Impurity Profile Research Articles

Разработка методики хроматографического разделения и установление профиля примесей для 5-(5-трифторметил-изоксазол-3-ил]фуран-2-сульфонамида – лекарственного кандидата для лечения глаукомы

Glaucoma is the leading cause of irreversible blindness. Early diagnosis and effective treatment can reduce the progression of the disease and prevent irreversible decay of visual functions. Indeed, human carboanhydrase isoform II is a classical target for glaucoma treatment. We proposed a compound molecule 5 (5 trifluoromethyl-isoxazol-3-yl)furan-2-sulfonamide under the project name B016, which showed high activity as a human carboanhydrase II inhibitor by in vitro tests. Our team is currently conducting development and preclinical studies of a medicinal product in the eye drops form based on this compound. One of the stages of pharmaceutical research is the development of quality control methods for both the pharmaceutical substance and the finished dosage form. We have developed a chromatographic separation procedure, which can be used for quality control of active pharmaceutical substance by quantification indicators. We also determined the impurity profile, i.e. the structure of the impurities found in the reaction product of compound B016.

Assessing the performance of new chromatographic technologies for the separation of peptide epimeric impurities: the case of Icatibant

The biopharmaceutical industry faces the challenge of efficiently characterising impurity profiles of therapeutical peptides, also due to their complex polar and ionisable attributes. This research explores the potential of advanced chromatographic techniques to address this challenge. The study compares dynamic electrostatic repulsion reversed phase (d-ERRP) to its counterparts (static ERRP and ion pair reversed phase IP-RP) in analysing Icatibant and its elusive epimeric impurity, [L-Arg]1-Icatibant and highlights its exceptional capabilities in generating symmetric peaks, mitigating the common tailing phenomenon, and serving as a steadfast guardian of column longevity. The result highlights d-ERRP as a pioneering tool in the domain of liquid chromatography, fostering its role as a reference technique for the analysis of therapeutic peptides.

Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification

Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual-binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size.Graphical abstract

Development of multiple heartcutting two-dimensional liquid chromatography with ion-pairing reversed-phase separations in both dimensions for analysis of impurities in therapeutic oligonucleotides

Oligonucleotides constitute an emerging and highly complex bioanalytical challenge and it is becoming increasingly clear that 1D methodologies are unable to fully resolve all possible impurities present in these samples. 2D-LC therefore constitutes a perfect solution wherein critical pairs can be sampled from a steep gradient 1D and separated in a shallower 2D gradient. Herein, we provide a facile 2D-LC method development approach to quickly generate high selectivity gradients utilizing ion pairing reverse phase (IPRP-IPRP). In particular we demonstrate how to iteratively generate a 12 % gradient from two training runs and then to utilize that data to predict retentions of analytes with a 2 % gradient with retention prediction errors as low as 3 and 11 %, respectively. This iterative method development workflow was applied to impurity profiling down to 1:1000 for the full-length product and phosphorothioate modified impurities. Additionally, we demonstrated the elucidation of critical pairs in complex crude pharmaceutical oligonucleotide samples by applying tailored high selectivity gradients in the second dimension. It was found that the iterative retention modeling approach allows fast and facile 2D-LC method development for complex oligonucleotide separations.

Exciting Advances in Sustainable Spectrophotometric Micro-Quantitation of an Innovative Painkiller "Tramadol and Celecoxib" Mixture in the Presence of a Toxic Impurity, Promoting Greenness and Whiteness Studies.

Tramadol (TRM) and celecoxib (CLX) form a novel mixture that helps relieve acute pain when other painkillers have no action. It is also reported that these drugs, TRM and CLX, are used to control COVID-19 symptoms. The current work highlights three important pillars of modern pharmaceutical analysis, which are as follows; impurity profiling, greenness/whiteness studies and simplicity accompanied by sensitivity. Since 4-methyl acetophenone inhibits the human carbonyl reductase enzyme (type I) and since this compound may pose a health risk, it is crucial to regulate its concentration in all dosage forms of CLX. Two simple and green spectrophotometric methods were developed, namely third derivative (D3) and Fourier self- deconvulation (FSD), for resolving severely overlapped spectra of TRM and CLX in the presence of 4-methyl acetophenone (4-MAP) as a process-related impurity in their novel tablet combination. The two approaches showed acceptable linearity with an excellent correlation coefficient. In both methods, TRM was measured when CLX and 4-methyl acetophenone were zero-crossing. The same procedure was applied for measuring CLX and its process-related impurity 4-MAP. The methodologies developed were thoroughly validated in compliance with ICH (International Council on Harmonisation) guidelines. Student t- and F-tests revealed no statistically significant variation among the current methods and the reported method. No spectrophotometric methods have been published previously for the simultaneous analysis of TRM and CLX along with 4-MAP. As a result, the newly developed spectrophotometric approaches have great relevance and originality in the field of pharmaceutical analysis.

Development of an Efficient Process for a Diagnostic Test Agent (Bentiromide) and Identification, Synthesis, Characterization, and Control Strategy for Potential Impurities

Abstract: The latest development studies provide a more effective and suitable process for Bentiromide (1), a diagnostic test substance. The enhanced procedure includes process modifications to get a better yield (76%) and high purity (above 99.8%) of Bentiromide (1). The current studies also outline a commercially viable method for removing important process-related impurities (A, B, C, D, E, F & G) in Bentiromide (1). During the impurity profile research, seven critical process-related impurities were found. These impurities were identified using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) techniques. The synthesis schemes and spectral analysis data (MS, IR, 1H NMR, and 13C NMR) were used to characterize and confirm these impurities' structures. All of these impurities sources were investigated, identified, and strict control measures were taken to reduce them to an acceptable level.

Overcoming drug impurity challenges in amorphous solid dispersion with rational development of biorelevant dissolution-permeation method

Hot-melt extrusion is often used to prepare amorphous solid dispersion to overcome low drug solubility and enhance bio-performance of the formulation. Due to the uniqueness of each drug – polymer combination and its physico-chemical properties, setting the appropriate HME barrel temperature, feed rate and screw speed ensures drug amorphization, absence of residual crystallinity, absence of water, and a suitable drug release profile. In this research, samples with BCS II/IV model drug and PVP/VA polymer were prepared to evaluate the impact of HME process parameters, incoming drug form (anhydrous vs. hydrate), and drug supplier (i.e., impurity profile), on biorelevant drug release. This study provides a relationship between observed in vitro supersaturation and precipitation behavior of amorphous solid dispersion formulation with in vivo results, on patients, by using the acceptor profile of side-by-side dissolution-permeation apparatus.An in vitro dissolution method, in small volumes, in an apparatus with paddles and dissolution-permeation side-by-side method was developed on the MicroFlux™ apparatus to assess if the differences observed in vitro bears relevance to the bioequivalence outcome in vivo. The former was used to guide the generic drug product development due to high discriminatory strength, while the latter was biorelevant, due to the inclusion of the second compartment assuring absorptive environment to capture the impact of supersaturation and subsequent precipitation on bioavailability. Bio-relevancy of the in vitro method was confirmed with the in vivo dog study and clinical study on patients, and an in vitro – in vivo correlation was established.For the investigated BCS II/IV drug, this research highlights the importance of considering supersaturation and formation of colloidal species during amorphous solid dispersion release testing to assure product quality, safety and efficacy.

A study of impurity profiling via method development and force degradation in hydrocortisone butyrate at low concentration

BackgroundSince long, triamcinolone and fluocinolone, fluorinated derivatives, have been used for treating various types of dermatitis. Rosacea and perioral dermatitis are the most common side effects observed with prolonged use of fluorinated derivatives. Recent studies have shown that these adverse effects are more severe at low doses of fluorinated derivatives compared to low doses of non-fluorinated derivatives. Therefore, this study focused on impurity profiling through force degradation studies of hydrocortisone butyrate in a 0.1% lotion.ResultsA precise and robust method with low concentration was established for the estimation of hydrocortisone butyrate in bulk and formulations was done using the RP-HPLC technique. The mobile phase consisted of a combination of acetonitrile and purified water (30:70% v/v) and acetonitrile and purified water (95:5% v/v), with detection at a wavelength of 254 nm and a total run time of 20 min. The method demonstrated linearity and accuracy within the concentration range of 0.1–250 µg/mL and 50–125 µg/mL, respectively, with an r2 value of 0.999. Stress stability studies were conducted on hydrocortisone butyrate, revealing 11% degradation in alkaline conditions and 18% degradation in photolytic conditions.ConclusionThe established method can be commercially used as it exhibits excellent linearity. Impurities were identified by injecting the reference standard, and their retentions were confirmed. The identified impurities included hydrocortisone, hydrocortisone-21-butyrate, hydrocortisone 3-methyl enol ether 17-butyrate, and hydrocortisone 17, 21-methylorthobutyrate, with retention times of 2.89, 9.14, 13.70, and 16.25 min, respectively. This precise method can be utilized in commercial applications for the accurate identification of hydrocortisone butyrate at low concentrations.

Separation and quantification of organic‐related impurities of beta‐adrenergic receptor blocking agent propranolol in pharmaceutical solid dosage forms: Impurity profiling using stability‐indicating HPLC method

AbstractPropranolol hydrochloride (PPH) is a medication of beta‐adrenergic receptors. It is used to treat pediatric hemangiomas that are growing and need systemic therapy. A reversed‐phase high‐performance liquid chromatography (HPLC) was made to separate and estimate the amounts of ten known organic impurities of propranolol in bulk drugs, tablets, and capsule dosage forms. The chromatographic separation was achieved on a C18 column (100 × 4.6 mm, 2.7 μm) using a binary gradient mixture of pH 2.3 phosphate buffer and organic modifiers of acetonitrile, methanol, and isopropyl alcohol as the mobile phase. The stability‐indicating character of the proposed method was proven using stress testing study. The test method was validated for specificity, limit of detection, limit of quantitation, linearity, precision, accuracy, and robustness. For the propranolol and its ten organic impurities, the limit of quantitation, linearity, and recoveries were found in a range of 0.0123–0.4266 μg/mL (R2 > 0.9982) and 89.2–98.9%, respectively. The proposed liquid chromatography method is found to be highly suitable for impurity profiling of propranolol in bulk drugs and pharmaceutical formulations (tablets and capsules).

Process intensification in biopharmaceutical process development and production – an industrial perspective

Abstract Process intensification aims to increase productivity in biologics manufacturing. Significant progress has been made in academia, the biopharmaceutical industry, and by the regulatory guidance since the 2000s. Process intensification can include all unit operations of a drug substance manufacturing process. The applied upstream concepts have consequences on the downstream process (DSP). The DSP process must manage larger product amounts while ensuring the required quality and impurity profiles, and cope with the available time frame as per scheduling requirements in a facility. Further, intensification in DSP is not based on a single technology only but rather on various technologies. This contribution provides an industry perspective on process intensification, describing basic concepts, technical and engineering aspects as well as the impact on the manufacturing process given existing facilities and a product portfolio to be manufactured. It also covers scientific approaches that support understanding and design of intensified bioprocesses. From an implementation perspective, the technologies used for intensification must be robust, scalable, and suitable for commercial manufacturing. Specific examples for a high seeding density fed batch (using N-1 perfusion) and a continuous process are provided for Chinese hamster ovary (CHO) cells producing therapeutic antibodies. Economic and sustainability aspects are addressed as well. Process intensification in an industrial environment is complex and many factors need to be considered, ranging from characteristics of a specific molecule to its commercial manufacturing at internal or external sites for global or regional markets.

Impurity profiling of an alternative pathway to ephedrine/pseudoephedrine and methamphetamine from the precursors benzaldehyde and nitroethane

Methamphetamine is produced using various routes from two predominant precursors, ephedrine/pseudoephedrine or phenyl-2-propanone (P2P). The multitude of synthetic routes results in many possible impurities (by-products or intermediates). Impurity profiling of methamphetamine by gas chromatography-mass spectrometry (GC–MS) is an important tool for the forensic chemist and investigators to assist in identification of the synthetic pathway and linking seizures. With the emergence of alternative routes, continued research into impurity profiles is important. One such alternative route is an ephedrine/pseudoephedrine-based pathway using the precursors typically associated with the phenyl-2-propanone (P2P) route, benzaldehyde and nitroethane. Benzaldehyde and nitroethane were condensed to form 2-nitro-1-phenyl-1-propanol. Subsequent reduction led to the corresponding 2-amino-1-phenyl-1-propanol. Cyclization-methylation with dimethyl carbonate to 3,4-dimethyl-5-phenyl-2-oxazolidinone, followed by catalytic hydrogenolysis or basic hydrolysis formed methamphetamine and ephedrine/pseudoephedrine, respectively. This paper presents the impurity profile of the alternative route. The conditions for the reduction of the intermediate 2-nitro-1-phenyl-1-propanol to 2-amino-1-phenyl-1-propanol was found to influence the by-products observed in the methamphetamine product. Notable by-products were amphetamine, N-ethylamphetamine and N-hydroxymethamphetamine.

Forced Degradation of an Anticancer Drug Apalutamide: Impurity Profiling and Structure Elucidation Study

Forced Degradation of an Anticancer Drug Apalutamide: Impurity Profiling and Structure Elucidation Study

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin.

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30mM sodium phosphate buffer, pH 3.0, containing 40mg/mL sulfated β-CD and 7mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.

Identification and Structural Characterization of Forced Degradation Impurities of Sacubitril by HRMS – Development and Validation of a Stability indicating RP-HPLC method

The current work identifies and characterises the stress degradation products of the anti-hypertensive drug sacubitril. The stability profile of sacubitril was evaluated under several stress settings including hydrolytic, oxidative, thermal and photolytic conditions in accordance with ICH recommendations. The degradation products of sacubitril were separated using reverse phase liquid chromatography and structural characterization was carried out using HRMS. The chromatographic separation was carried out by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) using a Fortis C18 column (150 mm X 4.6 mm; 5μm particle size) with mobile phase A (consisting of 0.1% TFA in Milli-Q water) and mobile phase B (100% Acetonitrile) in gradient mode. With a sample injection volume of 10μL, the mobile phase flow rate was 0.7 mL/min. A quadrupole time-of-flight (Q-TOF) mass spectrometer was used for the LC-MS analysis. Sacubitril is relatively stable in oxidative, thermal and photolytic conditions; however, considerable degradation was observed in acid, base and neutral hydrolysis. Three degradation products were identified and the structural characterization of these impurities was performed using the HRMS data and mass fragmentation. The proposed RP-HPLC method was robust, precise and specific in determining the impurity profile of sacubitril under different stress conditions.

Use of online sequential segmented heart cutting modulation and orthogonality approach for determination of isomeric impurity profile of a drug molecule using parallel 2-dimensional mass spectroscopy

Determination of impurity profile including chiral and achiral impurities is challenging as well as time consuming while analysis of the active moiety. With the recent evolution of 2-Dimensional separation modes, significant improvement can be made for simultaneous determination of both achiral and chiral impurities in a single set-up. Herein, the present work represents example of how both chiral and achiral impurities can be determined simultaneously. To represent the work, galantamine HBr was selected as the active moiety since it has achiral as well as many chiral impurities. Further, a 1D UPLC method with shorter runtime was developed to determine the achiral impurities and simultaneous heart cutting approach was used to cut the impurities and active compound and transfer to 2D chiral setup to separate out chiral impurities and perform its quantification. Both methods were proven to be orthogonal to each other with orthogonality factor of 0.241.

Additional degradants in the impurity profile of atropine low-dose ophthalmic solution

WHO has identified the increase in myopia as the world's greatest threat to vision health due to its association with vision-threatening complications like myopic macular degeneration, retinal detachment, cataract and open angle glaucoma. Pharmacological treatment with atropine is assessed as one of the best option in op-tical approaches to the control and inhibit of myopia progression. The aim of this study was to develop analytical method for quality control of 0.1 mg/mL atropine ophthalmic solution. UPLC analytical method for simultaneous determination of atropine sulfate monohydrate and its related substances described in Eur. Ph. and for additional impurities was developed. For method development low-dose of atropine formulations with a wide range of pH 4.5-6.6 were tested and the impact of formulation pH on the impurity profile was noted. The ionized form of tropic acid was observed in our studies in all buffered formulations with pH above 5. Additionally two isomers of atropine N-oxide were observed in the tested preparation with pH 6.6 in the presence of an oxidizing agent and were just possible to integrate with our new developed UPLC method. The ionized form of tropic acid was already defined in Berton et al. publication (1) and observed in the buffered formulation with pH 6.0. Our result complement and extend the impurity profile of atropine in wider pH range of atropine ophthalmic solutions.

Updating the European Pharmacopoeia impurity profiling method for cetirizine and suggesting alternative column, using design space comparison

The goal of the present study was to develop a generic workflow to evaluate the chromatographic resolution in a design space and find replacement column for the new method. To attain this objective, a limited number of initial experiments have been performed, and a modeling tool was employed to study and compare design spaces obtained with different columns. By overlaying the different individual resolution maps (design spaces), it is possible to quickly identify a robust zone where the different columns meet a given resolution criterion. This new feature of the modeling tool is very useful for finding alternative columns for a given separation, rather than the usual column tests. It was also demonstrated that two different columns can be used as equivalents (replacement columns), providing sufficient resolution at the same working point and with a high degree of robustness.

Challenges with retention and recovery of impurities containing acidic moieties during analytical UHPLC method development and validation for gefapixant freebase

Gefapixant citrate is a P2X3 purinergic receptor antagonist developed for the treatment of chronic cough. Gefapixant freebase is the penultimate intermediate in the commercial manufacturing route for gefapixant citrate and contains a complex impurity profile consisting of acidic and basic analytes. A UHPLC method was developed for assay and purity determination of gefapixant freebase utilizing a Waters Acquity Charged Surface Hybrid (CSH) C18 column (2.1 mm I.D. x 10 cm length, 1.7 µm particle size) with 0.1 % phosphoric acid and acetonitrile as the mobile phases. Method optimization was performed using ACD Labs LC Simulator to achieve baseline separation of all impurities and the method was successfully validated. During routine use and method transfer for gefapixant freebase, an increase in retention time for impurities containing strongly acidic functional groups, and poor recovery of a sulfinic acid impurity were observed. Subsequent investigation determined that the CSH column aging resulting from exposure of the column packing to the acidic mobile phase was the root cause for these behaviors. The mechanism of peak-shifting was further investigated using model compounds and determined to be due to an increase in ionic interactions with the CSH stationary phase with routine column use. The increase in ionic interactions was demonstrated to correlate with the charge state of the analyte. Poor recovery for the sulfinic acid impurity was attributed to increased peak tailing for this single impurity on older columns. This knowledge was leveraged to establish additional system suitability requirements to monitor column performance for the lifecycle of the analytical procedure.

A QbD Based RP-HPLC Method for Stability Indicating Impurity Profiling of Pyridoxine: Method Development, Validation, and Application

Pyridoxine impurity profiling in bulk and formulations was devised and validated using a reversed-phase high-performance liquid chromatography (RP-HPLC) strategy. The technique was fine-tuned using an Analytical quality by design (QbD) approach, ensuring its dependability and sturdiness. Linearity, accuracy, and precision were carefully examined as key performance indicators. With a relative standard deviation (RSD) < 2%, the approach showed exceptional precision, excellent recovery rates (100 and, 101.2%), and a strong correlation value (R2 = 0.9990). The technique has been used successfully for impurity profiling, and because it indicates stability, it can be used for long-term stability studies. The work contributes to the body of knowledge and has applications for pharmaceutical quality assurance.

Application of Quality by Design in RP-HPLC for Robust Impurity Profiling and Stability Assessment of Doxylamine

The primary purpose of this research was to create and validate a doxylamine detection method using reverse phase high performance liquid chromatography (RP-HPLC) for use in pharmaceutical formulations and bulk materials. Quality by design (QbD) served as the inspiration for this approach. The method was developed by running a C18 column at 1-mL/min through a mobile phase of 60 parts methanol to 40 parts 0.1% OPA water (pH 2.8). The detection was done with a UV detector set to 259 nm. All requirements for system applicability were satisfied by the suggested approach, including an acceptable asymmetry factor and an adequate number of theoretical plates. A correlation coefficient (R2) of 0.9990 was used to confirm linearity over a concentration of 10–50 g/mL. The approach showed good accuracy by a mean %recovery ranging from 99.59–101.45% and a %RSD between 0.11 and 0.81. Precision tests conducted both intraday and across days showed that the medication content stayed within allowable bounds. The approach was found to be robust, with only minor variations in flow rate, mobile phase composition, and detecting wavelength significantly affecting the accuracy and specificity. A 0.71 g/mL LoQ and a 0.23 g/mL LoD were determined to be analytical limits of detection and quantification, respectively. This study substantiates the development of a reliable, long-lasting, and cost-effective QbD-based RP-HPLC method suitable for the comprehensive analysis of doxylamine in tablet formulations and bulk dosage