Forced Degradation Research Articles

Analytical quality by design-based RP-HPLC method for quantification of pioglitazone and candesartan cilexetil in bilayer tablet and its forced degradation studies

Aim: The current project involves developing an RP-HPLC method for simultaneous quantification of Candesartan Cilexetil and Pioglitazone based on analytical quality by design (AQbD). Materials and methods: When analysed in the Design Expert application, the critical method parameters were systematically refined using Central Composite Design and contours were derived for significant variables. A contour plot has been used to discover the technique operable design region that governs response variation, which is then empirically tested. Results: Successful chromatographic separation of title analytes was achieved on kromasil C18 (150 × 4.6 mm, 5 µm) column at 30 °C with mobile phase comprising 60% 20 Mm Potassium dihydrogen orthophosphate and 40% acetonitrile (v/v), isocratic elution pattern, 0.9 mL/min flow rate, and UV detection at 220 nm. The linear model for Candesartan Cilexetil was from 4 to 24 µg/ mL and Pioglitazone at 7.5–45 µg/ mL, respectively. Conclusion: The method met all the ICH Q2 (R1) validation criteria. The current approach aided for analysing simultaneous drugs can be expanded into quantifying drugs in biological matrix predominance with maximum recovery.

Development and Validation of a Stability-Indicating Greener HPTLC Method for the Estimation of Flufenamic Acid

The literature on ecofriendly/greener high-performance thin-layer chromatographic (HPTLC) methods for quantifying flufenamic acid (FFA) is scant. In order to develop and validate a stability-indicating greener HPTLC densitometry assay for FFA determination in marketed products, this research was conducted. The ecofriendly eluent system was composed of ethanol–water (70:30 v/v). FFA was measured at 290 nm of wavelength. The greenness scale of suggested analytical assay was derived using “Analytical GREENness (AGREE)” methodology. The suggested stability-indicating HPTLC assay was linear for FFA determination in 25–1400 ng/band range with a determination coefficient of 0.9974. The suggested analytical assay for FFA analysis was simple, rapid, accurate, precise, robust, selective, stability-indicating, and greener. The AGREE scale for the developed stability-indicating HPTLC assay was derived to be 0.77 utilizing AGREE methodology, indicating an outstanding greenness characteristic of the suggested densitometry technique. The ecofriendly HPTLC technique was able to detect FFA degradation product under forced degradation studies, indicating its stability-indication characteristics and selectivity. The amount of FFA in marketed tablets brand A and B was determined to be 101.28 and 99.17%, respectively, indicating the suitability of the suggested analytical technique in the assay of FFA in marketed products. These results indicated that FFA in marketed products may be routinely measured using the stability-indicating greener HPTLC technique.

TLC–Densitometric Analysis of Selected 5-Nitroimidazoles

Metronidazole, ornidazole, tinidazole, and secnidazole are 5-nitroimidazoles. The purpose of this work was to propose a new economical TLC–densitometric method to evaluate the chemical stability of metronidazole, secnidazole, ornidazole, and tinidazole under stress conditions. A forced degradation study was performed on silica gel and aqueous solutions at various pH values; the metronidazole, secnidazole, ornidazole, and tinidazole solutions were prepared in saline and in hydrogen peroxide, respectively. The samples of the 5-nitroimidazoles were heated. TLC analyses were performed on silica gel 60F254 using chloroform–methanol (9:1, v/v) as the mobile phase. As the TLC–densitometric method can effectively separate the metronidazole, secnidazole, ornidazole, and tinidazole from their degradation products which formed as a result of the stress studies, it is considered to can be a good alternative and important tool in the routine quality control and stability testing of metronidazole, secnidazole, ornidazole, and tinidazole in pharmaceutical formulations. The results indicate that the proposed TLC–densitometric method is cost-effective, rapid, specific, accurate, and precise; the TLC–densitometric method also realizes the criterion of the linearity. A major advantage of the proposed method is its low cost and ability to analyze the 5-nitroimidazole which was investigated and all its degradation products simultaneously.

HPTLC-Densitometric Estimation of Anti-hypertensive Drug Combination Azilsartan Medoxomil and Cilnidipine in Combined Dosage Form

A combination of Azilsartan Medoxomil and Cilnidipine is prescribed in the treatment of Hypertension. The present work represents an accurate and precise high-performance thin layer chromatographic method for the estimation of Azilsartan Medoxomil (AZL) and Cilnidipine (CLN) in combined tablet dosage form. Pre-coated silica gel- G60 F254 aluminum sheet (100 × 100 mm, 0.2 mm layer thickness) were used as stationary phase and Ethyl Acetate: Toluene: Glacial Acetic Acid (5: 4.9: 0.1 %v/v/v) in the mixture was used as mobile phase. The method was linear in the concentration range of 200 - 2000 ng/band for AZL and 50 -500 ng/band for CLN with a correlation coefficient (r2) of 0.996 for AZL and 0.999 for CLN. The proposed method was validated with respect to linearity, accuracy, precision, and robustness as per ICH Q2 (R1) guideline. A forced degradation study was performed to assess the stability indicating the nature of the method. All the degradant peaks were well resolved from the peak of the drug without any interference. The method was successfully applied for the analysis of AZL and CLN in combined tablet formulation.

Method development and validation of Atorvastatin, Ezetimibe and Fenofibrate using RP-HPLC along with their forced degradation studies and greenness profiling

ABSTRACT The present study aimed to separate the three-drug combination and degradant products by applying stress studies in bulk and pharmaceutical using RP-HPLC green analytical chemistry principles to make the separation better and more effective without compromising the standard limits. The three drugs have separated using the Mobile Phase Mixture of 10 volumes of buffer (0.1% triethanolamine in water) and 90 volumes of ethanol on the stationary phase Inertsil ODS 3 (250 mm x 4.6 mm), 5 µm column, at a wavelength of 256 nm. The method has been performed on a linear concentration range of 12–28 µg/mL for ATR, EZB, and 192- 448 µg/mL for FNF. The forced degradation studies showed that the ATR had > 5% degradation in acid, peroxide and thermal degradation, as EZB tends to degrade more than 15% at alkali hydrolysis. Moreover, the methodology has been validated according to ICH Q2 R1 guidelines, assessed using green evaluation tools like GAPI, AES, and AGREE, and found eco-friendly according to the output of the greenness outcomes. The developed method employed for determining three drugs has shown within limits according to the guidelines, easily adaptable by the pharmaceutical industries for their regular quality control analysis of the combinations.

Persistence, sorption, and forced degradation of imidacloprid in environmental matrix

The enhanced utilization of insecticides over various crops has led the toxicity in the environmental matrix. In this study, the behavior of Imidacloprid is observed in soil and water. The adsorption–desorption characteristics of Imidacloprid are observed on three different soils (Bathinda, Moga, and Sangrur) of the Malwa Region. The soil of Bathinda has shown maximum adsorption followed by Sangrur and then by Moga soil. The reverse order is observed in the desorption process under optimum conditions of pH 6 and temperature 33 °C. The adsorption behavior is observed to be related to the mineral content of the soils. The various adsorption and kinetic models reveal the suitability of chemical adsorption. The persistence study indicates the presence of insecticide in water for more than 100 days. In the forced degradation studies, ZnO photocatalyst has shown higher degradation of Imidacloprid nearly 91 % after 5 h under UV light, whereas with TiO2, degradation has reached up to 85 % during the same period.

A new validated RP-HPLC-UV DAD method for assay of bisoprolol fumarate and related substances in tablets

The research focus of this work was development and validation of an efficient analytical method that can be used for separation and determination of related substances of bisoprolol in finished drug product using reversed-phase HPLC with UV-DAD. In a previous systematic study of various stationary phases and elution conditions it was found that several octadecylsilane phases can be successfully employed for separation of the critical pairs of bisoprolol and its related degradation products. Namely, method development led to conclusions that satisfactory resolution and peak shapes were obtained with gradient elution with water with 0.2% perchloric acid and acetonitrile and the reversed-phase columns: Hypersil 3 BDS C18 (100 x 4 mm, 3 m); Zorbax SB C18 (150 x 4.6 mm, 3.5 m); Acquity UPLC BEH C18 (50 x 2.1 mm, 1.7 m), and Xterra MS C18 (100 x 4.6 mm, 3.5 m). Since regular quality control includes the parameter related substances, which is of great importance for the overall quality of dosage forms, this study was oriented towards widening the available validated analytical methods for determination of this parameter. In this work, the results from the validation of the method for determination of related substances of bisoprolol using Zorbax SB C18 150 x 4.6 mm, 3.5 m, are presented. Validation parameters that were tested (linearity, accuracy, precision, selectivity) confirmed that the method is suitable for its intendance and it was used for characterization of the samples from a forced degradation study of bisoprolol tablets. Keywords: Bisoprolol, RP-HPLC-UV DAD, validation, related substances

Analytical Lifecycle Management (ALM) and Analytical Quality by Design (AQbD) based Analytical Method Development for Separation of Related Substances in Amodiaquine Hydrochloride along with its Degradation Products and Structural Elucidation by LC-Quadrupole-Time of Flight-Tandem Mass Spectrometry

The current study aimed to develop a robust, regulatory-flexible, stability-indicating ultra high performance liquid chromatography (UHPLC) analytical procedure compatible with mass spectrometry for the determination of impurities in amodiaquine hydrochloride using analytical lifecycle management (ALM) and analytical quality by design (AQbD) principles. The analytical target profile (ATP) and critical method attributes (CMAs) for the analytical method were identified. The pH of the mobile phase, flow rate and column oven temperature were identified as critical method parameters (CMPs) through risk assessment studies and they were screened and optimized using the design of experiments (DoE) to generate the design space (DS). Finally, all the impurities were well separated in a 15 min run time by using an Acquity UPLC BEH-C18 column with 20 mM ammonium acetate pH 8.0 as mobile phase A and acetonitrile as mobile phase B with a gradient program of time (min), % of B: 0/15, 1.0/15, 9/55, 12/55, 12.1/15 and 15/15. The flow rate was 0.3 mL min-1 and the column oven temperature were 30 ºC. Evaluated the robustness of the developed analytical method. Degradation products obtained from the forced degradation studies are well separated from process impurities and main compound. Based on the stress studies, impurity-3 was identified as a key degradation product in base degradation, while DP-1, impurity-3, DP-2, DP-3 and DP-4 were in the oxidative degradation. Total 9 impurities out of 4 process related and 5 degradation impurities were well separated in the developed method. Used the same method to LC coupled tandem mass spectrometer for separation and structure elucidation of degradation products. The method validation was carried out in accordance with ICH Q2 (R1) and USP <1225> guidelines.

STABILITY INDICATING METHOD DEVELOPMENT OF RPUPLC, VALIDATION OF SIMULTANEOUS QUANTITATION OF BUPIVACAINE and MELOXICAM IN PURE AND FORMULATION

An easy, accurate, and precise RP-UPLC was established and also validated for simultaneous quantitation of Bupivacaine and Meloxicam in the formulation as well as in pure form. An acceptable separation of Bupivacaine and Meloxicam was shown by the developed technique Bupivacaine and Meloxicam retention times were 1.359 min. and 3.687 min. correspondingly. From regression equations, LOQ and LOD were obtained and observed to be 0.2ppm and 0.6ppm - Bupivacaine, 0.06 and 0.0 I 8ppm - Meloxicam, respectively. Stability studies of the injectable dosage form under forced degradation conditions of acid, alkali, peroxide, thermal, hydrolysis, and ultraviolet rays showed that the technique was particular without interferences from any type of possible contaminations that might form over the shelf life. The newly established RP-UPLC technique was stability indicating, accurate, rapid, and sensitive, for active pharmaceutical ingredients and pharmaceutical dosage forms

Stability indicating HPLC method for the simultaneous analysis of Gatifloxacin and Loteprednol in eyedrop formulation using design of experiment approach

Design of experiment (DOE) assisted simple, rapid, precise and accurate stability indicating HPLC method has been developed for simultaneous estimation of Gatifloxacin (GTF) and Loteprednol (LOT) along with their forced degradation products. The developed method has been optimized and developed by using central composite design (CCD) in response surface methodology (RSM). Trails have been undertaken and ratio of phosphate buffer in mobile phase, pH of buffer and flow rate are selected as factors. Resolution, tailing factor (GTF) and tailing factor (LOTE) are selected for determining the system response in the process of method optimization. The responses have been optimized using the Derringer’s desirability function. The effective separation is achieved on Phenomenex EVO-C18 column (250 mm x 4.6 mm i.d, 5 μm particle size) with mobile composed of 10 mM phosphate buffer, pH 3.5 and organic phase composed of mixture of acetonitrile and methanol 60:40 % v/v, the flow rate was 1.0 mL/min, the signals were detected at 267 nm. The developed method was validated for linearity, accuracy, precision, and robustness. The method was applied successfully for stability samples.

An inclusive exploration of forced degradation analysis of some therapeutic categories of drugs in the last decade

An inclusive exploration of forced degradation analysis of some therapeutic categories of drugs in the last decade

The UHPLC-UV method applied for the forced degradation study of ixazomib and HRMS identification of its degradation products

Ixazomib is the only orally active proteasome inhibitor used in clinical practice as an anticancer drug. The novel, rapid UHPLC-UV assay for ixazomib was developed and applied to the forced degradation study followed by HRMS identification of the main degradation products. Oxidative deboronation and hydrolysis of the amid bond were found to be the principal degradation pathways. The chemical standards of the main degradation products were prepared. The method was validated for the simultaneous assay of ixazomib and its degradation products within the concentration ranges of 2.50–100.00 µg/mL (ixazomib); 0.75–60.00 μg/mL (Impurity A and B) and 1.25–60.00 μg/mL (Impurity C). The stability study revealed that ixazomib in solution is: 1) relatively stable in neutral and acidic environments, 2) its decomposition is accelerated at higher pH, 3) it is sensitive to the effects of oxidants and light, and 4) the degradation of ixazomib follows the first-order kinetics under neutral, acidic, alkaline, and UV stress. Contrary, the solid substance of ixazomib citrate was relatively resistant to heat (70 °C), heat/humidity (70 °C/75 % RH), and UV irradiation for 24 h. This study presents the first MS-compatible UHPLC method for the quantification of ixazomib and its degradation products. Furthermore, it provides data about the inherent stability and kinetics of degradation of ixazomib in a solution that may be useful in further investigation of this drug, or the development of novel proteasome inhibitors based on the ixazomib structure.

Overview on Development and Validation of Force Degradation Studies with Stability Indicating Methods

ABSTRACT: This article provides an overview on Development and Validation of Force degradation study with Stability Indicating Methods (SIMs) for drug substances. Different stress conditions (hydrolysis, oxidation, thermal, and photolytic conditions) are applied to drugs compounds during a process known as forced degradation, and as a result, various degradation products are created. The major purpose of these investigations is to assess a molecule's stability under accelerated settings. It is well recognized that the stability of molecules affects the regulatory documentation process, the choice of appropriate storage and packaging conditions, and the choice of formulation. The deficiencies of reported methods in terms of regulatory requirements are highlighted. This article is to provide appropriate International conference of harmonization (ICH) criteria for force degradation study and to discuss the methodical process for creating verified SIAMs. The aspects of Mass balance in SIMs are discussed and technique used in SIMs were highlighted. Recent advance in stability indicating methods include characterization of degrdant sample and in-vitro toxicity prediction are addressed. Background of force degradation study with stability indicating methods with respect to international author and national author are discussed.

Development and optimization of stability-indicating method of ethinylestradiol, levonorgestrel, and their main impurities using quality by design approach

The association of Ethinylestradiol 0.03 mg and Levonorgestrel 0.15 mg is a hormonal contraceptive that combines estrogen and progestogen. According to a bibliographic survey, these combined drugs present at least 18 known degradation products, which are required to control the potential impurities harmful to human health. The high number of impurities and the low concentrations of the active pharmaceutical ingredients (APIs) and their respective degradation products increase the complexity of the stability-indicating method development for this medicine. Thus, this work aimed to develop and optimize the stability-indicating method using the quality by design (QbD) approach and in-silico tools for application in samples of oral contraceptives sold in Brazil. The analysis samples were initially subjected to a forced degradation study through 7 days of exposure under acid and alkali hydrolysis, oxidative condition, and oxidation by metal ions. In addition to the chemical exposure, the sample was subjected to physical stress through 10 days of exposure under dry heat, moisture, and photolytic degradation. These exposure samples were analyzed in the development and optimization of chromatographic conditions. As a result, the developed method was able to separate 20 known substances, including the two APIs and their respective 18 degradation products, as well as unknown degradation products obtained by the forced degradation study. Finally, this stability-indicating method was successfully applied for comparative analysis of contraceptive drugs marketed in Brazil, newly purchased and subjected to accelerated stability condition at 40 °C and 75% RH over the 6-month period.

Unique Quality by Design Approach for Developing HPLC and LC-MS Method for Estimation of Process and Degradation Impurities in Pibrentasvir, Antiviral Agent for Hepatitis C.

Pibrentasvir (PIB) was approved for treating hepatitis C patients. A specific, accurate, linear, robust, and stability-indicating method was developed and validated for determining degradation impurities present in the PIB drug substance by studying the quality by design (QbD) principles. All identified degradation impurities were separated with the stationary phase HALO C18, 150 mm × 4.6 mm, 2.7 μm. Mobile phase A contains pH 2.5 phosphate buffer and acetonitrile in the ratio of (70:30, v/v), and mobile phase B contains water and acetonitrile in the ratio of (30:70, v/v), respectively. The chromatographic conditions were optimized, such as flow rate of 0.8 mL/min, UV detection at 252 nm, injection volume of 20 μL, and column temperature of 40 °C. The proposed method was validated per the current ICH Q2 (R1) guidelines. The recovery study and linearity ranges were established from limit of quantification (LOQ) to 300% optimal concentrations. The method validation results were between 98.6% and 106.2% for recovery, and linearity r 2 was more than 0.999 for all identified impurities. The method precision results achieved below 5% relative standard deviation (RSD). The forced degradation results demonstrated that the drug was sensitive to chemical stress conditions. During the stress study, degrading impurities were identified by the LC-MS technique and the mechanism pathway. A QbD-based experimental design (DoE) approach was used to establish the robustness of the method.

A Platform analytical method for intact polysorbates in protein-containing biopharmaceutical products via HPLC-CAD

Polysorbate (also known as “Tween”) 80 (PS80) is the most common surfactant in biopharmaceuticals. Here, we report a sensitive, and specific platform analytical method using reversed-phase high performance liquid chromatography employing a charged aerosol detector (HPLC-CAD), that offers the ability to quantitate intact PS80 and can distinguish between low recovery of PS80 and degraded PS80 by also monitoring several PS80 degradants which offers multiple improvements over various previously reported polysorbate analytical methods. The method employs protein/monoclonal antibody precipitation and is designed to be a quality control (QC) lab-friendly method. The method is distinguishable from other published methods because the quantification of the PS80 mono-esters is free from interference from known PS80 degradants. The method has demonstrated acceptable accuracy, precision, specificity, and sensitivity through qualification studies. The specificity afforded by the method allows for employment in forced degradation studies to discern kinetic information. Lastly, the method also has promising applications with other polysorbates (PS60, PS40, and all-laurate PS20) and shows acceptable performance with several types of biopharmaceutical proteins.

Development and validation of a stability-indicating, single HPLC method for sacubitril-valsartan and their stereoisomers and identification of forced degradation products using LC-MS/MS.

The aim of this research work was to develop and validate a stability-indicating, single reversed-phase HPLC method for the separation of five impurities, including enantiomers, diastereomers, and degradation products in sacubitril-valsartan tablets. The method was developed using a Chiralcel OJ-RH column (150 × 4.6mm, 5μm) at 45°C with a gradient program of (T/%B) 0.01/25, 10.0/25, 25/38, 37.0/45, 39.0/25, and 45.0/25 at a flow rate of 0.8ml/min. Mobile phase A consisted of 1ml of trifluoroacetic acid in 1000 ml of Milli-Q water. Mobile phase B consisted of 1ml of trifluoroacetic acid in a mixture of acetonitrile and methanol in the ratio of 950:50 (v/v). Sacubitril, valsartan, and their five impurities were monitored at 254 nm. Degradation was not observed when sacubitril-valsartan was subjected to heat, light, hydrolytic, and oxidation conditions. In acid degradation study (1N HCl/60°C/2h) impurity 1 (m/z 383.44) was formed, and in base degradation study (0.1N NaOH/40°C/1h) impurities 1 and 5 (m/z 265.35) were formed; both impurities were confirmed using LC-MS. The degradation products, enantiomers, and diastereomers were well separated from sacubitril and valsartan, proving the stability-indicating power of the method. The developed method was validated per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The inter- and intra-day percentage relative standard deviation for sacubitril, valsartan, and their five impurities was less than 5.2%, recovery of the five impurities was between 93 and 105%, and linearity was ≥0.999. The limit of detection was 0.030-0.048 μg/ml, and the limit of quantification was 0.100-0.160 μg/ml.

Development and Validation of Stability-Indicating RP-HPLC Method for the Estimation of Fisetin in Novel Cubosomal Nanoformulation: Application to the Marketed Formulation and Selected Plant Extracts

Background: Fisetin (FIS) is a bioactive flavonoid found in various plants, reported for many pharmacological activities, and presently marketed as a nutraceutical. To overcome less water solubility and bioavailability issues, FIS cubosomal nanoformulation has been prepared and characterized. Objective: To estimate FIS in prepared novel cubosomes, an RP-HPLC analytical method development with the most sensitivity, economical, robust, and wide applicability in marketed FIS formulations and plant extracts also. Method: An RP-HPLC method was developed and validated as per ICH Q2R1 guidelines by using C-18 Phenomenex Luna 5μ, 100A0 column, LC-20 AD pump, and Shimadzu LC solution 1.25 software. The combination of acetonitrile and formic acid (0.1%v/v) in the ratio of 25:75 v/v was used as a mobile phase for chromatographic separation using a PDA detector at 360 nm and a flow rate of 1 ml/min. Results: The developed method was remarkably linear in the range of 0.1 to 16 μg/ml (R2 ˃ 0.999). This method was found to be accurate (recovery 98.24 to 100.65 %), precise, robust (% RSD ˂ 2), and more sensitive than the earlier reported method with LOD and LOQ values of 17.26 and 52.31 ng/ml, respectively. The FIS estimation was also performed using the developed method in the marketed FIS formulation Doctor’s Best ® Fisetin, and different plant extracts such as strawberry, grapes, black tea, and green tea. The forced degradation study suggests that FIS was unstable in alkaline and oxidative stress conditions. Conclusion: For FIS estimation in cubosomal nanoformulation, a widely applicable, novel, robust, most sensitive, and economical RP-HPLC method was developed and validated and also applied to marketed formulations and plant extracts.

Development, Robustness by Design Expert and Validation of a Method for Enantiomeric Impurity Content Determination in Pretomanid Drug Substance and Pharmaceutical Dosage Form.

For the purpose of identifying enantiomeric impurities in the drug substance and pharmaceutical dosage forms of the novel anti-TB medication pretomanid, an RP-high-performance liquid chromatography method was devised. To ensure the robustness of the optimized approach, analytical quality by design was used. Studies on factor screening and risk evaluation helped pinpoint the critical method parameters (CMPs); resolution (R1), analyte retention time (R2) and tailing factor (R3) are those terms. Pareto charts, half-normal plots, 3D surface plots, 2D contour plots and 3D cube plots were used to study the effects of solo and interactive CMPs on critical analytical attributes. Analysis of variance (ANOVA) was used to verify the technique parameters' confirmation of significance (P = 0.05). With a Chiral Cel OJ-3R (150 × 4.6mm, 3µm) and a mobile phase consisting of 20mM of ammonium trifluoroacetate, pH= 2.5, and acetonitrile in a gradient mode, chromatographic separation was accomplished. At 30°C and 330 nm, the column's temperature and wavelength, respectively, were recorded. The procedure is stability-indicating and is LC-MS compatible. According to the International Conference on Harmonization tripartite guidelines, the method demonstrated appropriate specificity, sensitivity, linearity, accuracy, precision and robustness. The LOD and LOQ were, respectively, 0.09 and 0.3μg/mL. With a correlation coefficient of >0.990, it was discovered that the established method for enantiomeric impurity was linear over the concentration range of 0.3-2.25μg/mL. The approach exhibits adequate accuracy (%recovery = 85-115%), robustness (%RSD = 5.0) and precision (%RSD = 5.0). The method was also shown to be stability-indicating and was shown to provide effective separation in the presence of degradation products through the use of forced degradation tests.

Modeling study of long-term stability of the monoclonal antibody infliximab and biosimilars using liquid-chromatography-tandem mass spectrometry and size-exclusion chromatography-multi-angle light scattering.

Monoclonal antibodies (mAbs) represent a dynamic class of biopharmaceutical products, as evidenced by an increasing number of market authorizations for mAb innovator and biosimilar products. Stability studies are commonly performed during product development, for instance, to exclude unstable molecules, optimize the formulation or determine the storage limit. Such studies are time-consuming, especially for mAbs, because of their structural complexity which requires multiple analytical techniques to achieve a detailed characterization. We report the implementation of a novel methodology based on the accelerated stability assessment program (ASAP) in order to model the long-term stability of mAbs in relation to different structural aspects. Stability studies of innovator infliximab and two different biosimilars were performed using forced degradation conditions alongside in-use administration conditions in order to investigate their similarity regarding stability. Thus, characterization of post-translational modifications was achieved using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the formation of aggregates and free chain fragments was characterized using size-exclusion chromatography-multi-angle light scattering (SEC-MALS-UV/RI) analysis. Consequently, ASAP models were investigated with regard to free chain fragmentation of mAbs concomitantly with N57 deamidation, located in the hypervariable region. Comparison of ASAP models and the long-term stability data from samples stored in intravenous bags demonstrated a relevant correlation, indicating the stability of the mAbs. The developed methodology highlighted the particularities of ASAP modeling for mAbs and demonstrated the possibility to independently consider the different types of degradation pathways in order to provide accurate and appropriate prediction of the long-term stability of this type of biomolecule.