aqueous solution of formic acid or a solution of formic acid and acetonitrile, with a volume percentage of formic acid of 5% to 25%.

[0067] Programmed death molecule-1 (PD-1), also known as CD279, is a transmembrane glycoprotein with a molecular weight of 50-55 kDa. It is primarily expressed on the surface of activated T lymphocytes, B lymphocytes, monocytes, and other immune cells. In a healthy state, its normal function is to downregulate unwanted or excessive immune responses, including autoimmune responses. PD-1 is a member of the immunoglobulin (Ig) superfamily, and it has been confirmed that PD-1, when bound to its ligands (PD-L1 and / or PD-L2), can negatively regulate antigen receptor signaling. As a negative regulator of T cells, PD-1, along with its ligands PD-L1 / PD-L2, participates in T cell immune processes. Numerous studies have confirmed that PD-1 plays an important role in inflammation and tumor immunity in vivo. The PD-1 / PD-L1 regulatory axis plays a crucial role in controlling T cell activation and maintaining immune tolerance in the human body, and is therefore also utilized by tumor cells and in chronic viral infections. Although normal organs express only small amounts of PD-1, studies have confirmed that various tumor cells can express large amounts of this T-cell inhibitor. The PD-1 / PD-L1 regulatory axis has been shown to promote T-cell exhaustion, and tumor- or virus-induced PD-1 / PD-L1 signaling pathways can also achieve escape from host immune surveillance through various mechanisms, including promoting T-cell inactivation, exhaustion, sluggish response, and apoptosis, inducing Treg cell expansion, and enhancing the intrinsic resistance of tumors to killing and apoptosis.

[0068] Example 2 describes an LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples. This method uses PD-1-conjugated magnetic beads to enrich and purify the pembrolizumab monoclonal antibody in blood samples, optimizing the traditional operation steps (omitting reduction and alkylation steps, reducing the pretreatment process before enzymatic digestion to only 3 hours, omitting reduction and alkylation steps, and achieving ultra-fast enzymatic digestion in 1 hour).

[0069] Example 3

[0070] Based on the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to Example 1, the liquid chromatography conditions in step S5 are as follows:

[0071] Column type: One of C4, C8, C12 and C18 column types;

[0072] Mobile phase A: an aqueous solution containing formic acid (0.01%–0.5%); Mobile phase B: an acetonitrile or methanol solution containing formic acid (0.01%–0.5%); Column temperature: 30℃–60℃;

[0073] The mobile phase employs gradient elution, with the following elution program: mobile phase A + mobile phase B = 100%; 0–0.5 min, mobile phase B volume remains at 2–20%; 3.0–3.5 min, mobile phase B volume increases to 20–95%; 3.5–4.5 min, mobile phase B volume gradually increases to 70–95%; 4.5–5.0 min, mobile phase B volume decreases to 2–20%.

[0074] Flow rate: 0.3–1.0 mL / min;

[0075] Injection volume: 2–50 μL.

[0076] Preferably, in step S5, the mass spectrometry conditions are as follows:

[0077] Ion source: Electrospray ion source; Ion mode: Positive ion mode;

[0078] Monitoring mode: Multiple response monitoring;

[0079] The ion source temperature is 500℃~750℃, preferably 600℃;

[0080] The spray voltage for positive ions is 4500V to 5500V, preferably 5500V.

[0081] The air curtain gas pressure is 25–40 psi, preferably 35 psi;

[0082] The pressure of the atomizing gas Gas1 and the auxiliary heating gas Gas2 is 30-60 psi, preferably 55 psi.

[0083] Preferably, in step S5, the characteristic peptide information of the target monoclonal antibody drug is shown in Table 1.

[0084] Table 1

[0085]

[0086] In high-performance liquid chromatography (HPLC), the chromatographic column is a core component, and its performance directly affects the accuracy and efficiency of the analysis. C18, C8, and C4 columns are commonly used reversed-phase columns, each with its own characteristics and suitable for the analysis of different types of compounds. The C18 column, named for its octadecyl carbon chain, possesses high hydrophobicity and a long carbon chain. This structure gives the C18 column excellent separation performance in reversed-phase chromatography, making it particularly suitable for the analysis of long-chain fatty acids and pharmaceutical components. The alkane chain of the C8 column contains eight carbon atoms, exhibiting moderate hydrophobicity. Due to its shorter carbon chain, the C8 column has a relatively short retention time and sharp peak shape, making it suitable for the analysis of small organic compounds. The C4 column has a carbon chain length of four carbon atoms, exhibiting strong hydrophilicity. This characteristic makes the C4 column excellent for separating polar compounds, especially suitable for some difficult-to-separate polar acids and bases. The C18 column has the longest carbon chain and the highest hydrophobicity, making it suitable for the separation of non-polar and weakly polar compounds. C8 columns have moderate hydrophobicity and can be used for a wide range of compound analyses. C4 columns have strong hydrophilicity and are suitable for separating polar compounds. C18 columns have excellent separation performance and can effectively separate long-chain fatty acids, pharmaceutical components, and other compounds. While the separation performance of C8 columns is slightly inferior to that of C18 columns, they perform exceptionally well in the analysis of certain specific compounds. C4 columns have a unique advantage in separating polar compounds, especially suitable for some difficult-to-separate polar acids and bases. Due to the longer carbon chain of C18 columns, their retention time is relatively long, which gives them high resolution when separating complex samples. C8 columns have moderate retention times and are suitable for the analysis of most compounds. C4 columns have shorter retention times and are suitable for rapid analysis. C18, C8, and C4 columns all have good compatibility and can be used in different solvent systems; however, the separation performance of C18 columns may be affected to some extent in strongly polar solvents. C8 and C4 columns have good stability in polar solvents and are suitable for a wide range of solvent systems. C18, C8, and C4 columns all have high stability and durability, and can withstand high pressure and multiple uses without being easily damaged. However, due to the longer carbon chain of the C18 column, its stability under extreme conditions may be slightly inferior to that of the C8 and C4 columns.

[0087] Example 4

[0088] The LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples based on Example 1 includes step S1:

[0089] S11. Accurately transfer 20.0 μL of enrichment reagent I (self-prepared), 200.0 μL of buffer I working solution and 5.0 μL of calibration, quality control or blood sample to be tested into a 1.5 mL low-adsorption centrifuge tube and incubate at room temperature for 1 hour.

[0090] S12. Accurately transfer 50.0 μL of magnetic beads (self-prepared) and 100.0 μL of working buffer I into all the low adsorption centrifuge tubes in step S11. Incubate at 30°C for 30 min, then magnetically aspirate and let stand until the solution becomes clear. Then, aspirate and discard the supernatant to complete the capture of the target monoclonal antibody drug.

[0091] Step S2 includes:

[0092] S21. Accurately transfer 1000.0 μL of working solution of buffer I into all the low adsorption centrifuge tubes in step S12, incubate at 30°C for 2 min, then magnetically aspirate and let stand until the solution becomes clear, then aspirate and discard the supernatant.

[0093] S22. Accurately transfer 125.0 μL of buffer II solution into all the low-adsorption centrifuge tubes in step S21, incubate at 100°C for 5 min, then return to room temperature and centrifuge at 1000 rpm for 30 sec.

[0094] Step S3 includes:

[0095] S31. Accurately transfer 10.0 μL of internal standard and 3.0 μL of protease solution (self-prepared) into all the low adsorption centrifuge tubes in step S22, incubate at 37°C and 1100 rpm for 1 hour with shaking, and then centrifuge at 1000.0 rpm at room temperature for 30 seconds to perform enzymatic digestion.

[0096] Step S4 includes:

[0097] S41. Accurately transfer 7.0 μL of the enzymatic hydrolysis termination solution into all the low-adsorption centrifuge tubes in step S31, and shake at 1000 rpm for 5 min at room temperature to terminate the enzymatic hydrolysis reaction;

[0098] S42. After terminating the enzymatic digestion reaction, magnetically aspirate all centrifuge tubes and let them stand until the solution becomes clear. Then, transfer 120.0 μL of the supernatant to a new 96-well plate, shake at 1300.0 rpm at room temperature for 2 min, and then centrifuge at 4000.0 rpm at 4℃ for 5 min to obtain the peptide solution to be tested.

[0099] Step S5 includes:

[0100] S51. The 96-well plate was placed in the autosampler of a liquid chromatography-tandem mass spectrometer for analysis. The injection volume was 5.0 μL. The content of pembrolizumab in the blood sample was obtained by detecting the characteristic peptides of pembrolizumab.

[0101] Example 5

[0102] Based on the LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to Example 1, in step S5, the peptide solution to be tested is analyzed by liquid chromatography-mass spectrometry (LC-MS / MS):

[0103] Liquid chromatography conditions: AB SCIEX 4500MD was used;

[0104] Column type: C18;

[0105] Column temperature: 45℃;

[0106] Mobile phase A: an aqueous solution containing formic acid (0.1%); Mobile phase B: an acetonitrile solution containing formic acid (0.1%);

[0107] The mobile phase employs gradient elution, with the following elution program: Mobile phase A + Mobile phase B = 100%; 0–0.5 min, mobile phase B volume remains at 5%; 0.5–3.0 min, mobile phase B volume increases from 5% to 35%; 3.0–3.5 min, mobile phase B volume increases from 35% to 95%; 3.5–4.5 min, mobile phase B volume remains at 95%; 4.50–4.51 min, mobile phase B volume decreases from 95% to 5%; 4.51–5.0 min, mobile phase B volume remains at 5%.

[0108] Flow rate: 0.5 mL / min;

[0109] Injection volume: 5 μL.

[0110] Mass spectrometry parameters: AB SCIEX 4500MD was used;

[0111] Ion source: electrospray ionization source; Ion mode: positive ion mode; Monitoring mode: multiple reaction monitoring (parameters are shown in Table 2 for multiple reaction monitoring mass spectrometry parameters of pembrolizumab monoclonal antibody drug);

[0112] Table 2

[0113] 1 553.400 439.200 105 50.000 21 2 553.400 667.200 105 50.000 32 3 558.400 444.200 105 50.000 21

[0114] The ion source temperature is 600℃; the spray voltage for positive ions is 5500V.

[0115] The air curtain gas pressure is 35 psi; the atomizing gas Gas1 pressure is 55 psi; the auxiliary heating gas pressure Gas2 pressure is 55 psi.

[0116] Example 5 describes an LC-MS / MS method for detecting pembrolizumab in blood samples. The method involves detecting the characteristic peptides of pembrolizumab to determine the concentration of the drug in the blood sample. The total chromatograms of the monoclonal antibody and internal standard are shown below. Figure 1 As shown, the chromatogram of pembrolizumab is as follows. Figure 2 As shown, the qualitative chromatogram of pembrolizumab is as follows. Figure 3 As shown, the chromatogram of pembrolizumab internal standard is as follows. Figure 4 As shown; it can be seen that the chromatograms of pembrolizumab and internal standard obtained by the detection method of pembrolizumab in serum in Example 5 have good peak shapes, stable detection baselines, and no obvious interference. By using the indicated concentration (X) of each calibrator and the peak area ratio (Y) of the characteristic peptide of each monoclonal antibody in the series of calibrators to the characteristic peptide of the internal standard, calibration curves were plotted and the calibration curve equation was fitted to obtain the linear regression equation and correlation coefficient, as shown below. Figure 5 As shown in Table 3, the pembrolizumab drug concentrations obtained by this detection method all exhibit a good linear relationship, with a linear correlation coefficient r0. 2 All values ​​are greater than 0.995.

[0117] Table 3

[0118] 1 Pembrolizumab 2~80μg / mL y = 0.9575x + 0.7063 0.9984

[0119] Example 6, Accuracy of the detection method:

[0120] A mixed human serum and plasma sample (obtained by mixing a certain number of human serum and plasma samples to reflect the common biochemical properties of human serum and plasma and exclude the influence of individual differences; the specific number can be determined according to experimental needs) was selected as the routine sample. An equal volume of solvent without the analyte was added to the routine sample (mixed human serum and plasma) to prepare the baseline sample. Different amounts of the analyte standard were added to the routine sample (mixed human serum and plasma) (the volume of the added standard solution did not exceed 5% of the total sample volume) to prepare three recovery samples with different concentrations (low, medium, and high concentrations). The recovery samples and baseline samples were measured using the detection method for pembrolizumab monoclonal antibody in serum described in Example 1. The sample processing method was the same as in Example 4, and the chromatographic conditions and mass spectrometry parameters were the same as in Example 5. Each concentration level sample was analyzed three times in the same analytical batch, and the average value was used for calculation. The detection results of the LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples of the present invention are shown in Tables 4 and 5. The recovery rates of pembrolizumab monoclonal antibody are all within the range of 85% to 115%, which meets the technical requirements, indicating that the detection results of the detection method of the present invention are accurate and reliable.

[0121] Table 4. Recovery rate of pembrolizumab plasma spiked in table 4

[0122]

[0123] Table 5. Serum spiked recovery rate of pembrolizumab

[0124]

[0125] Example 7: Intra-batch and inter-batch precision of the detection method:

[0126] Quality control samples of a certain concentration were prepared by adding the analyte standard to a mixed human serum and plasma sample (the same as the mixed human serum and plasma sample in Example 6): low, medium, and high concentrations. Six replicates were performed daily for each concentration level, for a total of three batches over three consecutive days. The sample processing method was the same as in Example 4, and the chromatographic conditions and mass spectrometry parameters were the same as in Example 5. The coefficient of variation (CV%) for each concentration level was calculated, and this CV% represents the corresponding intra- and inter-batch precision. The detection results and calculation results are shown in Table 6. The intra- and inter-batch precision of the pembrolizumab monoclonal antibody drug in serum and plasma samples were both within 15.0%, indicating that the detection method of the present invention has good precision.

[0127] Table 6. Intra- and inter-batch precision of pembrolizumab

[0128]

[0129] Example 8, Contamination carried by the detection method:

[0130] High-concentration samples were prepared by adding the analyte standard to the mixed human serum and plasma sample (the same as the mixed human serum and plasma sample in Example 3).

[0131] Evaluation was conducted by injecting five consecutive injections of blank samples after injecting a high-concentration sample; measurements were performed continuously for three days. The sample processing method was the same as in Example 4, and the chromatographic conditions and mass spectrometry parameters were the same as in Example 5. The results are shown in Table 7. The residual peak areas of the compounds in the blank samples of the five monoclonal antibody drugs were all less than 20% of the peak area of ​​the compound at the lower limit of quantitation. This meets the technical requirements (peak area of ​​blank solution < 20% of C1 peak area), and the residual peak area of ​​the internal standard in the blank samples was less than 5% of the peak area corresponding to the working concentration of the internal standard.

[0132] Table 7. Pembrolizumab contamination

[0133]

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An LC-MS / MS method for detecting pembrolizumab monoclonal antibody in blood samples, characterized in that, Includes the following steps: S1. Take a blood sample, add enrichment reagent I for enrichment, and then use pre-washed magnetic beads to capture the target monoclonal antibody drug. The enrichment reagent I is PD-1 protein, with a concentration of 5–30 μg / ml; The magnetic beads used were one of Protein A, Protein G or streptavidin. Pre-washing was performed using one of phosphate buffer solution or Tris buffer solution, pH 6-9. The enrichment conditions were: temperature 8℃-40℃, time 10min-1.5hr, and shaking speed 500rpm-2000rpm. S2. After the enriched blood sample is separated by magnetic attraction, the supernatant is discarded, and buffer I is added for rinsing. After rinsing, buffer II is added for dilution. S3. After denaturing the diluted blood sample from step S2 at high temperature, internal standard solution and protease were added sequentially for enzymatic digestion. S4. After the enzymatic hydrolysis is completed, add the enzymatic hydrolysis termination solution to stop the enzymatic hydrolysis reaction and obtain the peptide solution to be tested; S5. Perform liquid chromatography-tandem mass spectrometry analysis on the peptide solution to be tested, and obtain the content of pembrolizumab in blood samples by detecting the characteristic peptides of pembrolizumab separately; Mass spectrometry conditions are: Ion source: Electrospray ion source; Ion mode: Positive ion mode; Monitoring mode: Multiple response monitoring; The ion source temperature is 500℃~750℃; The spray voltage for positive ions is 4500V~5500V; The air curtain gas pressure is 25–40 psi; The pressure of the atomizing gas Gas1 and the auxiliary heating gas Gas2 is 30-60 psi.

2. The LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to claim 1, characterized in that, The blood sample is either plasma or serum.

3. The LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to claim 1, characterized in that, In step S2, buffer I is either a phosphate buffer or a Tris buffer solution, with a pH of 6–9.

4. The LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to claim 1, characterized in that, In step S2, magnetic separation is achieved by using a magnetic rack to separate the magnetic beads from the supernatant and then discarding the supernatant. In step S2, buffer II is either ammonium bicarbonate solution or Tris buffer solution, with a concentration of 20 mM to 200 mM and a pH of 6 to 8. In step S3, the high-temperature denaturation conditions are: temperature 60℃~100℃; time 5min~20min; oscillation speed 500rpm~2000rpm; In step S3, the internal standard solution used contains pembrolizumab isotope peptide fragments at a concentration of 0.1–20 μg / mL; In step S3, the protease is either trypsin or lysine protease, and the enzymatic hydrolysis conditions are as follows: hydrolysis ratio: 1:10 to 1:200; hydrolysis time: 20 min to 16 hr; hydrolysis temperature: 15℃ to 60℃; shaking speed: 500 rpm to 2000 rpm. In step S4, the enzymatic hydrolysis termination solution is either an aqueous solution of formic acid or a solution of formic acid and acetonitrile, with a volume percentage of formic acid of 5% to 25%.

5. The LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to claim 1, characterized in that, Step S1 includes: S11. Accurately transfer 20.0 μL of enrichment reagent I, 200.0 μL of buffer I working solution and 5.0 μL of calibration, quality control or blood sample to be tested into a 1.5 mL low adsorption centrifuge tube and incubate at room temperature for 1 hour. S12. Accurately transfer 50.0 μL of magnetic beads and 100.0 μL of working buffer I into all the low adsorption centrifuge tubes in step S11. Incubate at 30°C for 30 min, then magnetically aspirate and let stand until the solution becomes clear. Then, aspirate and discard the supernatant to complete the capture of the target monoclonal antibody drug. Step S2 includes: S21. Accurately transfer 1000.0 μL of buffer I working solution into all the low-adsorption centrifuge tubes in step S12, incubate at 30°C for 2 min, then magnetically aspirate and let stand until the solution becomes clear, then discard the supernatant. S22. Accurately transfer 125.0 μL of buffer II solution into all the low-adsorption centrifuge tubes in step S21, incubate at 100°C for 5 min, then return to room temperature and centrifuge at 1000 rpm for 30 sec. Step S3 includes: S31. Accurately transfer 10.0 μL of internal standard and 3.0 μL of protease solution into all the low-adsorption centrifuge tubes in step S22, incubate at 37°C and 1100 rpm for 1 hour with shaking, and then centrifuge at 1000.0 rpm for 30 seconds at room temperature to perform enzymatic digestion. Step S4 includes: S41. Accurately transfer 7.0 μL of the enzymatic hydrolysis termination solution into all the low-adsorption centrifuge tubes in step S31, and shake at 1000 rpm for 5 min at room temperature to terminate the enzymatic hydrolysis reaction; S42. After terminating the enzymatic digestion reaction, magnetically aspirate all centrifuge tubes and let them stand until the solution becomes clear. Then, transfer 120.0 μL of the supernatant to a new 96-well plate, shake at 1300.0 rpm at room temperature for 2 min, and then centrifuge at 4000.0 rpm at 4℃ for 5 min to obtain the peptide solution to be tested. Step S5 includes: S51. The 96-well plate was placed in the autosampler of a liquid chromatography-tandem mass spectrometer for analysis. The injection volume was 5.0 μL. The content of pembrolizumab in the blood sample was obtained by detecting the characteristic peptides of pembrolizumab.

6. The LC-MS / MS detection method for pembrolizumab monoclonal antibody in blood samples according to claim 1, characterized in that, In step S5, the solution of the peptide to be tested is analyzed by liquid chromatography-tandem mass spectrometry: Liquid chromatography conditions: AB SCIEX 4500 MD; Column type: C18; Column temperature: 45℃; Mobile phase A: an aqueous solution containing 0.1% formic acid; Mobile phase B: an acetonitrile solution containing 0.1% formic acid; The mobile phase employs gradient elution, with the following elution program: Mobile phase A + Mobile phase B = 100%; 0–0.5 min, mobile phase B volume remains at 5%; 0.5–3.0 min, mobile phase B volume increases from 5% to 35%; 3.0–3.5 min, mobile phase B volume increases from 35% to 95%; 3.5–4.5 min, mobile phase B volume remains at 95%; 4.50–4.51 min, mobile phase B volume decreases from 95% to 5%; 4.51–5.0 min, mobile phase B volume remains at 5%. Flow rate: 0.5 mL / min; Injection volume: 5 μL; Mass spectrometry parameters: AB SCIEX 4500MD was used; Ion source: Electrospray ionization source; Ion mode: Positive ion mode; Monitoring mode: Multiple reaction monitoring; The ion source temperature is 600℃; the spray voltage for positive ions is 5500V. The air curtain gas pressure is 35 psi; the atomizing gas Gas1 pressure is 55 psi; the auxiliary heating gas pressure Gas2 pressure is 55 psi.

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