Process-related Impurities Research Articles

Toxic effect prediction of cefatirizine amidine sodium and its impurities by structure-toxicity relationship of cephalosporins

The three-dimensional (3D) structure-toxicity relationship of cephalosporins was explored by computing the most stable conformations of 33 kinds of cephalosporins in aqueous solution and using the teratogenicity and lethality of these compounds obtained in zebrafish embryo toxicity testing to evaluate their toxic effects. The toxic effect of cefatirizine amidine sodium, a novel cephalosporin which has finished preclinical study, was investigated. It is thought that the teratogenic effect of the triazine ring at the C-3 position is the main toxic effect of cefatirizine amidine. In addition, cefatirizine amidine is no more toxic than cefathiamidine and ceftriaxone. The results of the zebrafish embryo toxicity test combined with gene expression microarray technology were consistent with the prediction. The toxic effects of some potential process-related impurities of cefatirizine amidine were also predicted.

Development and Validation of an Analytical Method for Related Substances in N-acetyl–L-cysteine Effervescent Tablets by RP-HPLC

Background: The reported chromatographic methods for N-acetyl cysteine [NAC] are reverse phase HPLC and ion pair chromatography [IPC] for related substances test in bulk and in formulations. No reported stability indicating methods for the estimation of related substances in NAC effervescent formulation was found in literature. Objective: The present work was aimed at developing a selective, sensitive and reproducible stability indicating high-performance liquid chromatographic method for the quantitative determination of known, unknown impurities, degradation impurities and processrelated impurities of NAC effervescent formulation. Method: A reversed phase ion pair chromatographic method was developed employing Cadenza C18 column as the stationary phase and 0.01M octane sulphonate [pH 2.20], methanol and acetonitrile in the ratio 90:8:2 as the mobile phase. A gradient programme was followed with a run time of 55 minutes. 0.3 M hydrochloric acid was selected as the optimum diluent. The performance of the method was validated according to the ICHQ2R1 guidelines. Results: The method was found to be linear from 1.5 to 25μg/ml for impurities A, C and D and from 2.0 to 25 μg/ml for impurity B. The official impurities C and D were mapped in all stress conditions. Additionally, impurity B was also seen in acidic conditions. Conclusion: The results from the study demonstrate that the method is suitable for evaluating the stability of NAC effervescent tablet.

Stability-indicating HPLC method for simultaneous determination of degradation products and process-related impurities of avanafil in avanafil tablets

The objective of the current research is to understand the degradation behavior of avanafil under different stress conditions and to develop a stability-indicating high-performance liquid chromatography (HPLC) method for simultaneous determination of degradants observed during degradation. Avanafil tablets were exposed to acid, base, water, oxidative, thermal, and photolytic degradation conditions. In acid, oxidative, thermal, and humidity degradation, significant degradation was observed. All the degradants observed during degradation were separated from known impurities of avanafil by using reverse-phase (RP)-HPLC. Mobile phase A, 0.1% trifluoro acetic acid and triethylamine in water, and mobile phase B, water and acetonitrile in the ratio of 20:80 (v/v), were used at a flow rate of 1.2 mL/min in gradient elution mode. Separation was achieved by using Inertsil ODS 3 column (3 μm, 4.6 mm × 250 mm) at 45 °C. Peak responses were recorded at 245 nm. Method capability for detecting and quantifying the degradants, which can form during stability, was proved by demonstrating the peak purity of avanafil peak in all the stressed samples. Mass balance was established by performing the assay of stressed sample against reference standard. Mass balance was found >97% for all the stress conditions. The developed analytical method was validated as per International Conference on Harmonization (ICH) guidelines. The method was found specific, linear, accurate, precise, rugged, and robust.

Design and operation of a continuous integrated monoclonal antibody production process.

The realization of an end-to-end integrated continuous lab-scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter-based cell-retention, a continuous two column capture process, a virus inactivation step, a semi-continuous polishing step (twin-column MCSGP), and a batch-wise flow-through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity-yield trade-off of classical batch-wise bind-elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight-through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end-to-end integration. The steady-state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303-1313, 2017.

Synthesis and Physicochemical Characterization of the Process-Related Impurities of Eplerenone, an Antihypertensive Drug.

Two unknown impurities were observed during the process development for multigram-scale synthesis of eplerenone (Inspra®). The new process-related impurities were identified and fully characterized as the corresponding (7β,11α,17α)-11-hydroxy- and (7α,11β,17α)-9,11-dichloroeplerenone derivatives 12a and 13. Seven other known but poorly described in the literature eplerenone impurities, including four impurities A, B, C and E listed in the European Pharmacopoeia 8.4 were also detected, identified and fully characterized. All these contaminants result from side reactions taking place on the steroid ring C of the starting 11α-hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12) and the key intermediate (7α,17α)-9(11)-enester 7, including epimerization of the C-7 asymmetric center, oxidation, dehydration, chlorination and lactonization. The impurities were isolated and/or synthesized and fully characterized by infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry/electrospray ionization (HRMS/ESI). Their 1H- and 13C-NMR signals were fully assigned. The molecular structures of the eight impurities, including the new (7β,11α,17α)-11-hydroxy- and (7α,11β,17α)-9,11-dichloroeplerenone related substances 12a and 13, were solved and refined using single-crystal X-ray diffraction (SCXRD). The full identification and characterization of these impurities should be useful for the quality control and the validation of the analytical methods in the manufacture of eplerenone.

Soft sensor for monitoring biomass subpopulations in mammalian cell culture processes

ObjectivesBiomass subpopulations in mammalian cell culture processes cause impurities and influence productivity, which requires this critical process parameter to be monitored in real-time.ResultsFor this reason, a novel soft sensor concept for estimating viable, dead and lysed cell concentration was developed, based on the robust and cheap in situ measurements of permittivity and turbidity in combination with a simple model. It could be shown that the turbidity measurements contain information about all investigated biomass subpopulations. The novelty of the developed soft sensor is the real-time estimation of lysed cell concentration, which is directly correlated to process-related impurities such as DNA and host cell protein in the supernatant. Based on data generated by two fed-batch processes the developed soft sensor is described and discussed.ConclusionsThe presented soft sensor concept provides a tool for viable, dead and lysed cell concentration estimation in real-time with adequate accuracy and enables further applications with respect to process optimization and control.

Identification, Characterization, and Quantification of Impurities of Safinamide Mesilate: Process-Related Impurities and Degradation Products.

The characterization of process-related impurities and degradation products of safinamide mesilate (SAFM) in bulk drug and a stability-indicating HPLC method for the separation and quantification of all the impurities were investigated. Four process-related impurities (Imp-B, Imp-C, Imp-D, and Imp-E) were found in the SAFM bulk drug. Five degradation products (Imp-A, Imp-C, Imp-D, Imp-E, and Imp-F) were observed in SAFM under oxidative conditions. Imp-C, Imp-D, and Imp-E were also degradation products and process-related impurities. Remarkably, one new compound, identified as (S)-2-[4-(3-fluoro-benzyloxy) benzamido] propanamide (i.e., Imp-D), is being reported here as an impurity for the first time. Furthermore, the structures of the aforementioned impurities were characterized and confirmed via IR, NMR, and MS techniques, and the most probable formation mechanisms of all impurities proposed according to the synthesis route. Optimum separation was achieved on an Inertsil ODS-3 column (250 × 4.6 mm, 5 μm), using 0.1% formic acid in water (pH adjusted to 5.0) and acetonitrile as the mobile phase in gradient mode. The proposed method was found to be stability-indicating, precise, linear, accurate, sensitive, and robust for the quantitation of SAFM and its process-related substances, including its degradation products.

Analytical Separation and Characterisation of Degradation Products and the Development and Validation of a Stability-Indicating Method for the Estimation of Impurities in Ipratropium Bromide Respules Formulation

A short selective, precise, accurate and sensitive stability-indicating gradient LC-MS/MSn method was developed for the quantitative determination of process-related impurities and degradation products of Ipratropium bromide in pharmaceutical respules formulations. During the stress study, the degradation products of Ipratropium bromide were well-resolved from Ipratropium bromide and its impurities and the mass balances were found to be satisfactory in all the stress conditions, thus proving the stability-indicating capability of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, ruggedness, and robustness. During the stability analysis of the drug product, one unknown impurity was detected by the above stability-indicating method. The flow rate was 0.5 ml/min and effluent was monitored at 242nm. Retention time was found to be 5.0150.15 min. The LOD and LOQ values for were found to be 0.20996 (?g/ml) and 0.63624 (?g/ml) respectively.

Analytical Separation and Characterisation of Degradation Products and the Development and Validation of a Stability-Indicating Method for the Estimation of Impurities in Montelukast Oral Dosage Formulation

A short selective, precise, accurate and sensitive stability-indicating gradient LC-MS/MSn method was developed for the quantitative determination of process-related impurities and degradation products of Montelukast in pharmaceutical oral dosage formulations. During the stress study, the degradation products of Montelukast were well-resolved from Montelukast and its impurities and the mass balances were found to be satisfactory in all the stress conditions, thus proving the stability-indicating capability of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, ruggedness, and robustness. During the stability analysis of the drug product, one unknown impurity was detected by the above stability-indicating method. The flow rate was 0.5 ml/min and effluent was monitored at 226nm. Retention time was found to be 7.836±0.012 min. The LOD and LOQ values were found to be 0.2099 (μg/ml) and 0.6362 (μg/ml) respectively.

Analytical Development and Validation of a Stability-Indicating Method for the Estimation of Impurities in Budesonide Respules Formulation

A short selective, precise, accurate and sensitive stability-indicating LC-MS/MSn method was developed for the quantitative determination of process-related impurities and degradation products of Budesonide in pharmaceutical respules formulations. During the stress study, the degradation products of Budesonide were well-resolved from Budesonide and its impurities and the mass balances were found to be satisfactory in all the stress conditions, thus proving the stability-indicating capability of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, ruggedness and robustness. During the stability analysis of the drug product, all known impurities were detected by the above stability-indicating method. The flow rate was 0.8 ml/min and effluent was monitored at 247nm. Retention time was found to be 17.329±0.75 min and18.439±0.65 min of epimers (22R and 22S)respectively. The LOD and LOQ values for were found to be 0.20936 (μg/ml) and 0.6344 (μg/ml) respectively.

Analytical Separation and Characterisation of Degradation Products and the Development and Validation of a Stability-Indicating Method for the Estimation of Impurities in Levosalbutamol Respules Formulation

A short selective, precise, accurate and sensitive stability-indicating gradient LC-MS/MSn method was developed for the quantitative determination of process-related impurities and degradation products of Levosalbutamol in pharmaceutical respules formulations. During the stress study, the degradation products of Levosalbutamol were well-resolved from Levosalbutamol and its impurities and the mass balances were found to be satisfactory in all the stress conditions, thus proving the stability-indicating capability of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, ruggedness, and robustness. During the stability analysis of the drug product, one unknown impurity was detected by the above stability-indicating method. The flow rate was 0.8 ml/min and effluent was monitored at 242nm. Retention time was found to be 2.237±0.08 min. The LOD and LOQ values were found to be 0.20984 (μg/ml) and 0.6359 (μg/ml) respectively.

A simple, selective, and sensitive gas chromatography-mass spectrometry method for the analysis of five process-related impurities in atenolol bulk drug and capsule formulations.

An extremely sensitive and simple gas chromatography with mass spectrometry method was developed and completely validated for the analysis of five process-related impurities, viz., 4-hydroxy-l-phenylglycine, 4-hydroxyphenylacetonitrile, 4-hydroxyphenylacetic acid, methyl-4-hydroxyphenylacetate, and 2-[4-{(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy}phenyl]acetonitrile, in atenolol. The separation of impurities was accomplished on a BPX-5 column with dimensions of 50m×0.25mm i.d. and 0.25μm film thickness. The method validation was performed following International Conference on Harmonisation guidelines in which the method was capable to quantitate 4-hydroxy-l-phenylglycine, 4-hydroxyphenylacetonitrile, and 4-hydroxyphenylacetic acid at 0.3ppm, and methyl-4-hydroxyphenylacetate and 2-[4-{(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy}phenyl]acetonitrile at 0.35ppm with respect to 10mg/mL of atenolol. The method was linear over the concentration range of 0.3-10ppm for 4-hydroxy-l-phenylglycine, 4-hydroxyphenylacetonitrile, and 4-hydroxyphenylacetic acid, and 0.35-10ppm for methyl-4-hydroxyphenylacetate and 2-[4-{(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy}phenyl]acetonitrile. The correlation coefficient in each case was found ≥0.998. The repeatability and recovery values were acceptable, and found between 89.38% and 105.60% for all five impurities under optimized operating conditions. The method developed here is simple, selective, and sensitive with apparently better resolution than the reported methods. Hence, the method is a straightforward and good quality control tool for the quantitation of selected impurities at trace concentrations in atenolol.

Synthesis of potential impurities of dabigatran etexilate

ABSTRACTThe present work describes the origin, control, and synthesis of two potent impurities of dabigatran etexilate 1, dabigatran dimer 2, and dabigatran n-propyl ester 3 from the commercially available raw materials 2-[(4-cyanophenyl)amino]acetic acid (4) and N-[3-amino-4-(methylamino)benzoyl]-N-2-pyridinyl-β-alanine ethyl ester (5). These impurities are the process-related impurities and may affect the quality of drug substance, during its manufacturing in large scale. These impurities are not only the crucial components in determining the quality and safety of the drug substance 1 but also provide a better understanding of impurity profiling.

Synthesis, isolation, identification and characterization of new process-related impurity in isoproterenol hydrochloride by HPLC, LC/ESI-MS and NMR

One unknown impurity (Imp-II) during the analysis of laboratory batches of isoproterenol hydrochloride was detected in the level ranging from 0.04% to 0.12% by high performance liquid chromatography with UV detection. The unknown impurity structure was proposed as 4-[2-(propan-2-ylamino)ethyl]benzene-1,2-diol (Imp-II) using the liquid chromatography--mass spectrophotometry (LC--MS) analysis. Imp-II was isolated by semi-preparative liquid chromatography from the impurity-enriched reaction crude sample. Its proposed structure was confirmed by nuclear magnetic spectroscopy such as 1H, 13C, DEPT (1D NMR), HSQC (2D NMR) and infrared spectroscopy (IR), and retention time and purity with HPLC followed by the chemical synthesis. Due to less removable nature of Imp-II during the purification, the synthetic process was optimized proficiently to control the formation of Imp-II below to the limit<0.12% in the course of reaction. The new chemical route was developed for the preparation of this impurity in required quantity with purity to use as reference standard. The most probable mechanism for the formation of Imp-II was discussed in details.

Host Cell Protein Profiling by Targeted and Untargeted Analysis of Data Independent Acquisition Mass Spectrometry Data with Parallel Reaction Monitoring Verification.

Host cell proteins (HCPs) are process-related impurities of biopharmaceuticals that remain at trace levels despite multiple stages of downstream purification. Currently, there is interest in implementing LC-MS in biopharmaceutical HCP profiling alongside conventional ELISA, because individual species can be identified and quantitated. Conventional data dependent LC-MS is hampered by the low concentration of HCP-derived peptides, which are 5-6 orders of magnitude less abundant than the biopharmaceutical-derived peptides. In this paper, we present a novel data independent acquisition (DIA)-MS workflow to identify HCP peptides using automatically combined targeted and untargeted data processing, followed by verification and quantitation using parallel reaction monitoring (PRM). Untargeted data processing with DIA-Umpire provided a means of identifying HCPs not represented in the assay library used for targeted, peptide-centric, data analysis. An IgG1 monoclonal antibody (mAb) purified by Protein A column elution, cation exchange chromatography, and ultrafiltration was analyzed using the workflow with 1D-LC. Five protein standards added at 0.5 to 100 ppm concentrations were detected in the background of the purified mAb, demonstrating sensitivity to low ppm levels. A calibration curve was constructed on the basis of the summed peak areas of the three highest intensity fragment ions from the highest intensity peptide of each protein standard. Sixteen HCPs were identified and quantitated on the basis of the calibration curve over the range of low ppm to over 100 ppm in the purified mAb sample. The developed approach achieves rapid HCP profiling using 1D-LC and specific identification exploiting the high mass accuracy and resolution of the mass spectrometer.

Identification of a host cell protein impurity in therapeutic protein, P1

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody.

Development Considerations for Nanocrystal Drug Products.

Nanocrystal technology has emerged as a valuable tool for facilitating the delivery of poorly water-soluble active pharmaceutical ingredients (APIs) and enhancing API bioavailability. To date, the US Food and Drug Administration (FDA) has received over 80 applications for drug products containing nanocrystals. These products can be delivered by different routes of administration and are used in a variety of therapeutic areas. To aid in identifying key developmental considerations for these products, a retrospective analysis was performed on the submissions received by the FDA to date. Over 60% of the submissions were for the oral route of administration. Based on the Biopharmaceutics Classification System (BCS), most nanocrystal drugs submitted to the FDA are class II compounds that possess low aqueous solubility and high intestinal permeability. Impact of food on drug bioavailability was reduced for most nanocrystal formulations as compared with their micronized counterparts. For all routes of administration, dose proportionality was observed for some, but not all, nanocrystal products. Particular emphasis in the development of nanocrystal products was placed on the in-process tests and controls at critical manufacturing steps (such as milling process), mitigation and control of process-related impurities, and the stability of APIs or polymorphic form (s) during manufacturing and upon storage. This emphasis resulted in identifying challenges to the development of these products including accurate determination of particle size (distribution) of drug substance and/or nanocrystal colloidal dispersion, identification of polymorphic form (s), and establishment of drug substance/product specifications.

Development of a Practical Synthesis of ERK Inhibitor GDC-0994

The process development of a synthetic route to manufacture ERK inhibitor GDC-0994 on multikilogram scale is reported herein. The API was prepared as the corresponding benzenesulfonate salt in 7 steps and 41% overall yield. The synthetic route features a biocatalytic asymmetric ketone reduction, a regioselective pyridone SN2 reaction, and a safe and scalable tungstate-catalyzed sulfide oxidation. The end-game process involves a telescoped SNAr/desilylation/benzenesulfonate salt formation sequence. Finally, the development of the API crystallization allowed purging of process-related impurities, obtaining >99.5A% HPLC and >99% ee of the target molecule.

Experimental design of a twin-column countercurrent gradient purification process

As typical for separation processes, single unit batch chromatography exhibits a trade-off between purity and yield. The twin-column MCSGP (multi-column countercurrent solvent gradient purification) process allows alleviating such trade-offs, particularly in the case of difficult separations. In this work an efficient and reliable procedure for the design of the twin-column MCSGP process is developed. This is based on a single batch chromatogram, which is selected as the design chromatogram. The derived MCSGP operation is not intended to provide optimal performance, but it provides the target product in the selected fraction of the batch chromatogram, but with higher yield. The design procedure is illustrated for the isolation of the main charge isoform of a monoclonal antibody from Protein A eluate with ion-exchange chromatography. The main charge isoform was obtained at a purity and yield larger than 90%. At the same time process related impurities such as HCP and leached Protein A as well as aggregates were at least equally well removed. Additionally, the impact of several design parameters on the process performance in terms of purity, yield, productivity and buffer consumption is discussed. The obtained results can be used for further fine-tuning of the process parameters so as to improve its performance.

Application of calcium phosphate flocculation in high-density cell culture fluid with high product titer of monoclonal antibody.

The calcium phosphate [Ca3(PO4)2] precipitation was used for improving the clarification efficiency in harvest process of the monoclonal antibody (mAb) containing cell culture fluid (CCF) with high turbidity and product titer. The flocculation conditions (concentration, addition order of flocculants, pH, and operation time), and the effect of flocculants on the mAb physical chemical properties (such as distribution of charge variants and aggregates) and process-related impurities removal (such as DNA and CHOP) were evaluated in this study. The results showed that the turbidity of CCF supernatant was significantly reduced at pH 7, 120min with addition of phosphate ions first, while a high mAb recovery yield was kept in the CCF supernatant after flocculation. Addition of calcium ions at 15-60mM was sufficient for flocculation in this study. A relationship between turbidity/mAb recovery yield and the concentration of calcium ions was established. More than 85% DNA in the CCF were effectively removed by the addition of optimal concentration of flocculants. Flocculation process of Ca3(PO4)2 is an effective pretreatment method in purification processes of mAbs from the CCF with high turbidity and product titer.