Applications of Fourier Transform Infrared and UV-Visible Spectroscopy for the demonstrating sameness of Ganirelix peptide in Liquid Injection Formulation
*Corresponding Author E-mail: shanmukh_fed@kluniversity.in
ABSTRACT:
Ganirelix (or Ganirelixum) is a synthetic peptide molecule made up of covalently bonded 10 amino acids. The structural order of peptides determines their biological properties. The minor change in the structure of therapeutic peptides can have a significant impact on their biological behaviors. In this regard, the ganirelix drug product was sterilized at 121°C for 15 mins and further exposed to thermal and humidity at 25°C/60%RH and 40°C/75% RH for 6 months. The ganirelix was isolated using flash chromatography and freeze- dried. We applied Fourier transform infrared (FTIR) and Ultraviolet -Visible (UV-Vis) spectroscopic techniques to investigate temperature and humidity effects on the chemical structure of Ganirelix. FTIR scans were conducted in the wave number range of 4000 – 400cm-1 and UV scans were conducted in the wavelength range of 190 – 400nm. The attained IR stretching vibrations, maximum UV absorbance results, overlayed IR and UV-Vis spectra of Ganirelix in temperature stressed generic drug product is closely comparable to reference listed drug (RLD). In addition, it showed homology in the finger print region of the FTIR with those of the reference listed drug (RLD).
KEYWORDS: Ganirelix acetate, Thermal and humidity effect, Fourier transform infrared (FTIR) spectroscopy, Ultraviolet -Visible (UV-Vis) spectroscopy.
INTRODUCTION:
Ganirelix (Fig.1) is a gonadotropin- releasing hormone (GnRH) antagonist developed for fertility treatment in women to prevent premature ovulation during in vitro fertilization (IVF) procedure.1-3 It is a synthetic decapeptide having molecular mass of 1570.4g/mol. It is approved by the United States Food and Drug Administration (USFDA) under the trademark Antagon TM in 19994 and by the European commission (EC) under the trademark Orgalutran® in 2000.5
The route of biopharmaceutical products from research and development to customer readiness is a lengthy and systematic process, intended to ensure efficacy and quality control. Development of sameness requires evaluation of the active ingredient’s chemical structure in generic drug products with the reference listed drug (RLD). Moreover, the structure of therapeutic peptides is sensitive to modifications in the manufacturing procedure or storage environment such as change in temperature or humidity. Therefore, it is prerequisite to assess the chemical structure of therapeutic peptides sustained after any alterations in processing or storage.6-10
Fig.1 Chemical structure of Ganirelix
A broad choice of analytical tools can be used to demonstrate the sameness of the peptide in the generic drug product. Fourier transform infrared (FTIR)11-16 and Ultraviolet -Visible (UV-Vis) spectroscopy17-21 are the most versatile analytical tools used to determine the sameness of proteins and polypeptides in solution.
The endorsed storage temperature of reference listed drug Orgalutran® is 15°C (or 59°F) - 30°C (or 86°F). During transport, there is a chance of immoderate temperature changes. The goal of present study is to assess the thermal and humidity effect on the chemical structure of Ganirelix in the injectable formulation. In this study, we prepared ganirelix acetate injection generic product based on the authorized formulation composition of the Orgalutran® and exposed to thermal and humidity for 6 months at 25°C/60%RH and 40°C/75% RH. The effect of temperature and humidity on the chemical structure of ganirelix was monitored using FTIR and UV-visible spectrophotometer and assess the sameness with the Orgalutran®. This is the first paper to characterize Ganirelix with respect to chemical structure using FTIR and UV-Visible spectroscopy in an injectable formulation.
MATERIALS AND METHODS:
Chemicals and reagents:
Ganirelix acetate ingredient (Batch no. CY45160802, potency 88.2%) was procured from Hybio pharmaceutical company limited (Shenzhen, China). Acetic acid (Pharmaceutical grade, Catalogue no. 1370002500), Mannitol (Pharmaceutical grade, Catalogue no. 1053035000), Sodium hydroxide (Reagent grade, Catalogue no. 1064621000) and Acetonitrile (HPLC grade, Catalogue no. 1000302500) were procured from Merck (Mumbai, India). Potassium bromide (FTIR grade, Catalogue no. 221864) was procured from Sigma Aldrich (Bangalore, India). Water was purified using Ultra clear TP TWF UV TM system (Evoqua Water Technologies, Germany).
Sample solution:
The drug product formulation was manufactured as per the marketed pharmaceutical product Orgalutran® (Ravensburg, Germany), was composed of Ganirelix acetate (5.0mg Ganirelix), 470.0mg mannitol, 2.0mg glacial acetic acid and adjustment to pH 5.0 with sodium hydroxide and/or acetic acid and water up to 10.0mL.4,5 The drug product formulation was sterilized at 121°C for 15 mins to control microbes and exposed to thermal and humidity for 6 months at 25°C/60%RH and 40°C/75% RH (Temperature and Humidity chambers, Newtronic lifecare equipment Pvt. Ltd., Mumbai, India). After exposure, the samples were studied jointly with Orgalutran (Lot no. 0000906968, expiry date: 16th March 2021, Ravensburg, Germany) stored at 25°C and monitored the chemical structure of Ganirelix.
Placebo solution:
Placebo solution was prepared by dissolving an appropriate amount of acetic acid and mannitol to achieve the concentration of 0.2mg/mL acetic acid and 47mg/mL mannitol in water. The pH of the solution was adjusted to 5.0 (Seven Excellence pH meter S400, Mettler-Toledo, India) with acetic acid.
Isolation and purification of Ganirelix:
Ganirelix was fractionated on Ultra-performance Flash purification system (Model: Puriflash 430; Interchim, USA) using Interchim C18, 12g cartridge. Mobile phase contained mixture of 40% acetonitrile and 60% water (v/v). The flow rate was 15mL/min and the cartridge temperature was maintained at 25°C± 5°C. The injection volume was 3mL and the peak signals were monitored at 220nm. The chromatographic run time was 25min. The acetonitrile was evaporated present in fractions collected solution using Rotavapor R-300 evaporation system (Buchi, Switzerland) under vacuum at 25°C± 2°C. The remaining effluent was frozen at – 80°C and freeze- dried.
Fourier transform infrared (FTIR) Spectrometer:
FTIR spectroscopy has been used to estimate major functional groups in the organic molecule. Sample analysis were performed using a Nicolet iS10 FTIR Spectrometer (Thermo Scientific, India) with a Fast recovery deuterated triglycine sulfate (DTGS) detector. The sample chamber temperature was maintained at 20°C.
The potassium bromide (KBr) was dried in hot air oven (Equitron medica private limited, Model no. 7051-150-SS.AGK.140, 2000 Watts, temperature range: 25°C– 200°C, Mumbai, India) at 105°C for 60 minutes and then cooled the KBr to room temperature. Mixed 2mg of Ganirelix sample with 300mg of KBr into mortar, fully grinded the KBr and sample and pressed the mixture to obtain the sample tablet, scanned the sample tablet. FTIR scan was conducted in the wave number range of 4000 – 400cm-1 with 4 cm-1 scan resolution and 32 scans accumulation.
Ultraviolet -Visible (UV-Vis) spectrophotometer:
Sample analysis was performed using Jasco V-650 UV-Visible Spectrophotometer (Jasco international Co. Ltd. Japan) with a photomultiplier tube detector (wavelength range 190-900nm). The system was monitored with Spectra Manager software.
Blank solution was prepared by transferring 0.1mL of placebo solution into a 10mL volumetric flask, diluted with 35% (v/v) acetonitrile solution to the volume and mixed well. Sample solution was prepared by transferring 0.1mL of thermal stressed generic drug product and Orgalutran® into a separate10 mL volumetric flasks, diluted with 35% (v/v) acetonitrile solution to the volume and mixed well. UV scan was conducted in the wavelength range of 190 – 400nm.
RESULTS AND DISCUSSIONS:
The infrared spectrum region from 4000cm-1 to 1400 cm-1 shows absorption bands to a number of functional groups and is called functional group region. From the obtained IR spectra, temperature stressed generic product and reference listed drug (RLD) Orgalutran® injection have absorption bands at 3291 ± 1 cm-1, and 1632±1cm-1 which are corresponding with the stretching vibration of N-H and C=O in secondary amide. It demonstrates that the amino acids in Ganirelix Acetate in temperature stressed generic product and Orgalutran® are linked with amide bond, which is in accordance with that of the theoretical structure of Ganirelix Acetate. Furthermore, the absorptions bands at 1557±1 cm-1, 1516±1 cm-1 and 1447±1cm-1 are due to skeletal vibrations of benzene ring. The absorption band at 1404 ± 1 cm-1 is consistent with C-N stretching vibration (amide III band). The stretching vibrations at 2360 cm-1 and 2340cm-1 in IR spectrum of blank may be the interference of CO2 during test. The FTIR spectra results are mentioned in Table 1.
Table 1. Summarized data of FTIR spectra in Functional group region (4000 cm-1 to 1400 cm-1)
|
Wave number (cm-1) |
Nature of bond |
|
3291 ± 1 |
N-H (stretching vibration in secondary amide) |
|
1632 ± 1 |
C=O (stretching vibration in secondary amide) |
|
1557± 1, 1516± 1 and 1447 ± 1 |
Skeletal vibrations of the benzene ring |
|
1404 ± 1 |
C-N stretching vibration (amide III bond) |
The IR spectrum region from about 1400 cm-1 to 900 cm-1 is complex because it contains many IR bands resulting from the sum or variance of their vibrational frequencies apart from fundamental stretching and bending vibrations. Therefore, in this region, specific vibrational assignments are very difficult. Thus, this IR spectrum range is characteristic of a chemical compound and is called finger print region. Similar compound may show very similar spectra in the functional group region but certainly exhibit discernible differences in the finger print region. From the overlap, the IR spectra of the temperature and humidity stressed Ganirelix acetate finished product and the Orgalutran® demonstrates that the functional groups of Ganirelix acetate in developed finished product is similar with that of Orgalutran® in both higher wave number region (> 1400cm-1) as well as in finger print region (1400 to 900cm-1). Overlay of FTIR spectra of the Ganirelix active ingredient in Orgalutran®, thermal stressed generic drug product (25°C/60% RH-6M and 40°C/75% RH-6M) is shown in Fig. 2.
Fig. 2 Overlay of FTIR spectra of the Ganirelix active ingredient in Orgalutran® (annotated in blue), thermal stressed generic drug product (25°C/65%RH-6M) (annotated in red) and thermal stressed generic drug product (40°C/75%RH-6M) (annotated in violet).
In the ultraviolet region (190nm to 380nm), the excitation of electrons p and d orbitals, π-orbitals and particularly π conjugated systems occurs above 200 nm and gives rise to readily accessible and informative spectra. Ganirelix UV spectra has two UV maxima at wavelengths 193nm and 226nm. The overlayed UV spectra demonstrates that the UV absorption of thermal stressed Ganirelix acetate formulation sample is same as that of the Orgalutran®. The UV results are presented in Table 2. Overlay of UV spectra of the Ganirelix active ingredient in Orgalutran®, thermal stressed generic drug product (25°C/60%RH-6M and 40°C/75%RH-6M) is shown in Fig. 3.
Table 2. Summarized data of system suitability
|
Sample name |
λmax1 (nm) a |
Λmax2 (nm) b |
|
Orgalutran® (Lot no. 0000906968) c |
193.2 |
226.4 |
|
Thermal and humidity exposed generic drug product (25°C/75%RH-6M) |
193.0 |
226.2 |
|
Thermal and humidity exposed generic drug product (40°C/75%RH-6M) |
193.2 |
226.0 |
a λmax1: First maximum wavelength; b λmax2: Second maximum wavelength; a Expiry date: 16 th March 2021.
Fig. 3 Overlay of UV spectra of the Ganirelix active ingredient in Orgalutran®, thermal and humidity exposed generic drug product at 25°C/65%RH-6M and 40°C/75%RH-6M.
CONCLUSION:
The FTIR and UV-Visible spectrophotometry techniques are eminently selective for the confirmation of the Ganirelix’s chemical structure in the injectable formulation. These methods were employed successfully to demonstrate sameness of generic peptides with the reference listed drug. Comparable overlaid FTIR, UV spectral lines and finger print region in FTIR are confirming that up to 40°C and 75% RH, Ganirelix is not thermal and humidity critical with respect to the chemical structure.
ACKNOWLEDGEMENTS:
We thank Mr. Gyan Ranjan (Vice president of operations) and Dr. Ashish Dubewar (Director), Slayback pharma India LLP, Hyderabad, India, for providing ganirelix project and necessary facility to perform the work.
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Received on 20.02.2021 Modified on 13.04.2021
Accepted on 15.06.2021 © RJPT All right reserved
Research J. Pharm.and Tech 2022; 15(4):1709-1712.
DOI: 10.52711/0974-360X.2022.00286