Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of pharmaceutical analysis, in particular to a method for determining Epcoritamab, a bispecific monoclonal antibody that is used to treat adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) using a reverse-phased ultra-performance liquid chromatography technique.
Background of the invention:
[0002] Every year, 25,000 people have been affected with Diffuse Large B-Cell Lymphoma (DLBCL), the most common non-Hodgkin lymphoma. Adult patients with high-grade B-cell lymphoma and specific forms of DLBCL who have relapsed or did not improve after two or more previous therapies can use Epcoritamab to treat. Epcoritamab is a bispecific monoclonal antibody containing antigen-recognition sites for human CD3 and human CD20. CD20 is often over expressed in B-cell malignancies. Epcoritamab binds to T-cells and CD20-expressing B-lineage tumor cells. This may trigger a potent cytotoxic T lymphocyte response against the CD20-expressing tumor B-cells.

[0003] Epcoritamab is a medicine used to treat specific kinds of B-cell lymphomas. It is marketed under the names Epkinly in the US and Tepkinly in the EU. A brand-new full-length IgG1 bispecific antibody (bsAb) called Epcoritamab (DuoBody-CD3xCD20, GEN3013) directs CD3+ T-cells toward cells that express CD20. Epcoritamab shown a good safety profile in the dose-escalation phase of an ongoing first-in-human clinical trial, and first efficacious data indicated remarkable anticancer activity as a single drug, including full responses, in patients with R/R DLBCL or FL.

[0004] Traditional methods for determining the Epcoritamab levels in biological samples primarily rely on Enzyme-Linked Immunosorbent Assay (ELISA). ELISA is less sensitive and requires larger sample volumes to detect the Epcoritamab accurately. The equipment and reagents for ELISA are expensive. ELISA setup and processing often involve manual steps, increasing the risk of errors and inconsistencies.

[0005] A method to determine Epcoritamab by using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique is needed after considering its significance and the problems addressed in the previous discourse. There is also a necessitate for a method for determining Epcoritamab that is highly sensitive and enables precise quantification with smaller sample volumes, which is crucial for therapeutic drug monitoring. There is also a need for a method that separates Epcoritamab from other components based on their physical and chemical properties, minimizing interference and enhancing accuracy. Additionally, there is a requirement for an Epcoritamab determination method that reduces analysis, allowing for timely clinical decisions and drug modifications.

[0006] In order to obtain a complete picture of the drug's behavior, a method for identifying Epcoritamab that permits the simultaneous quantification of various analytes, including degradation products and co-medications, is also required. A method for determining the Epcoritamab that improves robustness is also required in order to produce consistent, reliable results that are less susceptible to external factors. A technique for identifying Epcoritamab that permits appreciable runtime savings with excellent resolution and minimal solvent use is also required.
Objectives of the invention:
[0007] The primary objective of the present invention is to provide a novel method for determining Epcoritamab, a bispecific monoclonal antibody that is used to treat adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique.

[0008] Another objective of the present invention is to provide a method for determining Epcoritamab that is highly sensitive and enables precise quantification with smaller sample volumes, which is crucial for therapeutic drug monitoring.

[0009] Yet another objective of the present invention is to provide a method for determining Epcoritamab that reduces analysis time, minimizing interference, enhancing accuracy, facilitating timely clinical decisions and medication adjustments.

[0010] Another objective of the present invention is to provide a method for determining Epcoritamab that enables simultaneous quantification of multiple analytes, including degradation products and co-medications, for a comprehensive picture of the drug's behavior.

[0011] Further objective of the present invention is to provide a method for determining Epcoritamab that enhances robustness for consistent, reliable results, enables significant reductions in runtime with high resolution and less solvent consumption.
Summary of the invention:
[0012] The present disclosure proposes a method for determining Epcoritamab using an RP-UPLC technique. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0013] Using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique, the present disclosure resolves the scientific issue in order to provide a novel method for determining Epcoritamab, a bispecific monoclonal antibody used to treat adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). This assists to overcome the aforementioned defects of the prior art.

[0014] At one step, 6.30 g of ammonium formate was dissolved in one liter of HPLC grade water to obtain a buffer solution and the obtained buffer solution is filtered through a filter paper of 0.45 micron. The buffer solution was adjusted to a pH value of 3.0 with orthophosphoric acid.

[0015] At another step, the filtered buffer solution is mixed in an acetonitrile (CH3CN) at a ratio of 6:4 to obtain a mobile phase solution and the obtained mobile phase solution is filtered through a filter membrane of 0.45 µm. In one embodiment herein, the mobile phase solution is used as a diluent.

[0016] At other step, 40 mg of Epcoritamab and diluent are added into a 100 ml volumetric flask, thereby dissolving the Epcoritamab and diluent to obtain a mixed solution through a sonication process.

[0017] At another step, 5.0ml of the obtained mixed solution is added into a 50 ml volumetric flask and the diluent is added to the mixed solution for obtaining 40 µg/ml Epcoritamab standard solution.

[0018] At other step, 0.8 ml of the obtained Epcoritamab sample solution is added into a 10 ml volumetric flask and the diluent is added to the Epcoritamab standard solution, thereby dissolving the Epcoritamab sample solution completely through the sonication process and centrifugation for a time period of at least 30 min, thereby obtaining a sample Epcoritamab solution.

[0019] At another step, 1 ml of the sample Epcoritamab solution is added into a 10 ml volumetric flask through a pipette and diluting the sample Epcoritamab solution to obtain 40 µg/ml of Epcoritamab.

[0020] Further, at other step the obtained Epcoritamab is analyzed through a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique, thereby achieving separation of Epcoritamab by an isocratic elution within a time period of at least 2 min of runtime with a flow rate of 0.3 mL/min.

[0021] In one embodiment, the reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique includes a binary solvent manager, sample manager and a photodiode array (PDA) detector. The RP-UPLC technique uses a BEH Shield RP-18 (100 x 2.1mm, 1.7µm) tool as an analytical column, which enables the rapid development of faster and more robust separations.

[0022] In one embodiment, the obtained Epcoritamab is validated for specificity, linearity, precision, accuracy, sensitivity and robustness. In one embodiment, the separation of Epcoritamab is achieved by Isocratic elution using the ammonium formate buffer solution and the acetonitrile (CH3CN) at the ratio of 6:4.

[0023] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0025] FIG. 1 illustrates a flowchart of a method for determining Epcoritamab in epkinly using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique in accordance to an exemplary embodiment of the invention.

[0026] FIG. 2 illustrates a network diagram of an Epcoritamab (epkinly), in accordance to an exemplary embodiment of the invention.

[0027] FIG. 3A illustrates a graph of a blank chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0028] FIG. 3B illustrates a graph of a standard chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0029] FIG. 3C illustrates a graph of a sample chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0030] FIG. 3D illustrates a graph of an acid degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0031] FIG. 3E illustrates a graph of an alkali degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0032] FIG. 3F illustrates a graph of an oxidative degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0033] FIG. 3G illustrates a graph of a reduction degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0034] FIG. 3H illustrates a graph of a photolytic degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0035] FIG. 3I illustrates a graph of a thermal degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0036] FIG. 3J illustrates a graph of a neutral degradation chromatogram of the Epcoritamab, in accordance to an exemplary embodiment of the invention.

[0037] FIGs. 4A-4F illustrate graphs representing linearity trails chromatograms of the Epcoritamab, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0038] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0039] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for determining Epcoritamab, a bispecific monoclonal antibody that is used to treat adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique.

[0040] According to one exemplary embodiment of the invention, FIG. 1 refers to a flow chart of a method for determining Epcoritamab in epkinly using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique. The RP-UPLC technique is highly sensitive and enables precise quantification with smaller sample volumes, which is crucial for therapeutic drug monitoring. The RP-UPLC technique separates Epcoritamab from other degraded components, minimizing interference and enhancing accuracy. The RP-UPLC technique reduces analysis time for a faster turnaround, facilitating timely clinical decisions and medication adjustments.

[0041] At step 102, 6.30 g of ammonium formate is dissolved in at least one liter of HPLC grade water to obtain a buffer solution and the obtained buffer solution is filtered through a filter paper of 0.45µm. The buffer solution is adjusted to a pH value of 3.0 with orthophosphoric acid.

[0042] At step 104, the filtered buffer solution is mixed in an acetonitrile (CH3CN) at a ratio of 6:4 to obtain a mobile phase solution and the obtained mobile phase solution is filtered through a filter membrane of 0.45 µm. In one embodiment herein, the mobile phase solution is used as a diluent.

[0043] At step 106, 40 mg of Epcoritamab and diluent are added into a 100 ml volumetric flask, thereby dissolving the Epcoritamab and diluent to obtain a mixed solution through sonication.

[0044] At step 108, the obtained mixed solution 5ml was added into a 50 ml volumetric flask and the diluent is added to the mixed solution for obtaining 40 µg/ml Epcoritamab standard solution.

[0045] At step 110, 0.8 ml of the obtained Epcoritamab sample solution is added into a 10 ml volumetric flask and the diluent is added to the Epcoritamab sample solution, thereby dissolving the Epcoritamab sample solution completely through sonication and centrifugation for a time period of at least 30 min, thereby obtaining an Epcoritamab sample solution.

[0046] At step 112, 1 ml of the Epcoritamab sample solution is added into a 10 ml volumetric flask through a pipette and diluting the Epcoritamab sample solution to obtain 40 µg/ml Epcoritamab.

[0047] Further, at step 114 the obtained Epcoritamab is analyzed through a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique, thereby achieving separation of Epcoritamab by an isocratic elution within a time period of 2 min of runtime with a flow rate of 0.3 mL/min.

[0048] In one embodiment herein, the reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique includes a binary solvent manager, sample manager and a photodiode array (PDA) detector. The RP-UPLC technique uses a BEH Shield RP-18 (100 x 2.1mm, 1.7µm) analytical column, which enables the rapid development of faster and more robust separations.

[0049] In one embodiment herein, the obtained Epcoritamab is validated for specificity, linearity, precision, accuracy, sensitivity and robustness. In one embodiment herein, the separation of Epcoritamab is achieved by Isocratic elution using the ammonium formate buffer solution and the acetonitrile (CH3CN) at the ratio of 6:4. In one embodiment herein, the Waters ‘H- Class Acquity-UPLC’, a system with the PDA- detector is used to do the data acquisition. An Empower Software is used for the method screening and optimization.

[0050] According to another exemplary embodiment of the invention, FIG. 2 refers to a network diagram 200 of the Epcoritamab (epkinly). The Epcoritamab is a bispecific monoclonal antibody containing antigen-recognition sites for human CD3 and human CD20. CD20 is often over expressed in B-cell malignancies. The Epcoritamab binds to T-cells and CD20-expressing B-lineage tumor cells. This may trigger a potent cytotoxic T lymphocyte response against the CD20-expressing tumor B-cells.

[0051] In one embodiment herein, the RP-UPLC technique is a relatively a good technique that enables significant reductions in runtime with high resolution and less solvent consumption. Moreover, the RP-UPLC technique provides highly narrow and sharp peaks, which simplify the proper analysis of complex mixtures. The Epcoritamab is primarily evaluated in a single chromatographic run having short run time along with degradation products. The method is aimed to develop a single compatible method for the determination of Epcoritamab by using the RP-UPLC technique and followed by force degradation studies.

[0052] In one embodiment herein, the Epcoritamab is validated for specificity and a stress study is performed on the Epcoritamab solution to establish inherent stability characteristics and exhibits the stability-indicating capability of the proposed method. The Epcoritamab solution is exposed to one or more sample solutions, which includes alkaline (0.1N NaOH, room temperature, 15 min), acidic (1N HCl, room temperature, 15 min), reduction (10% Sodium bisulfate solution and leave it for 15 min), neutral (water, 25 °C, 15 min), thermal degradation conditions (105 °C, 24 hrs), strong oxidizing (10 % H2O2, for 15 min), and a photolytic stress study is conducted on sample solution (72 lux hrs). The blank solutions are subjected to stress in the same manner as the Epcoritamab solution. In one embodiment herein, the one or more sample solutions are analysed using at least one suggested procedure.

[0053] In one embodiment herein, the linearity of the Epcoritamab is shown from 25 % to 150 % of standard concentration utilizing a minimum of six calibration levels (25, 50, 75, 100, 125, and 150 %). To assess the linearity, linear regression is applied to the Epcoritamab. Plots are made between the peak areas of standard solutions and the corresponding concentrations. The correlation coefficient is calculated and an equation being used to describes the linearity of the Epcoritamab.

[0054] In one embodiment herein, the precision of the Epcoritamab is investigated by using the sample preparation procedure for six real samples of a commercial brand of injection and analysing by the proposed method. Intermediate precision is studied using different equipment and columns and performing the analysis on alternative days.

[0055] In one embodiment herein, the accuracy of the Epcoritamab is validated by executing recovery experiments using the standard addition technique. The Epcoritamab injection samples that had already undertaken are pre-analysed at three distinct levels (50, 100, and 150 %). The Epcoritamab % of recoveries are calculated for each level and replicated. Relative standard deviation (% RSD) is determined for each level with a mean of percentage recoveries (n = 9).

[0056] In one embodiment herein, the sensitivity of the Epcoritamab is validated by the signal-to-noise approach, as described in the international council for harmonisation of technical requirements of pharmaceuticals for human use (ICH) guideline, is used to assess the limit of detection (LOD) and limit of quantitation (LOQ) of Epcoritamab. The signal-to-noise (S/N) ratio is calculated at each concentration after injecting progressively diluted solutions of the Epcoritamab into the RP-UPLC technique.

[0057] In one embodiment herein, the robustness of the Epcoritamab is validated by examining the effects of tiny changes in the organic content of the mobile phase (+5%, absolute) and flow rate (+10%), as a measure of the method's capacity to remain unaffected by modest but deliberate changes in chromatographic conditions, the robustness is investigated at ambient column temperature.

[0058] In one embodiment herein, the retention of the Epcoritamab is increased with an increase in column length, so a shorter column BEH Shield RP-18 (100 x 2.1mm, 1.7µm) is selected to provide the shortest possible run time without compromising to increase symmetry shape of the drug. The retention time of Epcoritamab drug is significantly impacted by a change in the percentage of acetonitrile (ACN) in the mobile phase solution. The final isocratic run is chosen with regards to the peak shape, analysis time and tailing.

[0059] Regarding backpressure in addition to run time, the flow rate of 0.3 mL/min is carefully chosen as the most effective approach. The standard and sample concentration of the Epcoritamab 40 µg/mL and injection volume of 5 µL are also optimized regarding peak response, resolution and tailing. The Epcoritamab standard solution and Epcoritamab sample concentrations responded satisfactorily at 242 nm, making it possible to determine their assay results in a single run. The Epcoritamab is eluted in reasonable time of approximately 2 min with retention time of 0.925 min. According to another exemplary embodiment of the invention, FIG. 3A refers to a graph 300 of a blank chromatogram of the Epcoritamab. Typical chromatograms of the blank, standard, sample, of drug and acid degradation are shown in graphs (300, 302, 304, and 306) of FIGs. 3A-3D.

[0060] The final optimized chromatographic conditions are achieved with a BEH Shield RP-18 (100 x 2.1mm, 1.7µm), and a mobile phase (400:600) made up of acetonitrile and 0.1% ammonium formate by adapting isocratic elution at ambient column temperature. The Epcoritamab is detected (PDA-detector) at 242 nm, and a satisfactory separation was achieved within a time period of at least 2.0 min of runtime with a flow rate of 0.3 mL/min., in ‘H Class- Waters acquity ultra performance liquid chromatography’.

[0061] In one embodiment herein, the method is put through a method validation in accordance with ICH principles. The proposed RP-UPLC technique is validated using the approved procedure to assess acceptable validation properties. The method validation results of the Epcoritamab is shown in Table 1.

[0062] Table 1:
Epcoritamab
Details Acceptance Criteria Results
Retention time Different retention time No interference
Range (µg/mL) Range (µg/mL) 1.25-7.5
Minimum 6 concentration levels in the range 25%–150 R2> 0.999 0.99982
Slope M 66898.78
Intercept C 532.39
Determinations on same day-Repeatability (% Assay) 100.1
% RSD < 2% 0.82
Determinations on different day-Intermediate precision (% Assay) 100.2
%RSD < 2% 1.11
Determinations at 50% Recovery at each level: 90–110% Minimum Maximum
100.0 101.0
Determinations at 100% 100.0 100.8
Determinations at 150% 99.8 101.8
LOD: lowest detectable conc. S/N ratio > 3 0.12
LOQ: lowest quantitatable conc. S/N ratio > 10 0.4
Std. Flow Rate:0.3 mL /Min. & Std. Mobile Phase: Acetonitrile and Buffer in the ratio of 40:60
Change Actual Change Limit Epcoritamab
Resolution -0.10% Flow rate (mL/Min): 0.28 > 2 -
0.10% Flow rate (mL/Min): 0.32 -
MP: - 5% MP: Ammonium Formate buffer and CH3CN:-65:35 -
MP: + 5% MP: Ammonium Formate buffer and CH3CN:-55:45 -
USP tailing -0.10% Flow rate (mL/Min): 0.28 < 2 1.10
0.10% Flow rate (mL/Min): 0.32 1.11
MP: - 5% MP: Ammonium Formate buffer and CH3CN:-65:35 1.13
MP: + 5% MP: Ammonium Formate buffer and CH3CN:-55:45 1.16
Column efficiency -0.10% Flow rate (mL/Min): 0.28 < 2000 7849
0.10% Flow rate (mL/Min): 0.32 7566
MP: - 5% MP: Ammonium Formate buffer and CH3CN:-65:35 7705
MP: + 5% MP: Ammonium Formate buffer and CH3CN:-55:45 7598

[0063] In one embodiment herein, the stress study results of the Epcoritamab are shown in Table 2.

[0064] Table 2:
Degradation condition Area Assay % Degradation Purity Angle Purity Threshold
Control 2746472 100 0 4.625 11.562
Acid 2378549 86.6 13.4 4.647 11.544
Alkali 2396953 87.3 12.7 4.629 11.578
Peroxide 2345896 85.4 14.6 4.686 11.536
Reduction 2420563 88.1 11.9 4.654 11.542
Photo 2645716 96.3 3.7 4.673 11.558
Hydrolysis 2705231 98.5 1.5 4.681 11.561
Thermal 2455173 89.4 10.6 4.639 11.559

[0065] According to one exemplary embodiment of the invention, FIG. 3D refers to a graph 306 of an acid degradation chromatogram of the Epcoritamab. In one embodiment herein, the Epcoritamab is found to be sensitive to acid hydrolysis. Less than 10 % degradation of the Epcoritamab is found when using 0.1N HCl. Hence, 1N HCl is selected for degradation studies, which yielded one major degradation product at RT 0.647 min with degradation of 13.4 % with respect to the Epcoritamab. Table 2 summarizes the results of the stress study. In one embodiment herein, the graph 306 of the FIG. 3D displays an acid degradation chromatogram of the Epcoritamab solution.

[0066] According to one exemplary embodiment of the invention, FIG. 3E refers to a graph 308 of an alkali degradation chromatogram of the Epcoritamab. In one embodiment herein, the Epcoritamab is found to be sensitive to alkali hydrolysis. The reaction in 0.1N NaOH at room temperature resulted in not more than 10 % degradation of both drugs. Subsequently, studies are performed in 1N NaOH at room temperature (25 °C). The Epcoritamab degradation is associated with a rise in a slight degradation product (DP1) at RT 0.588, with degradation of 12.7 % (as shown in the graph 308 of the FIG. 3E).

[0067] According to one exemplary embodiment of the invention, FIG. 3F refers to a graph 310 of an oxidation degradation chromatogram of the Epcoritamab. In one embodiment herein, the Epcoritamab is stable to H2O2 at room temperature. When the Epcoritamab solution allowed in hydrogen peroxide (10 %) at 25 °C for 15 min, yielded one minor degradation product at RT 0.359 min, with degradation of 14.6 %, with respect to the Epcoritamab (as shown in the graph 310 of the FIG. 3F).

[0068] The Epcoritamab is found to be sensitive to the effects of temperature. When the mixture of Epcoritamab solution is exposed to dry heat (as shown in a graph 316 of FIG. 3I) at 105 °C for one day, 10.6% degradation of the drug is observed and yielded one minor degradation product at room temperature (RT) 1.339 min; additionally. The Epcoritamab is stable in photolytic and neutral degradation as shown in graphs 314, 318 of FIG. 3H and FIG. 3J).

[0069] In one embodiment herein, the Epcoritamab is sensitive in reduction degradation at room temperature. When the drug solution is added to 10% Sodium bisulfate solution and allowed it for 15 min and yielded one minor degradation product at RT 1.171 min with degradation of 11.9%, and the corresponding chromatogram is shown in the graph 312 of the FIG. 3G. The Epcoritamab is stable to the neutral hydrolysis at room temperature and the corresponding chromatogram is shown in the graph 318 of the FIG. 3J.

[0070] According to another exemplary embodiment of the invention, FIGs. 4A-4F refer to graphs (400, 402, 404, 406, 408, and 410) representing linearity trails chromatograms of the Epcoritamab. The linearity is proven by linear regression analysis through constructing a calibration curve graph for a specific concentration range for the Epcoritamab. The linearity of the Epcoritamab is evaluated in the range of 1.25–7.5 µg/mL. The R2 was < 0.9998 for the Epcoritamab. Table 3 shows the linearity equations and correlation coefficients for this drug.

[0071] Table 3:
Linearity level Epcoritamab
Concentration
(µg/mL) Peak’s
area Slope Intercept CC
EPM@1 10.00 669531 66898.78 532.39 0.99982
EPM@2 20.00 1350264
EPM@3 30.00 1964548
EPM@4 40.00 2725639
EPM@5 50.00 3332121
EPM@6 60.00 4010368

[0072] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure a method for determining Epcoritamab, is disclosed. The proposed invention provides a method for determining Epcoritamab, a bispecific monoclonal antibody that is used to treat adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique.

[0073] The method uses RP-UPLC technique, which is highly sensitive and enables precise quantification with smaller sample volumes, which is crucial for therapeutic drug monitoring. The method separates Epcoritamab from degradation products, minimizing interference, and enhancing accuracy. The method reduces analysis time for a faster turnaround, facilitating timely clinical decisions and medication adjustments. The method enables simultaneous quantification of multiple analytes, including degradation products and co-medications, for a comprehensive picture of the drug's behavior.

[0074] The method enhances robustness for consistent and reliable results with less susceptibility to external factors. The method enables significant reductions in runtime with high resolution and less solvent consumption.

[0075] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.

, Claims:CLAIMS:
I/We Claim:
1. A method for determining Epcoritamab in epkinly using a reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique, comprising:
dissolving 6.30 g of ammonium formate in at least one liter of HPLC grade water to obtain a buffer solution and filtering the obtained buffer solution through a filter paper;
mixing the filtered buffer solution in an acetonitrile (CH3CN) at a ratio of 6:4 to obtain a mobile phase solution and filtering the obtained mobile phase solution through a filter membrane, thereby using the mobile phase solution as a diluent;
adding 40 mg of Epcoritamab into a 100 ml volumetric flask and adding the diluent to the volumetric flask, thereby dissolving the Epcoritamab to obtain a mixed solution through a sonication process;
adding 5 ml of the obtained mixed solution into a 50 ml volumetric flask and adding the diluent to the mixed solution for obtaining a 40 µg/ml Epcoritamab standard solution;
adding 0.8 ml of obtained Epcoritamab sample solution into a 10 ml volumetric flask and adding the diluent to the Epcoritamab sample solution, thereby dissolving the Epcoritamab sample solution completely through the sonication process and centrifugation for obtaining a sample Epcoritamab solution;
adding 1 ml of the sample Epcoritamab solution into a 10 ml volumetric flask through a pipette and diluting to obtain 40 µg/ml Epcoritamab; and
analyzing the obtained Epcoritamab through the RP-UPLC technique, thereby achieving separation of Epcoritamab by an isocratic elution within a time period of 2 min of runtime with a flow rate of 0.3 mL/min.
2. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the RP-UPLC technique includes a binary solvent manager, sample manager and a photodiode array (PDA) detector.
3. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the reverse-phased ultra-performance liquid chromatography (RP-UPLC) technique uses a BEH Shield RP-18 (100 x 2.1mm, 1.7µm) an analytical column, which enables the rapid development of faster and more robust separations of Epcoritamab.
4. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the obtained Epcoritamab is validated for specificity, linearity, precision, accuracy, sensitivity and robustness.
5. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the separation of Epcoritamab is achieved by Isocratic elution using the ammonium formate buffer solution and the acetonitrile (CH3CN) at the ratio of 6:4.
6. The method for determining Epcoritamab in epkinly as claimed in claim 1, wherein the Epcoritamab standard solution is subjected through the sonication process for a time period of at least 30 min.
7. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the Epcoritamab standard solution is subjected through centrifugation for a time period of at least 30 min.
8. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the buffer solution is adjusted to a pH value of 3.0 with orthophosphoric acid.
9. The method for determining Epcoritamab in Epkinly as claimed in claim 1, wherein the buffer solution is filtered through the filter paper of 0.45 µm and followed by mobile phase solution.