INTRODUCTION:

Merging Pembrolizumab with Trastuzumab and cytotoxic chemotherapy is substantiated by preclinical and clinical data for managing HER2-positive cancers^1-5. This combination demonstrates the capability to eliminate tumors in a murine model of HER2-positive breast cancer and elicits enduring inducing clinical responses in a subset of individuals diagnosed with HER2-positive metastatic breast cancer that is resistant to Trastuzumab Pembrolizumab is a type of drug used to treat various types of cancer. It's a PD-1-blocking antibody and a type of targeted therapy drug called an immune checkpoint inhibitor.^6-9

Figure 1: Molecular arrangement of Trastuzumab

Figure 2: Molecular arrangement of Pembrolizumab

MATERIALS AND METHODS:

Waters Alliance HPLC system, featuring a 2695 pump, controlled auto-injector by Empower 2 software, UV detector, and controlled auto-injector. Additionally, instrumental support included a UV–visible spectrophotometer made by Shimadzu, an Electronic balance (Ohus), an Eutech pH Meter, and a digital ultrasonic cleaner of a Phoenix 4.5 L used in the study. Pembrolizumab with Trastuzumab API samples obtained from Pharma Life Research facility in Hyderabad, India and other chemicals were of AR-grade (Rankem Chemicals, India).

Procedure

Precisely measure transfer 44 mg of Trastuzumab, and 5mg of Pembrolizumab working standard into a 10 ml clean, volumetric flask. Appropriately added the diluent and sonicated the mixture to ensure complete dissolution then adjust the volume to the mark using the diluent. to create the stock solution. Subsequently, pipette out 1 ml from the aforementioned stock solutions to a 10 ml volumetric flask and dilute it up to the mark with the specified diluent. (440ppm of Trastuzumab, 50ppm of Pembrolizumab)

Sample solution preparation

0.1 ml of Trastuzumab sample and 5mg of Pembrolizumab sample transfer into a 10 ml clean, dry volumetric flask, add the appropriate diluent, and sonicate it for up to 30 minutes to achieve complete dissolution. Adjust the volume up to the mark with the same solvent. Subsequently, filter the solution through a 0.45 μ injection filter to obtain the stock solution. Next, pipette 1 ml from the prepared stock solution into a 10 ml volumetric flask and dilute it to the mark with the specified solvent. This results in a solution containing 440 ppm of Trastuzumab and 50 ppm of Pembrolizumab.

Method validation¹⁰

The process of method validation involves conducting a series of tests that are based on the analytical method. These tests are used to determine and document the performance characteristics of the method, as well as to determine if the method is suitable for a specific analytical purpose. The performance characteristics of LC methods include specificity, selectivity, precision, linearity, robustness, recovery, range, limit of detection, limit of quantification, and ruggedness. The focus of validation should be on the precision of the analytical system, rather than just the method itself. This includes having a well-defined method protocol, concentration ranges for the analyte, and a specified type of test material. The method validation protocol will be prepared by carefully considering the analytical system as a whole and the analytical procedure.

Linearity, Precision, Accuracy, Robustness

The specificity of the proposed method was confirmed by the absence of interfering peaks in the blank, placebo, and sample during the retention periods of Trastuzumab and Pembrolizumab. In Figures 5 and 6, the chromatograms of a placebo and blank are displayed. The retention times for Trastuzumab and Pembrolizumab were found to be 2.730 min and 4.468 min respectively. Figure 4 displays the optimized chromatogram.

Assay and Forced degradation studies¹¹

The HPLC was used to acquire the chromatogram by injecting 5 µL of the standard solution and sample solution separately. The quantification of the medicines in solution was accomplished by evaluating the peak regions of the obtained chromatograms.

Stress testing was suggested by ICH guidelines as a means to determine the intrinsic stability of drug substances. Acid degradation (2 N HCl, refluxed at 60 °C for 30 minutes), alkali degradation (2 N NaOH, refluxed at 60 °C for 30 minutes), peroxide degradation (20% hydrogen peroxide heated at 60 °C for 30 minutes), thermal degradation (samples arranged in a hot air oven at 105 °C for 1 hour), photolytic degradation (sample in an ultraviolet chamber for one day), and hydrolysis were all applied to the solution of the standard in these investigations. The peak areas of the so-stressed samples were determined and compared to the standard's peak areas.

RESULTS AND DISCUSSION:

A new stability-indicating RP-HPLC technique has been developed and validated for the simultaneous assay of Trastuzumab and Pembrolizumab using the Luna Phenyl Hexyl, and a mixture of acetonitrile: Ammonium acetate pH-3.0/OPA (30:70v/v) with a run time of 7 min. The retention time of Trastuzumab and Pembrolizumab was found to be 2.730 min and 4.468 min (Figure 3) respectively.

Linearity, Precision, Accuracy, Robustness studies

The linearity of the proposed method was observed within the concentration ranges of 110-660 μg/ml (R² = 0.9991) and 12.50-75.00 μg/ml (R² = 0.99956) (Table 1). Linear regression equations were determined as y = 4623.70x + 14577.21 and y = 4718.92x + 1338.89 for Trastuzumab and Pembrolizumab, respectively. The limit of Quantification values was 2.2 μg/ml and 0.25 μg/ml, while Limits of Detection (LOD) were determined to be 0.66 μg/ml and 0.075 μg/ml, respectively (Figure 4). Trastuzumab and Pembrolizumab eluted at 2.730 mins and 4.468 mins, presenting sharp peaks with theoretical plates of 3173 and 6509, and tailing factors of 1.07 and 0.98, meeting system suitability criteria were evaluated (Figure 3). System precision (n=6) and method precision (n=6) were assessed by injecting drug solutions (440 μg/ml and 50 μg/ml), and % RSD was calculated (Table 2). The accuracy study, based on spiked samples of 50%, 100%, and 150%, demonstrated % recovery with % RSD < 2.0 (Table 3). Results from the robustness study also indicated % RSD (0.61-0.55 and 1.41-0.56) below 2.0 indicating that the proposed method is precise, accurate, and robust.

Table. 1. Linearity study

Sample	Trastuzumab		Pembrolizumab
Sample	Conc. (µg/ml)	*Peak area	Conc. (µg/ml)	*Peak area
1	110. 00	525768	12.50	60080
2	220.00	1035788	25.00	120208
3	330.00	1569797	37.50	184611
4	440.00	2040308	50.00	230807
5	550.00	2545380	62.50	295964
6	660.00	3065754	75.00	356418
Slope	4623.70		4718.92
Intercept	14577.21		1338.89
R 2	0.99991		0.99956

* Mean of three replicates

Blank

Placebo

Figure 3: Optimized chromatogram


A) Trastuzumab	B) Pembrolizumab
Figure 4: Calibration curves

Table 2: Precision studies

S. No.	Repeatability (For sample)		Intermediate precision (Method precision)
S. No.	Trastuzumab	Pembrolizumab	Trastuzumab	Pembrolizumab
1	2097743	231565	2017743	231565
2	2058124	232315	2050814	2072174
3	2047959	232897	2072174	234315
4	2078154	233102	2028318	234433 5
5	2036487	233354	2048514	234748
6	2024748	236848	2059154	235190
Mean	2057203	233347	2046120	233767
SD	27023.01	1830.85	19993.13	1454.99
RSD	1.314	0.785	0.977	0.622

Table 3: Accuracy studies

Level	Peak area		% Recovery (% RSD)
	Trastuzumab	Pembrolizumab	Trastuzumab	Pembrolizumab
50%	1035788 1036487 1016554	115208 114911 117356	100.1	98.8
100%	2053743 2025484 2068794	231565 232312 234145	99.6	98.4
150%	3065754 3026145 3047153	346418 344154 345648	98.7	100.4

Table 4: Assay for Trastuzumab and Pembrolizumab

Drug

Avg sample

area (n=5)

Std. Conc. (µg/ml)

Sample Conc. (µg/ml)

Label

amount (mg)

Std purity

*Amount

found (µg/ml)

Trastuzumab

2051236

440

99.8

99.4

Pembrolizumab

233390

99.9

100.3

* mean of three replicates

Table 5: Stress degradation studies

Degradation condition	% Drug degradation*		Peak purity of Trastuzumab		Peak purity of Pembrolizumab
Degradation condition	Trastuzumab	Pembrolizumab	Purity angle	Purity threshold	Purity angle	Purity threshold
Acid degradation	12.8	12.1	3.431	17.563	6.835	18.542
Alkali degradation	13.7	11.8	3.456	17.527	6.857	18.567
Peroxide degradation	14.8	13	3.472	17.548	6.859	18.524
Thermal degradation	9.4	4.3	3.447	17.562	6.878	18.552
Photolytic degradation	1.4	2	3.442	17.528	6.883	18.566
Hydrolysis degradation	1.8	1.3	3.454	17.579	6.849	18.543

* mean of three replicates


Acid Degradation	Alkali Degradation

Hydrolysis Degradation	Peroxide Degradation

Thermal Degradation	Photolytic Degradation
Figure 5: Typical chromatograms obtained during the forced degradation studies

CONCLUSIONS:

The HPLC technique that was developed is characterized by its simplicity, speed, cost-effectiveness, specificity, and reliability in estimating the quantities of Trastuzumab and Pembrolizumab. The approach was deemed satisfactory and may be effectively utilized in normal laboratory analysis to simultaneously estimate Trastuzumab and Pembrolizumab in both bulk and pharmaceutical dose forms.

REFERENCES:

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Received on 05.02.2024 Modified on 12.03.2024

Research J. Pharm. and Tech 2024; 17(5):2241-2246.

DOI: 10.52711/0974-360X.2024.00353