INTRODUCTION:

The development of analytical methods¹ is an essential component of any scientific study. To get precise and trustworthy results for Inmazeb², it entails developing, refining, and validating sample analysis techniques.^{3, 4} A thorough grasp of the characteristics of the material being examined, as well as the tools and methods employed for analysis, is necessary for the process of developing analytical methods.⁵Successfully developing a method for reverse-phase HPLC⁶ requires an understanding of the sample being analyzed (in this case, mAbs), as well as the synthetic process, associated impurities, potential degradation pathways, and degradation products.⁷

Atoltivimab (ALT), Maftivimab (MFT), and Odesivimab (ODE) are human monoclonal antibodies (mAbs) designed to target glycoproteins of the Zaire ebolavirus (EBOV). These antibodies specifically bind to a glycoprotein on the Ebola virus's⁸ surface that normally attaches to a host cell receptor, facilitating membrane fusion and viral entry. By binding to three distinct sites on this glycoprotein simultaneously, they block the virus from attaching and entering the cell. Atoltivimab (ALT), a human monoclonal antibody⁹ targeting the Zaire ebolavirus glycoprotein, is part of the fixed-dose combination with Odesivimab (REGN 3471) and Maftivimab (REGN 3479), collectively known as REGN-EB3 (InmazebTM), which is used to treat infections caused by Zaire ebolavirus.

Figure 1. Cryo-EM at 3.1A^oStructural Overview of Inmazeb Cocktail (REGN - EB3) bound to EBOV GP.¹⁰

Note: From “Structure of the Inmazeb cocktail and resistance to Ebola virus escape,” by Rayaprolu et al., 2023, Cell Host and Microbe 31, 260–272, Introduction Section, Figure 1 (https://doi.org/10.1016/j.chom.2023.01.002).

Each of these three monoclonal antibodies (mAbs) binds to a unique region of the GP 1,2 glycoprotein, offering both neutralizing effects and Fc-arbitrated immune responses against EBOV in vitro, and providing protection from EBOV condition in vivo. The FDA authorised Regeneron Pharmaceuticals' INMAZEB™ on October 14, 2020.¹¹ There are only a few studies in the literature that test the stability of this combination. When designing stability-indicating procedures, forced deterioration¹² or stress testing is used to demonstrate specificity, especially when there is minimal information about probable degradation products. This research seeks to embark on a precise, specific, and reproducible method of Reverse phase - HPLC to assess the stability of ALT, MFT, and ODE. The proposed approach for analysing ALT, MFT, and ODE in combination parenteral preparations was concurrent estimation^13-17and was validated^16-19using ICH guidelines and the new international convention. And to determine the precise nature of the approach, forced degradation investigations were conducted by exposing the sample solution of mAbs to a variety of stress conditions such as acidulant²⁰, acrid, hydrolytic²¹, oxidative, high temperature, and exposure to sun light.^{22, 23}

MATERIALS AND METHODS:

Drugs and Chemicals:

Atoltivimab, Odesivimab, Maftivimab were obtained from HETERO, Hyderabad, India. The formulation INMAZEB^TM by Regeneron Pharmaceuticals was purchased from the market. The chemicals utilized, including sodium bisulfate, HCl, potassium dihydrogen phosphate, and methanol, were of AR grade. The eluent solvents required for HPLC are of HPLC grade²⁴ and were purchased from Merck.

Instruments:

The Waters HPLC system, Model E2695, is equipped with a 2998 Photodiode Array Detector (PDA) and features an automated sampling injecting system.. The resultant signal is processed and analyzed using Empower 2 software. For the separation processes, a Waters C18 column (250 mm x 4.6 mm, 5 μm) were employed.

Chromatographic conditions:

The separation in this chromatographic instrument was performed using an INERTSIL C18 column (250 x 4.6 mm, 5 µm). The eluent consisted of a buffer containing a mixture of aqueous sodium bisulfate, adjusted to a pH of 3.8, and acetonitrile in a 50:50 v/v ratio. This eluent was screened through a 0.45 µm membrane strainer. The rate of the eluent flow was set at 1.0 mL/min, and the spectrum was monitored at a wavelength of 236 nm. An aliquot of 10 µL was injected in the system. Peak similitude was assessed and expressed in the form of peak purity, derived right through the spectrographic analysis record generated by specified tool. A similarity curve for the peaks of ALT, MFT, and ODE was plotted over time during the elution process.

Preparation of working standard solutions:

Precisely measure and transfer 16.67 mg each of Atoltivimab, Maftivimab, and Odesivimab in a 100 ml volumetric flask. Add 10 ml of acetonitrile, then agitate on a sonicator for 10 minutes or waggle the flask for 5 minutes. Complete the volume with HPLC grade water. Take an ml of this agitated solution, transfer it to a 10 ml volumetric flask, and make the volume with water to prepare the working standard.

Preparation of Solution for Marketed Formulation:

A commercially available formulation, equivalent to 50.01 mg of active ingredients (16.67 mg each of Atoltivimab, Maftivimab, and Odesivimab), was placed in a 100 ml flask. To this, 10 ml of acetonitrile was included, and the resultant mixture was either agitated using a sonicator for 20 minutes or shaken for 10 minutes. The final volume has to be made to 100 ml with water. Next, an ml of resultant mixture was fetched to a 10 ml vol. flask and the volume was made with acetonitrile. This solution has to be screened through a 0.45 μm filter before introducing it to the chromatographic system.

RESULTS AND DISCUSSION:

Chromatographic Method Development and Optimization:

The trials for developing a method²⁵ were conducted by various buffers, buffer ratios, and acetonitrile across various columns²⁶. Trials on a SUPELCO C18 column (150x4.6mm, 5µm particle size) with a K₂HPO₄: Methanol (70:30) mobile phase showed tailing²⁷ greater than 2, and system suitability parameters were unsatisfactory. To optimize²⁸ mobile phase conditions, factors²⁹ such as analysis time, flow rate, peak symmetry, assay sensitivity, solvent noise, and compatibility of the solvent system for drug extraction from formulation matrices were considered. Additional trials with a Phenomenex C18 column (150x4.6mm, 5µm) using K₂HPO₄:Methanol (60:40) improved peak shape and reduced tailing. The final optimized mobile phase, NaHSO₄: Acetonitrile (50:50) at pH 3.8 and a 1.0 mL/min flow rate, produced sharp peaks for ALT, MFT, and ODE with retention times of 2.7, 3.9, and 6.2 minutes, respectively, detected at 236 nm. The temperature at which the column had to be maintained was 25°C (±2°C) with a thermostat, which provided optimal peak separation, minimized tailing, and achieved the best resolution³⁰ compared to other tested combinations (Fig. 1). The optimized chromatographic conditions are detailed in Table 1.

Table 1. RP-HPLC Chromatographic Conditions

Instrument	Waters HPLC E2695 with automated sample Injector
Column	Inertsil , C18, 250X4.6mm, 5µm
Detector	Photodiode array detector (PDA) 2998
Mobile phase	NaHSO4: Acetonitrile (50:50 %v/v)
Flow rate	1.0m l/ Min
Detection wavelength	236 nm
Run time	8 minutes
Sample Temperature	25oC
Column Temperature	25oC
The volume of Injection loop	10µl
pH	3.8

Figure 2. Chromatogram of ALT, MFT, and ODE.

Method Validation:

Specificity:

Inmazeb^TM31 solution containing 16.67 μg/mL each of ALT, MFT, and ODE was prepared using a diluent composed of 0.1M NaHSO₄ (pH 3.8) and pure acetonitrile in a 50:50 (v/v) ratio. A working solution with the same concentrations of ALT, MFT, and ODE (16.67 μg/mL each) was then analyzed according to specified criteria. Chromatograms for ALT, MFT, and ODE peaks were recorded, with no peaks related to excipients or diluents detected during the retention times for LDE, DRE, or TFE. The analysis provided accurate values for the combined quantification of ALT, MFT, and ODE.

System Suitability:

This parameter is quite important to confirm a quality performance of the system used for chromatographic aalysis. Previously developed aliquots for chromatographic conditions were evaluated through this testing. The theoretical plate count for ALT, MFT, and ODE peaks was maintained at 2500 or higher, analyte peak tailing factors were kept at or below 2.00, and the RSD across the 5 injections in replication of the working standard preparation didn’t exceed 2.00.

Table 2. System suitability data

Parameter	Atoltivimab	Maftivimab	Odesivimab	Acceptance Criteria
Retention time	2.742	3.866	6.132	±10
Theoretical plates	4744	5803	3967	> 2500
Tailing factor	1.32	1.19	1.28	< 2.00
% RSD	0.2	0.2	0.1	< 2.00

Precision:

Six replicate injections were performed with ALT, MFT, and ODE solutions, each at a concentration of 16.67 μg/mL. Chromatograms were generated under the optimized chromatographic conditions, and the standard deviation and RSD of the responses by the peaks were calculated, all detailing given in the Table. The percentage relative standard deviation (%RSD) was below 2%, confirming that the method is precised.

Accuracy:

Six consecutive injections of solutions containing ALT, MFT, and ODE of 16.67 μg/mL concentration each were performed. The evaluation was based on the gist outlined in the "Settings for ALT, MFT, and ODE collective assessment" section. Response of the peak, assay, and chromatograms for ALT, MFT, and ODE were calculated, with test results confirming the accuracy of the combined analysis for ALT, MFT, and ODE.

Linearity:

Stock solutions of ALT, MFT, and ODE (each at 16.67 μg/mL) were precisely diluted with a diluent (50:50 v/v NaHSO₄ and acetonitrile) to achieve concentrations of 8.335, 12.50, 16.67, 20.83, and 25.005 μg/mL, as outlined in Table 3. Standard curves³² were generated by plotting areas of the peak versus these concentrations. The slope and Y-intercept of the standard curve were then obtained by calculation.

Table 3. Linearity Data

Concentration (μg/ml)	Peak Areas for
Concentration (μg/ml)	Atoltivimab	Maftivimab	Odesivimab
8.335 (50%)	1341089	1169049	1750275
12.50 (75%)	2023435	1758769	2632059
16.67 (100%)	2712651	2356016	3524847
20.83 (125%)	3399223	2944298	4412694
25.005 (150%)	4071512	3539413	5298639
CC (R²)	0.999	0.999	0.999

Figure 3. Linearity Plot for ALT, MFT, and ODE.

LOD and LOQ:

The detection limit (LOD) and quantitation limit (LOQ) for ALT, MFT, and ODE was determined using the calibration curve method, on the basis of SD (σ) and the slope (S) of the regression line. This involved a series of injections of solutions which were diluted whose concentrations were known. The detection limit and quantitation limit values obtained for ALT, MFT, and ODE are tabulated in Table 4.

Table 4. LOD and LOQ and Precision data

Inmazeb (REGN-EB3’s)	LOD (µg / ml)	LOQ (µg / ml)	Precision - % RSD
ALT	0.055	0.185	0.1
MFT	0.100	0.333	0.1
ODE	0.088	0.293	0.1

Recovery:

To assess the recoveries of ALT, MFT, and ODE, standards of each were added to the working InmazebTM solution at concentrations of 16.67 μg/ml. Recovery data are presented in Table 5.

Table 5. Recovery data

Recovery stage	Inmazeb (REGN-EB3’s)
Recovery stage	ALT	MFT	ODE
50%	99.83	99	100.01
100%	99.05	100.02	100.05
150%	99.03	100.05	100.06

Figure 4. Recovery data

Robustness:

To assess robustness, key chromatographic settings were adjusted, and the compatibility profile of the chromatographic equipment was tracked and recorded simultaneously. The main parameters adjusted included pH level, detector wavelength, rate of flow, and temperature for the column. Robustness testing had been conducted using working standard solutions of ALT, MFT, and ODE at 16.67 μg/ml concentrations each. The response of the prak, equipment suitability profile, and elution profile for ALT, MFT, and ODE were analyzed, and the results demonstrated strong robustness for the combined analysis of these compounds.

Degradation Studies:

The stock Inmazeb^TM solution, containing ALT, MFT, and ODE at 16.67 μg/ml concentrations, was subjected to stress testing following ICH guidelines. Degradation data are presented in Table 6. No stress-induced degradation compounds were detected during the LDE, DRE, or TFE retention times. The test results showed high specificity and confirmed stability-indicating properties for the combined analysis of ALT, MFT, and ODE.

Table 6. Degradation studies

Condition	% Assay			% Degradation
Condition	ALT	MFT	ODE	ALT	MFT	ODE
0.1 N HCl	89.37	90.66	89.86	10.63	9.34	10.14
0.1 N NaOH	93.59	94.95	92.18	6.41	5.05	7.82
30% H₂O₂	91.61	93.49	95.24	8.39	6.51	4.76
105^oC	90.24	87.89	87.48	9.76	12.11	12.52
Sunlight	94.91	91.71	93.24	5.09	8.29	6.76
Water	99.04	98.31	98.84	0.96	1.69	1.16

CONCLUSION:

The objective of this research is to build and validate an HPLC method to estimate Atoltivimab, Odesivimab, and Maftivimab in parenteral dosageform, using an RP-HPLC approach. The Inmazeb combination method, incorporating ALT, MFT, and ODE, was optimized and demonstrated to be efficient, linear, reliable, robust, stable, and sensitive in measuring ALT, MFT, and ODE together. For routine analysis, methods were developed to handle large sample volumes quickly, with high robustness, accuracy, and precision, eliminating the need for preliminary separation steps.

ACKNOWLEDGEMENT:

Sincere thanks are extended by the authors to the Deccan School of Pharmacy at Darussalam, Aghapura, Hyderabad, India, for providing exceptionally good laboratory research facilities that were crucial to the accomplishment of this work.

REFERENCES:

1. Ghosh M.K. HPLC methods on drug analysis. Berlin: Springer Verlag. 1992. 1-22.

2. FDA Approved Drug Products: INMAZEB (atoltivimab, maftivimab, and odesivimab-ebgn) injection.

3. Day R.A., Underwood AL. Quantitative analysis, 5th ed. New Delhi: Prentice- Hall India Pvt Ltd. 1986. 1-6.

4. Lindsay S. High Performance Liquid Chromatography. 1st ed. New York: Wiley Interscience. 1991. 1-24.

5. Bassett J., Denney R.C., Jeffrey G.H., Mendham J. Vogel’s text Book of Quantitative Inorganic Analysis. 4th ed. England: Longman Group. 1986. 2.

6. Lough W.J., Wainer I.W. High performance liquid chromatography: fundamental principles and practice. Glasgow (UK): Blackie Academic and Professional. 1995. 2-28.

7. Prasad Babu N., Ramachandran D. Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Ornidazole and its Impurities in Ornidazole Injection. Research J of Pharm and Tech. 2022; 15(1): 82-88. https://doi.org/10.52711/0974-360X.2022.00015

8. Jacob ST, Crozier I, Fischer WA 2nd, Hewlett A, Kraft CS, Vega MA, Soka MJ, Wahl V, Griffiths A, Bollinger L, Kuhn JH: Ebola virus disease. Nature Reviews Disease Primers. 2020; Feb 20. 6(1). 13. https://doi.org/10.1038/s41572-020-0147-3

9. Saphire EO, Schendel SL, Gunn BM, Milligan JC, Alter G: Antibody-mediated protection against Ebola virus. Nat Immunol. 2018; Nov; 19(11): 1169-1178. https://doi.org/10.1038/s41590-018-0233-9. Epub 2018 Oct 17.

10. Vamseedhar Rayaprolu et al., Structure of the Inmazeb cocktail and resistance to Ebola virus escape. Cell host and microbe. 31. 2023. 260-272. https://doi.org/10.1016/j.chom.2023.01.002

11. Pascal KE, Dudgeon D, Trefry JC, Anantpadma M, Sakurai Y, Murin CD, Turner HL, That Treat Advanced Ebola Virus Disease in Nonhuman Primates. J Infect Dis. 2018 Nov 22. 218 (suppl_5):S612-S626. https://doi.org/10.1093/infdis/jiy285.

12. https://www.ejbps.com/ejbps/abstract_id/9090

13. Soanki, Renu et. al. Development and Validation of Simultaneous Estimation Method for Amoxycillin Trihydrate and Tinidazole in Tablet Dosage Form by RP-HPLC. Asian J. Pharm. Ana. 2013; Vol. 3: Issue 2, Pg 66-71. https://ajpaonline.com/Abstract View.aspx?PID=2013-3-2-8

14. Swapna, Jajow. Madhu, Chandaka. Srivani, Mallepelli. Sumalatha, M. Nehalatha, Y. and Anusha, Y. Analytical Method Development and Method Validation for the Simultaneous Estimation of Metformin hydrochloride and Pioglitazone hydrochloride in Tablet Dosage Form by RP-HPLC. Asian J. Pharm. Ana. 2012; Vol. 2: Issue 3, Pg 85-89. https://ajpaonline.com/AbstractView.aspx?PID=2012-2-3-6

15. Sebaiy, Mahmoud M. A. Abdullah et. al. Rapid RP-HPLC Method for Simultaneous Estimation of Norfloxacin and Tinidazole in Tablet Dosage Form. Asian J. Pharm. Ana. 2011; Vol. 1: Issue 4, Pg 79-84. https://ajpaonline.com/AbstractView.aspx?PID=2011-1-4-4

16. Eswarudu, M.M. et. al. RP-HPLC Method Development and Validation for Simultaneous Estimation of Irbesartan and Hydrochlorothiazide in Pharmaceutical Dosage Form. Asian J. Research Chem. 5(4): April 2012. https://www.ajrconline.org/AbstractView.aspx?PID=2012-5-4-6

17. Dr. Pant, Shalini et. al. Development and Validation of a Simultaneous HPLC Method for Assay and Dissolution of Bisoprolol fumarate and Amlodipine besylate in Pharmaceutical Dosage. Research J. Ph. Dosage Forms and Tech. 2012; 4(1): 62-663. https://rjpdft.com/AbstractView.aspx?PID=2012-4-1-11

18. Vaishnani, Bhavin. Kimbahune, Ritu. Kabra, Prachi. Surani, Sanjay. Nargund, L.V.G. Validated RP-HPLC Method for Estimation of Rasagiline in Tablet Dosage Form. Asian J. Research Chem. 2012; 5(4). https://www.ajrconline.org/AbstractView.aspx?PID=2012-5-4-14

19. M. M., Maste et. al. Validation and Application of a High-Performance Liquid Chromatography Method for Estimation of Sitagliptin Phosphate from Bulk Drug and Pharmaceutical Formulation. Asian J. R. Chem. 2012; 5(1). https://www.ajrconline.org/AbstractView.aspx?PID=2012-5-1-24

20. Gupta, Akhilesh. Rawat, Swati. Gandhi, Mayuri. And Yadav, Jaydeep Singh. Method Development and Acid Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Ph. Ana. 2011; 1(1); 10-13. https://ajpaonline.com/AbstractView.aspx?PID=2011-1-1-4

21. Gupta, Akhilesh. Yadav, Jaydeep Singh. Rawat, Swati. Gandhi, Mayuri. Method Development and Hydrolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 2011; 1(1): 14-18. https://ajpaonline.com/AbstractView.aspx?PID=2011-1-1-5

22. Fegade Bharti S. et.al., Development and Validation of a Stability indicating RP-HPLC method for Estimation of Deferiprone in its capsule Dosage Form. Research Journal of Pharmacy and Technology. 2024; 17(6). https://doi.org/10.52711/0974-360X.2024.00427

23. Gupta, Akhilesh. Rawat, Swati. Pandey, Arun. Method Development and Photolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 2011; 1(2): 29-33. https://ajpaonline.com/AbstractView.aspx?PID=2011-1-2-3

24. Kasture AV, Mahadik KR, Wadodker SG, More HN. Instrumental methods of Pharmaceutical analysis.Vol-II, 14th ed. Pune: Nirali Prakashan; 2006. 1-30.

25. USP 28, NF 23, The United State Pharmacopoeial Convention: Asian Edition. 2005.

26. Macek Karel et al., Pharmaceutical applications of thin-layer and paper chromatography. Journal of Pharmaceutical Sciences. American Elsevier. 1973.

27. Meyer VR. Practical High Performance Liquid Chromatography. 2nd ed. New York: Wiley Inter Science. 1994. 12.

28. Massart D.L., Vandeginste B.G.M., Deming S.N., Michotte Y., Kaufman L. Chemometrics: A Text Book. Amsterdam: Elsevier Science. 1988. 1-50.

29. Kirkbright G.F. Development and publication of new spectrophotometric methods of analysis. Talanta. 1966. 1-14. https://doi.org/10.1016/0039-9140(66)80119-4

30. Fong G.W, Lam S.K. HPLC in the Pharmaceutical Industry. Vol-47, New York: Marcel Dekker Inc. 1991. 16-56.

31. Zaire Ebolavirus Drugs of Today. 57 (8): 483–490. https://doi.org/10.1358/dot.2021.57.8.3280599 PMID 34405205. S2CID 237198823.

32. Pattengill Merle D., Sands Donald E. Statistical significance of linear least-squares parameters. Journal of Chemical Education. 1979. 56. 4. 244. https://doi.org/10.1021/ed056p244

Received on 12.11.2024 Revised on 18.03.2025

Accepted on 20.05.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):308-312.

DOI: 10.52711/0974-360X.2026.00044

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