Forced Degradation Conditions Research Articles

Streamlined Multi-Attribute Assessment of an Array of Clinical-Stage Antibodies: Relationship Between Degradation and Stability.

Clinical antibodies are an important class of drugs for the treatment of both chronic and acute diseases. Their manufacturability is subject to evaluation to ensure product quality and efficacy. One critical quality attribute is deamidation, a non-enzymatic process that is observed to occur during thermal stress, at low or high pH, or a combination thereof. Deamidation may induce antibody instability and lead to aggregation, which may pose immunogenicity concerns. The introduction of a negative charge via deamidation may impact the desired therapeutic function (i) within the complementarity-determining region, potentially causing loss of efficacy; or (ii) within the fragment crystallizable region, limiting the effector function involving antibody-dependent cellular cytotoxicity. Here we describe a transformative solution that allows for a comparative assessment of deamidation and its impact on stability and aggregation. The innovative streamlined method evaluates the intact protein in its formulation conditions. This breakthrough platform technology is comprised of a quantum cascade laser microscope, a slide cell array that allows for flexibility in the design of experiments, and dedicated software. The enhanced spectral resolution is achieved using two-dimensional correlation, co-distribution, and two-trace two-dimensional correlation spectroscopies that reveal the molecular impact of deamidation. Eight re-engineered immunoglobulin G4 scaffold clinical antibodies under control and forced degradation conditions were evaluated for deamidation and aggregation. We determined the site of deamidation, the overall extent of deamidation, and where applicable, whether the deamidation event led to self-association or aggregation of the clinical antibody and the molecular events that led to the instability. The results were confirmed using orthogonal techniques for four of the samples.

Analytical quality by design aided stability indicating a robust ultra‐performance liquid chromatographic technique for the quantification of sonidegib and its organic impurities in bulk drug substance

AbstractA simple quality by design aided stability indicating method was developed for quantification of sonidegib (SONI) and its process related impurities using on ultra‐performance liquid chromatography in bulk drug substance. AutoChrom and Design‐Expert software were used to predict physicochemical properties, draw Ionization graphs, and generate analytical target profile. SONI was subjected to forced degradation conditions, such as oxidative, acid hydrolysis, base hydrolysis, hydrolytic, thermal, and photolytic hydrolysis. All degradation products and process contaminants were separated using an Acquity Ethylene Bridged Hybrid C18 column in gradient elution mode with a mobile phase containing 0.02 M ammonium acetate buffer and acetonitrile: methanol (80:20 v/v). The predicted physicochemical properties are accurate and they facilitated for selection of robust conditions in development of chromatographic method with minimal trials. The developed method can be used for quantification of drug and its process related impurities in bulk drugs.

A stability-indicating liquid chromatography - diode array detection method for quantification of febuxostat in pharmaceuticals: a recent application to chemometrics based method development and comparative greenness assessment

With an increased demand for anti-gout drugs, the need for a quality-controlled drug product also arises. In this context, a new liquid chromatography method was developed for quantifying febuxostat that integrates chemometrics principles and assesses the method's eco-friendly nature. Systematized risk assessment identified that organic proportion, flow rate, and pH are the critical method variables and were subsequently optimized using a Box-Behnken design. The validation studies were performed using the optimized chromatographic conditions of 80% organic solvent, 1.0 mL/min flow rate, and a pH value of 6.8 on a C-18 column. The analyte eluted at 2.2 min and was monitored at 262 nm using photodiode array detection. After applying the forced degradation conditions, febuxostat degraded from 1.32 to 11.98% of its initial content. Statistical evaluation found that accuracy, precision, detection and quantitation limits, solution stability, and system suitability were acceptable. The lack of interfering degradation peaks ensured the stability-indicating nature of the method and method specificity. The application of three greenness metrics tools, such as the Green Analytical Procedure Index (GAPI), Analytical Greenness (AGREE) metrics, and analytical eco-scale (AES), revealed excellent method greenness. The current chromatographic method is apt for the routine quality control of febuxostat present in pharmaceutical formulations.

Identification, characterisation and in silico ADMET prediction of ozenoxacin and its degradation products using high‐performance liquid chromatography‐photodiode array and liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectrometry

RationaleOzenoxacin (OXC) is an antibiotic used topically to treat impetigo. This study aimed to evaluate the degradation products (DP) of OXC drug substance under different stress conditions, including hydrolysis, oxidation, thermal and photolysis, in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q1A(R2) and Q1B. The analytical technique was validated in compliance with ICH Q2(R1) requirements.MethodsThe drug substance underwent degradation under various forced degradation conditions, including thermal, oxidative, photolytic and hydrolytic (neutral, acidic and basic) degradation. Overall, four DPs were formed under oxidative stress conditions with AIBN. The formed DPs were identified and separated using a Shimadzu LC system with a reversed‐phase Phenomenex Kinetex C18 column (4.6 × 250 mm, 5 μm), using 10 mM NH4CH3COOH buffer (pH −5.0) as mobile phase A and acetonitrile as mobile phase B at a detection wavelength of 254 nm.Results and ConclusionThe drug was found to be stable in neutral, acidic, basic and oxidative degradation conditions with hydrogen peroxide. Liquid chromatography‐electrospray ionisation‐quadrupole time‐of‐flight‐tandem mass spectrometry‐ was employed in positive ionisation mode to analyse both the drug and the mass of the identified DP. The mechanism and the pathway of mass fragmentation have been proposed. The developed method was accurate, repeatable, linear and selective for further research. The ADMET Predictor software was applied to predict the in silico toxicity of the drugs and its DPs as well as their physicochemical characteristics.

Development of potentiometric immunosensor for determination of live attenuated Varicella Vaccine: Potency and stability studies

Determination of Varicella vaccine's potency; containing live attenuated strain (Oka) of Varicella Zoster virus, has been limited to in vitro cell culture methods. In this study, a label free potentiometric biosensor has been developed for the first time and optimized to determine the content of varicella zoster virus. A passive ion-flux sensing platform has been developed using an anti-varicella monoclonal antibody and tetrabutyl ammonium bromide as a marker ion. The immunosensor has been optimized with respect to membrane diameter and concentration of the immobilized antibody. Linearity was achieved over a concentration range of 2.5–3.2 log PFU/dose with a LOD of 1.9 log PFU/dose. Potentiometric results were compared to the plaque-forming assay using the cell culture technique. The developed immunosensor was superior with respect to analysis time and cost without affecting critical analytical performance characteristics. Furthermore, in order to evaluate the stability indicating ability of the immunosensor, the effect of pH and temperature was investigated. Vaccine samples were subjected to forced degradation conditions; pH and elevated temperatures. Stability results showed the ability of immunosensor to differentiate between intact and degraded viral content. This would demonstrate the reliability of the immunosensor for evaluating the efficacy and stability of the vaccine.

Application of NMR and Chemometric Analyses to Better Understand the Quality Attributes in pH and Thermally Degraded Monoclonal Antibodies

PurposeNuclear magnetic resonance (NMR) spectroscopy provides the sensitivity and specificity to probe the higher order structure (HOS) of monoclonal antibodies (mAbs) for potential changes. This study demonstrates an application of chemometric tools to measure differences in the NMR spectra of mAbs after forced degradation relative to the respective unstressed starting materials.MethodsSamples of adalimumab (Humira, ADL-REF) and trastuzumab (Herceptin, TRA-REF) were incubated in three buffer-pH conditions at 40°C for 4 weeks to compare to a control sample that was left unstressed. Replicate 1D 1H and 2D 1H-13C HMQC NMR spectra were collected on all samples. Chemometric analyses such as Easy Comparability of HOS (ECHOS), PROtein FIngerprinting by Lineshape Enhancement (PROFILE), and Principal Component Analysis (PCA) were applied to capture and quantitate differences between the spectra.ResultsVisual and statistical inspection of the 2D 1H-13C HMQC spectra of adalimumab and trastuzumab after forced degradation conditions shows no changes in the spectra relative to the unstressed material. Chemometric analysis of the 1D 1H NMR spectra shows only minor changes in the spectra of adalimumab after forced degradation, but significant differences in trastuzumab.ConclusionThe chemometric analyses support the lack of statistical differences in the structure of pH-thermal stressed adalimumab, however, it reveals conformational changes or chemical modifications in trastuzumab after forced degradation. Application of chemometrics in comparative NMR studies enables HOS characterization and showcases the sensitivity and specificity in detecting differences in the spectra of mAbs after pH-thermal forced degradation with respect to local and global protein structure.

Pharmaceutical Forced Degradation (Stress Testing) Endpoints: A Scientific Rationale and Industry Perspective

Forced degradation (i.e., stress testing) of small molecule drug substances and products is a critical part of the drug development process, providing insight into the intrinsic stability of a drug that is foundational to the development and validation of stability-indicating analytical methods. There is a lack of clarity in the scientific literature and regulatory guidance as to what constitutes an “appropriate” endpoint to a set of stress experiments. That is, there is no clear agreement regarding how to determine if a sample has been sufficiently stressed. Notably, it is unclear what represents a suitable justification for declaring a drug substance (DS) or drug product (DP) “stable” to a specific forced degradation condition. To address these concerns and to ensure all pharmaceutically-relevant, potential degradation pathways have been suitably evaluated, we introduce a two-endpoint classification designation supported by experimental data. These two endpoints are 1) a % total degradation target outcome (e.g., for “reactive” drugs) or, 2) a specified amount of stress, even in the absence of any degradation (e.g., for “stable” drugs). These recommended endpoints are based on a review of the scientific literature, regulatory guidance, and a forced degradation data set from ten global pharmaceutical companies. The experimental data set, derived from the Campbell et al. (2022) benchmarking study,1 provides justification for the recommendations. Herein we provide a single source reference for small molecule DS and DP forced degradation stress conditions and endpoint best practices to support regulatory submissions (e.g., marketing applications). Application of these forced degradation conditions and endpoints, as part of a well-designed, comprehensive and a sufficiently rigorous study plan that includes both the DS and DP, provides comprehensive coverage of pharmaceutically-relevant degradation and avoids unreasonably extreme stress conditions and drastic endpoint recommendations sometimes found in the literature.

Validation of stability-indicating LC method, degradation study, and impact on antioxidant and antifungal activities of Eugenia uniflora leaves extract.

The aim of this study is to demonstrate the stability-indicating capacity of an analytical method for Eugenia uniflora, enhance understanding of the stability of myricitrin, and assess the effect of degradation of spray-dried extract (SDE) on antioxidant and antifungal activities. Validation of the stability-indicating method was carried out through a forced degradation study of SDE and standard myricitrin. The antioxidant and antifungal activities of SDE were evaluated both before and after degradation. The quantification method described was found to be both accurate and precise in measuring myricitrin levels in SDE from E. uniflora, with excellent selectivity that confirmed its stability-indicating capability. The forced degradation study revealed that the marker myricitrin is sensitive to hydrolysis, but generally stable under other stress conditions. By contrast, the standard myricitrin displayed greater susceptibility to degradation under forced degradation conditions. Analysis of the antioxidant activity of SDE before and after degradation showed a negative impact in this activity due to degradation, while no significant effect was observed on antifungal activity. The method described can be a valuable tool in the quality control of E. uniflora, and the findings can assist in determining the optimal conditions and storage of products derived from this species.

A Different Perspective on the Characterization of a New Degradation Product of Flibanserin with HPLC-DAD-ESI-IT-TOF-MSn and its Pharmaceutical Formulation Analysis with Inter-Laboratory Comparison.

Flibanserin (FLB) was firstly synthesized as an antidepressant drug; however, due to its enhancing effects on sexual activity, it was approved for treatment of hypoactive sexual desire disorder in women in 2015. The aim of this study was to develop a new and fully validated HPLC method for analysis of FLB in pharmaceutical formulations besides its degradation products, and identification of possible formation mechanisms by using HPLC-DAD-ESI-IT-TOF-MSn. The HPLC separation was achieved in an Supelco Ascentis® Express series phenyl hexyl column (100 × 4.6 mm, ID 2.7µm). The mobile phase was acetonitrile-ammonium acetate solution (50:50, v/v, 10 mM, pH 5.4) mixture, which was pumped at the rate of 0.5 mL/min. Chromatography, detection and structural identification was performed by using LCMS-IT-TOF instrument (Shimadzu, Japan). 1-(2-(4-(3-hydroxy-5-(trifluoromethyl)phenyl)piperazine-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one is proposed as a novel degradation product, with a mass of 407.1695, and a formula of C20H21F3N4O2 with a margin of error about 0.001 ppm. The developed method is applicable with 98% accuracy within 2.5-50.0 µg/mL range. The LOD and LOQ was about 500 ng/mL and 1.50 µg/mL, respectively. The transferability and variation between laboratories were tested by inter-laboratory comparison and evaluated with one-way analysis of variance. A novel FLB degradation product, which was produced under oxidative forced degradation condition was observed and identified for the first time; in addition, the formation kinetics of the degradation product, besides decomposition of FLB was studied. Furthermore, an inter-laboratory comparison was carried out and application of the proposed method on pseudo Addyi® sample was tested using both instrument configurations. A novel stability indicating assay method was developed and fully validated according to ICH (Q2)R1 for the analysis of FLB in the pharmaceutical preparations. A new degradation product was identified in the oxidative forced degradation condition and characterized using HPLC-DAD-ESI-IT-TOF-MS3. Moreover, the possible mechanism and the formation kinetic of the degradation product were revealed. In addition, the developed method was transferred to another LC-PDA instrument for inter-laboratory comparison. Finally, the current method was applied to pseudo formulation of Addy® in both instruments and ANOVA was applied for evaluation.

Degradants of Tenofovir Disoproxil Fumarate Under Forced Yet Mild Thermal Stress: Isolation, Comprehensive Structural Elucidation, and Mechanism.

In addition to understanding the mechanism of action for a specific drug candidate, information regarding degradation pathways/products under various stress conditions is essential to know about their short- and long-term effects on health and environment. In line with that, tenofovir disoproxil fumarate (TDF, a co-crystal form of the prodrug tenofovir with fumaric acid), particularly used as an antiretroviral drug for treatment of HIV and hepatitis-B among others, is subjected to primarily thermal and other ICH-prescribed forced degradation conditions and their various degradation products are identified. Upon thermal degradation at 60°C for 8h, five different degradants (namely DP-1 to DP-5) are isolated, and their structures are unambiguously confirmed using advanced analytical and spectroscopic techniques including ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), high-resolution mass spectrometry (HRMS), state-of-the-art 1- and 2-dimensional nuclear magnetic resonance (1D and 2D NMR), and Fourier-transform infrared spectroscopic (FT-IR) techniques. Among fully characterized five degradants, two new degradants (DP-2 and DP-4) are identified which can potentially impact the stability of TDF via different pathways. Plausible mechanisms leading to all five thermal degradation products are also proposed including the generation of carcinogenic formaldehydefor some cases. The present systematic structural study especially combining MS and advanced NMR investigations unequivocally confirms the structures of the degradants and opens opportunities for connecting the various degradation pathways especially for the TDF-related pharmaceutical candidates.

Identification and enrichment of a UV-induced degradant of Anagrelide drug substance

Anagrelide (ANG) is a widely used drug for the treatment of essential thrombocytosis and myeloproliferative neoplasms. Recently, a new oxidative degradant was identified when the drug product capsule underwent stress testing. Full structural characterization of this previously unidentified degradant was conducted. Preliminary LC-MS analysis indicated the targeted degradant as a mono-oxygenated product of ANG. For the purpose of facile isolation and purification, various forced degradation conditions were screened to enrich the desired degradant, among which, pyridinium chlorochromate (PCC)-treatment effectively afforded a yield of 55 % unknown degradant. Following isolation by prep-HPLC, 1D and 2D NMR studies and HRMS characterization assigned the products as a pair of 5-hydroxy-Anagrelide (5-OH-ANG) enantiomers. A plausible mechanism of formation is proposed.

A Validated, Stability-Indicating, Eco-Friendly HPTLC Method for the Determination of Cinnarizine

The eco-friendly high-performance thin-layer chromatographic (HPTLC) approaches for measuring cinnarizine (CIN) are scant in reported databases. As a result, the current work has developed and validated an eco-friendly HPTLC technique for assessing CIN in commercial formulations. The proposed approach was based the use of ethyl alcohol-water (90:10 v/v) as the eco-friendly mobile phase. A wavelength of 197 nm was used to detect CIN. The greenness score of the current approach was measured using the Analytical GREENness (AGREE) approach. The current approach was linear for CIN measurement in 50–800 ng band−1 range. The current approach for CIN measurement was validated successfully using ICH guidelines and was found to be linear, accurate (% recovery = 99.07–101.29%), precise (% CV = 0.80–0.95%), robust, sensitive (LOD = 16.81 ng band−1 and LOQ = 50.43 ng band−1), specific, selective, stability-indicating, and eco-friendly. The AGREE score for the current approach was calculated to be 0.80, showing an excellent greenness characteristic of the present approach. Under forced degradation conditions, the current approach was successful in separating the CIN degradation product, demonstrating the stability-indicating qualities/selectivity of the present approach. The % assay of CIN in commercial tablet brands A and B was found to be 98.64 and 101.22%, respectively, suggesting the reliability of the present approach in the pharmaceutical analysis of CIN in commercial dosage forms. The obtained findings indicated that CIN in commercial formulations could be routinely determined using the current approach.

Analytical Quality by Design-Assisted HPLC Method for Quantification of Canagliflozin and Stability Studies.

The present study is intended to develop the high-performance liquid chromatography (HPLC) method for the analysis of Canagliflozin using the analytical quality by design (AQbD) approach. The key parameters were methodically optimized with the help of factorial experimental design, and contours were plotted when investigated using Design Expert software. A stability-indicating HPLC technique was developed and validated for the quantitative estimation of Canagliflozin, and its stability was assessed using various forced degradation conditions. Successful separation of Canagliflozin was accomplished using a Waters HPLC system with a photo diode array (PDA) detector and Supelcosil C18 column (250 × 4.6 mm, 5 μm) and 0.2% v/v solution of trifluoroacetic acid in water/acetonitrile (80:20% v/v) as the mobile phase maintaining the flow rate at 1.0 mL/min. The detection wavelength was 290 nm, and Canagliflozin got eluted at 6.9 min with a run time of 15 min. Canagliflozin peak purity values in all degradation conditions indicated that the peak is homogeneous, and therefore this method can be considered stability-indicating. The proposed technique was found to be specific, precise (% RSD about 0.66%), linear (12.6-37.9 μg/mL), rugged (overall % RSD about 0.50%), and robust. The standard and sample solutions were stable after 48 h (cumulative % RSD about 0.61%). The developed AQbD-based HPLC method can be used for the assay of Canagliflozin in Canagliflozin tablets of regular production batches and stability samples.

Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary electrophoresis coupled to mass spectrometry (mCZE-MS).

Fragmentation of therapeutic proteins is a potential critical quality attribute (CQA) that can occur in vivo or during manufacturing or storage due to enzymatic and non-enzymatic degradation pathways, such as hydrolysis, peroxide mediation, and acid/metal catalysis. Characterization of the fragmentation pattern of a therapeutic protein is traditionally accomplished using capillary gel electrophoresis with UV detection under both non-reducing and reducing conditions (nrCGE and rCGE). However, such methods are incompatible with direct coupling to mass spectrometry (MS) due to the use of anionic surfactants, e.g., sodium dodecyl sulfate (SDS). Here, we present a novel method to characterize size-based fragmentation variants of a new biotherapeutic kind using microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS) to determine the molecular masses of fragments. A new modality of immuno-oncology therapy, bispecific antigen-binding biotherapeutic, was chosen to investigate its fragmentation pattern using mCZE-MS for the first time, according to our knowledge. Bispecific antigen-binding biotherapeutic samples from different stages of downstream column purification and forced degradation conditions were analyzed. The results were cross-validated with denaturing size-exclusion chromatography-mass spectrometry and conventional rSDS-CGE. In this study, we demonstrated that mCZE-MS could separate and characterize 12-40 kDa bispecific antigen-binding biotherapeutic fragments rapidly (within ≤12 minutes), with higher resolution and better sensitivity than traditional LC-MS methods.

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

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.

Analytical comparability demonstrated for an IgG4 molecule, inclacumab, following transfer of manufacturing responsibility from Roche to Global Blood Therapeutics

ABSTRACT Background Inclacumab is a recombinant, fully human, immunoglobulin IgG4 monoclonal antibody that selectively binds to P-selectin. Initially discovered and developed by Roche through phase 2 clinical studies in peripheral arterial disease and coronary artery disease, inclacumab has been in-licensed by Global Blood Therapeutics (GBT) as a potential treatment to reduce the frequency of vaso-occlusive crises in individuals with sickle cell disease. Research design and methods GBT sought to demonstrate the analytical comparability between material produced by Roche and material produced by GBT to ensure that no meaningful differences in identity, safety, purity, potency, or bioavailability exist between the GBT and Roche lots. Results Inclacumab samples produced by GBT were found to be comparable to the Roche v0.2 inclacumab samples based on (1) comparable primary and higher-order structures; (2) comparable purity profiles; (3) comparable potency, in vitro functional activities, and in vivo plasma exposures and pharmacokinetic profiles; and (4) comparable degradation patterns and kinetics under forced degradation conditions. Conclusions Based on the design of this comparability study and the results obtained, the US Food and Drug Administration approved the changes to the manufacturing process and gave clearance for GBT to proceed with phase 3 clinical trials.

Characterization of outcomes of amino acid modifications using a combinatorial approach to reveal physical and structural perturbations: A case study using trastuzumab biosimilar

Biopharmaceuticals, such as monoclonal antibodies, are considered as life-saving drugs for autoimmune diseases, cancer and infectious diseases. However, biotherapeutics tend to undergo chemical degradation during various stages of manufacturing. The conditions of chemical degradation, along with the physical degradation pathways, have a direct influence on the overall stability, safety and efficacy of these therapeutics. While site-specific chemical changes have been well-explored and investigated using various analytical approaches, the resulting conformational and structural changes have not been much studied. Thus, we explored various biophysical techniques for assessing the influence of three representatives forced degradation conditions viz. oxidation, deamidation, and glycation, in a model therapeutic trastuzumab biosimilar. The site-specific modifications caused by these stress conditions were analysed using high resolution mass spectrometry. While their thermodynamic and conformational consequences were investigated by using differential scanning colorimetry (Nano-DSC), circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), and dynamic light scattering (DLS). The investigated stress conditions resulted in reduced thermodynamic stability of mAb, as confirmed using Nano-DSC. Secondary structure analysis performed with CD spectroscopy indicated detectable structural alterations in the beta sheets of stressed samples. DLS and SV-AUC studies demonstrated an enhanced level of aggregation and fragmentation in presence of all stress conditions. Thus, the biophysical analytical toolkits, when used simultaneously, could offer deeper insights into the subtle conformational changes that result from site-specific chemical modifications in mAbs. Hence, these analytical approaches may serve as significant additions to the battery of techniques used for forced degradation analysis of biopharmaceuticals.

Stability-indicating HPLC method development and validation of rivaroxaban impurities and identification of forced degradation products using LC-MS/MS.

The current aim of this study is to develop a simple, sensitive, and robust analytical method that can separate the Rivaroxaban and its related impurities by using HPLC. This new method can separate enantiomers and all the process-related impurities of rivaroxaban. This method can also be used to determine the assay of rivaroxaban in drug substances and drug products. This new method was developed using a Chiralpak IC (250 × 4.6mm, 5μm) column at 27°C with a gradient program of 25.0min at a flow rate of 0.8mL/min. Mobile phase A consisted of acetonitrile, ethanol, and n-butyl amine in the ratio of 95:5:0.5 (v/v/v), respectively. Mobile phase B consisted of a mixture of Milli-Q water, methanol, and n-butyl amine in the ratio of 50:50:0.5 (v/v/v), respectively. The detector wavelength was 254 nm. Rivaroxaban was subjected to the forced degradation conditions (stress conditions) of hydrolysis, base, acid, thermal, photolysis, and oxidation. The degradation products were well separated from rivaroxaban peak and enantiomer peak, and its potential impurities prove the stability-indicating power of the method. The proposed new stability-indicating method was validated with respect to specificity, precision, accuracy, limit of quantification, limit of detection, linearity robustness, and roundness per International Conference on Harmonization guidelines. The %RSD (relative standard deviation) for six sample preparations for rivaroxaban and impurities less than 10% and the recovery is between 90 and 110%. The current method can be used in quality control labs for analysis of commercial products.

Identification and characterization of degradation products of Nateglinide

Nateglinide (NTG) was evaluated under a variety of ICH-recommended forced degradation settings and found to be degraded under acid and alkali hydrolysis but stable under other forced degradation conditions. Under acid hydrolytic conditions, three degradation impurities (I-III) were produced. All three contaminants were separated using a C-18 column with ammonium acetate buffer (20 mM), methanol, and acetonitrile as mobile phases provided in isocratic mode, and characterisation was done utilising +ESI-MSn and LC-MS-QTOF investigations. All three degrading impurities (I-III) were discovered to be novel. The most likely pathways of deterioration were presented and debated.