Quantification of Tezepelumab in rat plasma by Bio-analytical Stability Indicating Method Development and Validation using LCMS and its Implementation to Pharmacokinetics

Nagaraju Bandaru¹, Kranthi Yalla², Naga Venkata Indira Devi Jajula³*,

Anusha Gandi⁴, Rajanand Swamy⁵

¹Associate Professor, School of Pharmaceutical Sciences (SOPS),

Sandip University, Nashik, Maharashtra, India - 422213.

²Assistant Professor, Shri Vishnu College of Pharmacy, Bhimavaram, Andhra Pradesh, India - 534202.

³Assistant Professor, Emmanuel College of Pharmacy, Visakhapatnam, Andhra Pradesh, India - 531162.

⁴Assistant Professor, Vignan Institute of Pharmaceutical Technology,

Visakhapatnam, Andhra Pradesh, India - 530049.

⁵Principal, Emmanuel College of Pharmacy, Visakhapatnam, Andhra Pradesh, India - 531162.

*Corresponding Author E-mail: indiradevijajula18@gmail.com

ABSTRACT:

Tezepelumab, marketed under the trade name Tezspire, is a human monoclonal antibody used for the treatment of asthma. The present study aimed to develop a simple, economical, robust, and highly sensitive analytical method for the estimation of Tezepelumab in plasma samples obtained from male Wistar rats, using Abciximab as the internal standard. The chromatographic analysis was performed with a total runtime of 7.0 minutes, with Tezepelumab eluting at 3.115 minutes. The method demonstrated excellent linearity over the concentration range of 12.5–100.00 ng/mL, with a correlation coefficient (R²) of 0.999. The intra- and inter-assay precision showed %CV values below 15% across five quantification levels, indicating high reproducibility. All bioanalytical validation parameters were in accordance with USFDA guidelines and found to be within acceptable limits. Stability studies conducted at various time intervals confirmed the method’s reliability under different conditions. Furthermore, the peak plasma concentrations were determined as a function of time during the intravenous pharmacokinetic study, validating the suitability of the method for pharmacokinetic evaluation of Tezepelumab.

KEYWORDS: LC-MS, Tezepelumab, Bio-Analytical method development and validation, Stability studies and Pharmacokinetics.

INTRODUCTION: 

Tezepelumab¹ is sold under the brand name called Tezspire for asthmatic treatments. This formulation is a human monoclonal antibody¹. It blocks TSLP, an epithelial cytokine believed to be critical in the initiation and persistence of airway inflammation. The estimated clearance is 0.17 L/d¹. United States granted this drug for usage in the month of December². Tezepelumab possess the chemical formula as C₆₄₀₀H₉₈₄₄N₁₇₃₂O₁₉₉₂S₅₂ and molecular weight of 144590.4g/mol and the structure is shown in figure 1. The internal standard is Abciximab and its structure is represented in figure 2.

Figure 1: Crystal structure of human TSLP in complex with the Fab-fragment of the Tezepelumab antibody (PDB 5J13)

Figure 2: Chemical structure of Abciximab

A review of the literature^3-13 on Tezepelumab concerning its physical and chemical properties, as well as various analytical methods reported for the estimation of Tezepelumab served as the foundation for the development of a new stability indicating bioanalytical LC-MS method according to USFDA guidelines for its intended application.

EXPERIMENTAL PART:

Instrumentation:

HPLC Waters 2695 with an Auto sampler of high speed, column oven and degasser with software classAB SCIEX.

Chemicals used:

Tezepelumab sample is procured from Bangalore and all the organic solvents like Methanol, Water, Acetonitrile of LC-MS grade were bought from Mumbai.

Preparation of reagents:

1.   Stock solution (200ng/mL): 5mg of Tezepelumab was weighed and transferred into a 10mL volumetric flask and made upto the mark with the diluent. 0.1mL of the solution was made till 10mL volume with the diluent. Later 0.4mL of this solution was again diluted to 10mL with the diluent in volumetric flask.

2.   Internal standard stock (200ng/mL): A weight of 5mg of Abciximab working standard was measured and placed into a 10mL volumetric flask, followed by the addition of the diluent. A 0.1mL portion of this solution was further diluted to 10mL using the diluent in a separate 10mL volumetric flask. Subsequently, 0.4mL of this solution was brought to volume with diluent in another 10mL volumetric flask.

3.   Standard solution (50ng/mL of Tezepelumab): A volume of 500µL from the standard stock solution was placed into a 2mL centrifuge tube. To this tube, 200µL of plasma, 500µL of internal standard, 300µL of acetonitrile, and 500µL of diluent were introduced. Following centrifugation for 20 minutes, the supernatant was moved into an LC vial.

4.   Preparation of solutions for linearity: The linearity solutions of concentration 12.5-100ng/mL were prepared in the same way as above. The supernatant solution after centrifugation for 20min at 4000 RPM was injected into the chromatograph after it was collected in the LC.

Extraction procedure:

The samples that are centrifuged and treated were named in like manner to their time interims. To approximately 200μL of plasma, 500μL of diluent was added and mixed thoroughly. Then, 300μL of ACN was introduced to precipitate the proteins, followed by vortex mixing. The mixture was centrifuged at 4000 RPM for 15 to 20 minutes, and the supernatant was collected into the LC vial for infusion into the chromatograph.

Buffer preparation: 1mL of formic acid was transferred into a 1000mL volumetric flask and diluted to the mark with water, then mixed thoroughly. Later, the solution was sifted through 0.22µ film channel paper.

RESULTS AND DISCUSSION:

Chromatographic conditions:

1. Column: Waters X-Bridge Phenyl column of 150 x 4.6mm, 3.5µm.

2. Infusion Volume and Run time: 10µL and 7min respectively

3. Column and sample temperature: 25^oC

4. Stream rate: 1.0mL/min

Conditions for the mass spectrometer:

Conditions of mass spectrometer were overseen in positive particle electro spray ionization interface mode. MRM mode has been connected to measure Tezepelumab. The ion transitions observed were m/z 161250.86→57120.65 for Tezepelumab, m/z 63895.48→14258.34 for Abciximab (Internal standard). Working parameters have been set as Collision energy: 15 V for Tezepelumab and Abciximab, Ion spray voltage: 5500 V, Source temperature: 550ºC, Drying gas temperature: 120-250ºC, Collision gas: Nitrogen, Flow stream of drying gas: 5L/ min, De-clustering, entrance and exit potential: 40V, 10V and 7V respectively and Dwell time: 1 second

Table 1: Results of System Suitability

Tezepelumab MQC (50ng/mL)	Area in cps	Retention time	Internal standard area (50ng/mL)	RT of ISTD (min)	Ratio of area
Mean for 6 values	3.235x10^5	3.114	3.461x10^5	4.735	0.9346
SD	0.012	0.003	0.014	0.002	0.004
%CV	0.38	0.08	0.41	0.05	0.48

The Bio-analytical method validation is done according to USFDA guidelines¹⁴.

QC levels: HQC: 75ng/mL, MQC: 50ng/mL,  and LQC: 25ng/mL

1. System suitability:

Assurance of instrument execution by investigation of a set of reference benchmarks is conducted earlier to the explanatory run.

RESULTS:

The Tezepelumab and ISTD area ratio met system suitability with a %CV of 0.48.

Figure 3: Chromatogram of System suitability (50ng/mL)

2. Auto sampler carryover:

The method passed the carryover effect as it was found to be less than 20%.

3. Specificity and Evaluation of biological matrix:

Six random blank Rat plasma samples were analyzed with no interfering peaks.

Figure 4: Blank chromatogram

Figure 5: Internal standard chromatogram

4. Sensitivity:

The %CV of Tezepelumab was determined to be 0.83%, and the average accuracy % was 112.83, suggesting that it successfully passed the sensitivity assessment.

5. Matrix effect:

The plasma constituents’ effect was estimated by the comparison of post-extracted MQC (50ng/mL) level for six times each with 3 replicate neat samples, using chromatographically screened rat plasma. The %CV of HQC and LQC levels were 0.26 and 0.74 and %mean accuracy of the HQC and LQC levels were 99.93%, and 107.08% respectively.

6. Linearity:

The standard curve for the drug is linear in the range 5-100ng/mL. The R² value was 0.999. The concentration of the sample was determined by comparing the peak area of the sample to that of the internal standard.

Table 2: Back calculated standard curve data for Tezepelumab in rat plasma.

Final Concentration (ng/mL)	Response	Area response ratio
0	0	0.0
12.50	0.885	0.255
25.00	1.743	0.503
37.50	2.493	0.724
50.00	3.241	0.937
62.50	4.056	1.169
75.00	4.862	1.400
100.00	6.467	1.881
Slope	0.0184
Intercept	0.02763
R^2	0.99961

Figure 6: Linearity plot for Tezepelumab

7. LOD and LOQ:

LOD (S/B) and LOQ (S/B) was found to be 3 and 10 respectively.

8. Precision and Accuracy:

These values of intra assay were evaluated with 6 replicates at 3QC levels. The inter assay determined with 4 replicates at 3QC levels and are within the limits.

Table 3: Intra and inter-run precision and accuracy

		Conc.(ng/mL)
		LQC (25)	MQC (50)	HQC (75)
Batch-1 (n=6)	Intra-run	24.89	50.02	75.06
	SD	0.348	0.230	0.221
	%CV	1.40	0.46	0.29
	%Accuracy	99.56%	100.03%	100.09%
Inter-Batch (n=6)	Inter-run mean	25.02	50.13	75.05
	SD	0.177	0.204	0.249
	%CV	0.71	0.41	0.33
	%Accuracy	100.08%	100.25%	100.07%

Analyte recovery: 

The study evaluated drug and IS recovery at low, medium, and high concentration levels for six times comparing responses in the replicating samples with that of responses.

Internal standard:

The %CV of unextracted data and extracted data were 2.76% and 0.39% respectively. The mean recovery 5 was found to be 100.36%.

Limit of acceptance:

The recovery %CV at every QC levels of both Tezepelumab and internal standard must be≤15.00%, with mean recovery %CV for all QC levels is ≤ 20.00%.

9. Ruggedness:

a) On precision accuracy:

The ruggedness test was passed based on the %CV of tezepelumab as the %CV of the three HQC, MQC and LQC levels were 0.35, 0.41, and 0.65 and %mean accuracy of the three HQC, MQC and LQC levels were 100.07%, 100.25% and 108.07% respectively.

b) Ruggedness on reproducibility of reinjection: Tezepelumab's %CV ranged from 0.36%-1.13%, indicating its ability to pass the ruggedness test on reinjection reproducibility.

Limit of acceptance:

The QC samples should achieve a minimum of 67% (16 out of 24) and 50% (3 out of 6) at each level, with the exception of LLOQ QC, which should be between 80.00 and 120.00%. The average accuracy for low QC(LQC), medium QC(MQC), and high QC(HQC) samples should be within the range of 85.00-115.00%, and for the LLOQ QC sample, it should be between 80.00-120.00%.

BIO-ANALYTICAL STABILITY STUDIES:

1. Bench Top Stability:

The average accuracy has met the benchmark for stability with the coefficient of variation (CV) at HQC and LQC levels. The %CV of the three HQC, MQC and LQC levels were 0.34, 0.40, and 0.58 and %mean accuracy of the three HQC, MQC and LQC levels were 100.05%, 100.40% and 100.40% respectively.

Limit of acceptance: The QC samples should meet a standard deviation of 67.5% (8 out of 12) and 50% (3 out of 6) at each level. The LQC and HQC levels mean accuracy must be in 85-115.00% and % CV of the same must be ≤ 15.00%.

2. Auto sampler stability:

The auto sampler stability was successfully maintained for Tezepelumab, as the % CV of the three HQC, MQC and LQC levels were 0.26, 0.46, and 0.87 and %mean accuracy of the three HQC, MQC and LQC levels were 100.24%, 100.25% and 97.61% respectively.

Table 4: Recovery of Tezepelumab from rat plasma

S. No.	Peak response
	LQC		MQC		HQC
	Extracted	Unextracted	Extracted	Unextracted	Extracted	Unextracted
Mean	1.735x10^5	1.841x10^5	3.245x10^5	3.371x10^5	4.853x10^5	4.965x10^5
% CV	0.55	0.72	0.44	0.33	0.27	0.23
%Recovery	107.26%	113.82%	100.31%	104.20%	100.01%	102.32%

Limit of acceptance (for ruggedness and auto sampler stability): The QC samples should achieve a minimum of 67% (16 out of 24) and 50% (3 out of 6) at each level, with the exception of LLOQ QC, which should be between 80.00 and 120.00%. The average accuracy for low QC (LQC), medium QC (MQC), and high QC (HQC) samples should be within the range of 85.00-115.00%, and for the LLOQ QC sample, it should be between 80.00-120.00%.

3. Freeze Thaw, Wet and Dry extract stability:

The average accuracy has met the benchmark for stability with the coefficient of variation (CV) at HQC and LQC levels. The % CV and %mean accuracy of the three HQC, MQC and LQC levels were found to be within the acceptable range

Limit of acceptance: QC samples should meet a standard deviation of 67.5% (8 out of 12) and 50% (3 out of 6) at each level. The LQC and HQC levels mean accuracy must be in 85-115.00% and % CV of the same must be ≤ 15.00%.

4. Short-term and Long-term stability:

The %CV and average accuracy of Tezepelumab have demonstrated stability in the short term as the %CV of the three HQC, MQC and LQC levels were 0.21, 0.41, and 0.20 and %mean accuracy of the three HQC, MQC and LQC levels were 98.07%, 98.21% and 99.29% respectively. The long-term stability data was tabulated below.

Table 5: Long-term stability data of Tezepelumab

Day	LQC(25ng/mL)		MQC (50ng/mL)		HQC (75ng/mL)
	% CV	% Mean accuracy	% CV	% Mean accuracy	% CV	% Mean accuracy
1	1.35	99.76	0.46	100.06	0.27	99.75
7	0.22	99.10	0.28	98.30	0.27	98.11
14	0.75	98.24	0.33	96.85	0.27	97.23
21	0.90	96.07	0.42	95.15	0.35	96.07

APPLICATION OF PROPOSED METHOD TO PHARMACOKINETIC STUDIES:

Tezepelumab from the rat plasma was extracted with liquid-liquid extraction method by adding 200µL of plasma sample to labelled polypropylene tubes and briefly vortexed. The tubes were vortexed for 10 minutes with 500µL of acetonitrile, then centrifuged at 20ºC for 4000rpm. The supernatant from each sample was transferred and evaporated at 40°C until it reached dryness. A single dose of Tezepelumab was administered to rats via intravenous route, and samples were collected at various (0, 0.5, 1, 2, 3, 10, 15, 20, 25, and 30, days) post-dose intervals. At each designated time point, 5 mL of blood was drawn into K2 EDTA vacutainer tubes, along with a pre-dose sample to assess any possible plasma interferences. The collected plasma samples were then centrifuged, stored at temperatures between −70°C and −10°C, spiked with internal standards (IS), and processed alongside quality control (QC) samples at four different concentrations. The plasma samples underwent spiking with IS and were processed at four different concentrations, with pharmacokinetic parameters for Tezepelumab calculated using WinNonlin software version 5.2. The stability of the study samples was evaluated through incurred sample reanalysis (ISR), involving two samples taken near the Cmax and during the elimination phase in the pharmacokinetic profile, ensuring a percentage difference of 20% or lesser than that value.

Figure 7: The graph illustrates the average plasma concentration and time (hrs) of Tezepelumab in rat plasma.

Table 7: Pharmacokinetic parameters of Tezepelumab         

Pharmacokinetic parameters	Tezepelumab Intravenous
AUC0-t	729ng -hr/mL
Cmax	-
AUC0-∞	750ng-hr/mL
t1/2	0 Hours

Table 6: Pharmacokinetic studies

Time interval in hours	0.5	1.0	2.0	3.0	10.0	15.0	20.0	25.0	30.0
Tezepelumab IV response (ng/mL)	0	45	22	10	5	0	0	0	0

CONCLUSION:

A new Liquid Chromatography-Mass Spectrometry method has been developed for determining Tezepelumab in male Wistar rat plasma with internal standard Abciximab. The method has a 7.0-minute runtime and a retention time of 3.115 for Tezepelumab, and has been applied to pharmacokinetic studies.

ETHICAL APPROVAL:

Approval No. 1074/PO/Re/S/05/CPCSEA.

AUTHORS CONFLICT OF INTEREST:

The authors declared that they have no conflict of interest.

ACKNOWLEDGMENT:

Authors are thankful to the Shree Icon Laboratories, Vijayawada, Andhra Pradesh and Manisha Analytical Laboratories, Mumbai for carrying out Bio-analytical work and providing facilities for a smooth run of this research work.

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Received on 01.06.2024      Revised on 12.04.2025

Accepted on 14.10.2025   Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):45-50.

DOI: 10.52711/0974-360X.2026.00007

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