Process-related Impurities Research Articles

A UPLC method development and validation study of Upadacitinib and its impurities in extended – release oral tablet dosage forms

The primary objective was to develop a concomitant isocratic ultra-performance liquid chromatographic photo-diode array detection method to estimate Upadacitinib and its process-related impurities: impurity-1 and impurity-2. Further validation was conducted and studied for possible degradants under stress environments. All the chemicals and reagents used were of HPLC (acetonitrile, methanol) and analytical grade (trifluoro acetic acid). The ultra-performance liquid chromatography (Agilent 1290 Infinity II LC system) consists of a quaternary pump, a BEH C18 (50×2.1mm, 1.7μ) column, and photo-diode array detector. The method was developed with acetonitrile: methanol: 0.1% v/v trifluoro acetic acid (50:20:30 v/v/v) mobile phase at 0.2mL/min flow rate within a run time of 5.5min The detection was carried at 231.2nm. The respective retention times achieved were 2.289min (Upadacitinib), 0.972min (Upadacitinib impurity-1), and 3.508min (Upadacitinib impurity-2). The optimized method was validated further, and the linearity range was best fit at 15.0-180.0μg/mL for Upadacitinib and 1.0-12.0μg/mL for both Upadacitinib impurity-1 and 2 respectively. The detection and quantification limits were 4.50μg/mL, 15.00μg/mL (Upadacitinib) and 0.30μg/mL, 1.0μg/mL (Upadacitinib impurity-1 and 2). A fast, isocratic, specific, and reproducible ultra-performance liquid chromatographic method was developed and validated for various parameters according to the ICH Q2 (R1) guidelines studies. Stress studies were conducted exposing the sample dilution to various treatments (acid, alkali, peroxide, HPLC water, heat, and UV light). The degradants were well-separated apart from the peaks of the active substance. The stability indicating nature was observed during the degradation. The optimized method can be applied for the separation and estimation of Upadacitinib and its process-related impurities in pharma sector in tablet dosage forms.

Host cell proteins in monoclonal antibody processing: Control, detection, and removal.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Characterization of Degradation Products of Selpercatinib by Mass Spectrometry: Optimization of Stability-Indicating HPLC Method for Separation and Quantification of Process Related Impurities of Selpercatinib

The study aimed to investigate a novel approach by utilizing liquid chromatography in coupled with mass spectrometry (LC-MS) to resolve, analyze and characterize significantly less quantity of stress degradation products (DPs) of selpercatinib along with its process related impurities. The analytes resolved on ZORBAX Eclipse (250 mm) stationary phase that was maintained employing 0.5 M sodium perchlorate at pH 5.4 methanol and acetonitrile in 50:20:30 (v/v) pumped at 0.8 mL/min isocratic flow and 241 nm wavelength. The method produces very high correlate linear curve in 30-240 μg/mL for selpercatinib and 0.03-0.24 μg/mL for its impurities with significantly low detection limit of 0.01 μg/mL for impurities. Selpercatinib pure compound was subjected to stress studies and chromatographic results confirm the formation of four DPs, which were characterized with the interpretation of their mass fragmentation pattern. The DPs were identified as 6-hydroxy-4-(6-(6-((6-methoxypyridin3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (DP 1), 6′-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)-1,2-dihydro-[3,3′-bipyridin]-5-ol (DP 2), (6-hydroxy-4-(6-(6-(pyridin-3-ylmethyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (DP 3), 1-amino-6′-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)-1,2-dihydro-[3,3′-bipyridin]-5-ol (DP 4), 6-(2-hydroxy-2-methyl-propoxy)-4-(6-(3-methylpiperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (DP 5) and 6-(2-hydroxy-2-methyl-propoxy)-4-(6-(3-methylpiperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (DP 6). Based on findings, it was concluded that this method was effectively applied for the regular quantification of impurities of selpercatinib in formulations. Additionally, it demonstrated applicability for the identification of both known and unknown impurities of selpercatinib.

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 NOVEL VALIDATED STABILITY INDICATIVE UPLC METHOD FOR RELUGOLIX FOR THE DETERMINATION OF PROCESS-RELATED AND DEGRADATION IMPURITIES

A NOVEL VALIDATED STABILITY INDICATIVE UPLC METHOD FOR RELUGOLIX FOR THE DETERMINATION OF PROCESS-RELATED AND DEGRADATION IMPURITIES

Demonstration of physicochemical and functional similarity between Stimufend (pegfilgrastim-fpgk) and Neulasta (pegfilgrastim): A comparative analytical assessment.

Pegfilgrastim is a long-acting recombinant human granulocyte colony-stimulating factor biologic that is indicated to reduce the incidence of infections, manifested by febrile neutropenia, in patients receiving myelosuppressive anti-cancer drugs and to increase survival in patients acutely exposed to myelosuppressive doses of radiation. Due to the high cost of biologic therapy and the scarcity of biosimilar alternatives, there is an unmet medical need for targeted biologics. This comparative analytical investigation aimed to confirm the similarity of biosimilar Stimufend® (pegfilgrastim-fpgk) to reference product Neulasta® (pegfilgrastim). The analysis was designed using state-of-the-art orthogonal techniques and side-by-side testing to compare the physicochemical and biological properties of these two products. The measured quality attributes included the primary structure and higher order structure of the molecule, purity/impurity profiles, product variants, process-related impurities, composition, content, and biological activity. The statistical analysis was based on risk ranking of the critical quality attributes (very low, low, moderate, high, very high), and scientific considerations in combination with the characteristics of the assay (sensitivity, selectivity, and variability). In addition, non-quantitative parameters were compared using a descriptive assessment of the product profile. Analytical similarity was concluded by quality attributes falling within the defined range of the originator product. The results of this study confirm that Stimufend® is biosimilar to Neulasta® for all measured quality attributes. There are no clinically significant differences between Stimufend® and Neulasta®, which was confirmed by the marketing approval for Stimufend® by the Food and Drug Administration and the European Medicines Agency. The findings of this study provide robust evidence supporting the structural and functional biosimilarity between Stimufend® and Neulasta®.

Characterization of Forced Degradation Products of Netarsudil: Optimization and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Process-Related Impurities.

The aim of this study is to examine resolution, identification, and characterization of forced degradation products of netarsudil by liquid chromatography-tandem mass spectrometry by validating a simple and sensitive high-performance liquid chromatography method for the resolution, identification, and quantification of two process-related impurities in netarsudil. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB C18 (250 x 4.6 mm; 5 µ id) column at room temperature as the stationary phase and 257 nm as the detector wavelength with the mobile phase consisting of acetonitrile, methanol, and pH 4.6 phosphate buffer in 45:35:20 (v/v) at 1.0 mL/min flow rate in isocratic elution. The method reported very sensitive detection limits of 0.008 µg/mL for impurity 1 and 0.003 µg/mL for impurity 1. The method produces a calibration curve linear in the concentration level of 25-200 for netarsudil and 0.025-0.2 µg/mL for impurities. The proposed method gives acceptable results for other validation parameters such as accuracy, precision, ruggedness, and robustness. The drug was subjected to various stress conditions such as acid, base, peroxide, and thermal and ultraviolet light to investigate the stability-indicating ability of the method. Considerable degradation was observed in stress studies, and the degradation products were well resolved from process-related impurities. The characterization of degradation products was performed on the basis of collision-induced dissociation mass spectral data, and the possible structures of the six degradation compounds of netarsudil were proposed. The outcomes of other validation studies were likewise satisfactory and proven adequate for the regular analysis of netarsudil and its process-related impurities in bulk drug and pharmaceutical dosage forms and can also be applied for the evaluation of the stress degradation mechanism of netarsudil.

An effective HPLC method for evaluation of process related impurities of Letermovir and LC-MS/MS characterization of forced degradation compounds

An effective HPLC method for evaluation of process related impurities of Letermovir and LC-MS/MS characterization of forced degradation compounds

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

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.

Predictive mechanistic modeling of loading and elution in protein A chromatography

Protein A chromatography is an enabling technology in current manufacturing processes of monoclonal antibodies (mAbs) and mAb derivatives, largely due to its ability to reduce the levels of process-related impurities by several orders of magnitude. Despite its widespread application, the use of mathematical modeling capable of accurately predicting the full protein A chromatographic process, including loading, post-loading wash and elution stages, has been limited. This work describes a mechanistic modeling approach utilizing the general rate model (GRM), the capabilities of which are explored and optimized using two isotherm models. Isotherm parameters were estimated by inverse-fitting simulated breakthrough curves to experimental data at various pH values. The parameter values so obtained were interpolated across the relevant pH range using a best-fit curve, thus enabling their use in predictive modeling, including of elution over a range of pH. The model provides accurate predictions (< 3% mean error in 10% dynamic binding capacity predictions and ∼ 5% mean error in elution mass and pool volume predictions, both on scale-up) for various residence times, buffer conditions and elution schemes and its effectiveness for use in scale-up and process development is shown by applying the same parameters to larger columns and a wider range of residence times.

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 &amp; 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.

In-house CHO HCPs platform: A promising approach for HCPs ELISA monitoring

A key aspect that must be supervised during the development of recombinant therapeutic products is the potential presence of impurities.Residual host cell proteins (HCPs) are a major class of process-related impurities derived from the host organism that even in trace amount have the potential to affect product quality, safety, and efficacy. Therefore, the product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible. The workhorse of HCP monitoring and quantitation during bioprocessing manufacturing is sandwich ELISA (enzyme‐linked immunosorbent assay), which employs polyclonal anti-HCP antibodies for both capture and detection. Commercial ELISA kits developed from Chinese Hamster Ovary (CHO) cell lines are widely applied in early drug development stages (preclinical, phase I, and phase II), but are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency. For later development stages, the upstream process-specific method is preferred to guarantee an improved sensitivity and coverage. In agreement with the USP General Chapter 〈1132〉, a platform assay can be used in place of the commercial one through all stages of product development, if already available when product development starts.This proof-of-concept study was carried out to demonstrate the feasibility and the advantages of the development of a proprietary CHO HCPs platform ELISA. Different proprietary mock materials have been characterized and compared by orthogonal bidimensional electrophoresis techniques (SDS-PAGE coupled to SS/WB and 2D DIGE) with the scope of selecting the best antigen-antibody couple for setting up the in-house ELISA. A preliminary evaluation of the in-house method performance has been done in comparison with the commercial assay, demonstrating that the platform method is promising for an accurate and precise CHO HCPs quantification during the early phase product and process development.

Development of novel gradient RP-HPLC method for separation of dapagliflozin and its process-related impurities: insight into stability profile and degradation pathway, identification of degradants using LCMS

BackgroundIn the dapagliflozin (DPF) synthesis, 5-Bromo-2-chlorobenzoic acid (5-BC impurity) and 4-Bromo-1-chloro-2-(4-ethoxybenzyl) benzene (4-BC impurity) are used as starting and reagent sources, respectively. The presence of 5-BC and 4-BC impurities in DPF could potentially affect the effectiveness of the final DPF product. The purpose of this investigation was to develop a stability indicating HPLC methodology for the separation of DPF, its process-related impurities and degradants. The method of analysis was developed on Xbridge Phenyl C18 column of dimensions, 250 × 4.6 mm, 5 μm with gradient elution using mobile phase made up of 0.05% aqueous trifluoroacetic acid and acetonitrile (AcN).ResultsThe method proposed indicates a good linearity (R2 = 0.9996 and 0.9993), good system precision (RSD ≤ 2%), good method precision (RSD ≤ 2%), accuracy (50–150%), LOD (0.000053 ppm and 0.0000165 ppm) and LOQ (0.00016 ppm and 0.00005 ppm) for 4-BC impurity and 5-BC impurity, respectively. LC–MS was used to detect and characterise degradants that were obtained in acid and base condition were identified and characterised. A comparison of the fragmentation pattern of the [M + H] + ions of DPF and its degradation products revealed the most likely processes for the generation of degradation products.ConclusionDPF sample quality can be evaluated using the suggested method for the presence of 4-BC impurity, 5-BC impurity and 2-(3-(4-ethoxy benzyl)-4-chloro phenyl)-tetrahydro-6-(hydroxy methyl)-2H-pyran-3,4,5-triol.

Development of stability-indicating HPLC method for quantification of pharmacopeia impurities of Zuclopenthixol and characterization of its stress degradation products by LCMS/MS

BackgroundThe present study focused to develop a simple and sensitive HPLC method for resolution and estimation process-related impurities of zuclopenthixol and further assessment of forced degradation behavior of zuclopenthixol.ResultsThe chromatographic separation of drug substance, process-related impurities and its degradation products (DPs) was achieved on KNAUER C18 (250 mm × 4.6 mm, 5µ id) column at that was maintained at 35 °C temperature using 0.1 M sodium acetate buffer at pH 4.3 and methanol in 20:80 (v/v) as mobile phase A, 0.1% formic acid and acetonitrile in 75:25 (v/v) as mobile phase B. Equal volume of mobile phase A and B was pumped in isocratic elution at 0.8 mL/min. Detection wavelength was selected as 257 nm. In the proposed conditions, zuclopenthixol is identified at 6.91 and 1.91 min and 2.89 min, respectively, for impurity B and A min with acceptable system suitability and specificity. The method produces LOD at 0.009 for impurities with calibration range of 30–180 µg/mL for zuclopenthixol and 0.03–0.18 µg/mL for impurities. The other validation parameters were notices to be with in the acceptable levels for zuclopenthixol and its impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in ESI positive mode.ConclusionThe analysis involved a comparison of collision-induced dissociation mass spectrometry data between the degradation products and zuclopenthixol. As a result, potential structures for six degradation compounds were suggested. The results from additional validation studies were similarly pleasing and demonstrated their suitability for the routine analysis of zuclopenthixol and its associated impurities in both bulk drug and pharmaceutical dosage forms. Additionally, these findings can be extended to assess the mechanism of stress degradation in zuclopenthixol.

Optimization of Stability-Indicating HPLC Method for Analyzing Process Related Impurities of Penfluridol and Structural Elucidation of Stress Degradation Products by LCMS/MSucidation of Stress Degradation Products by LCMS/MS

This study focused on the development of a simple and sensitive HPLC method for resolution and quantification of process-related impurities of penfluridol and further assessment of forced degradation behavior of penfluridol. The chromatographic separation was achieved on XTerra™ C18 (250×4.6 mm, 5.0μm) column and UV detection at 245nm. The mobile phase comprises of methanol and tetrahydrofuran in 55:45 (v/v) as solvent A and acetonitrile and tetrahydrofuran in 80:20 (v/v) as solvent B. The 60:40 (v/v) composition of solvent A and B were pumped isocratically at 1.0mL/min. In the proposed conditions, the retention time identified as 5.29 min for penfluridol, 4.51 min, 9.95 min and 7.64 min respectively for impurity 1, 2 and 3 with acceptable system suitability. The method produces sensitive detection limit of 0.008μg/mL for impurity 1, 2 and 0.004 μg/mL for impurity 3 with calibration range of 25-150 μg/mL for penfluridol and 0.025-0.150 μg/mL for impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The Degradation Products (DPs) formed during the stress study was characterized by LCMS/MS in ESI positive mode and the possible structures of five DPs with possible degradation pathways were proposed. The outcomes of other validation studies were likewise satisfactory and proven adequate for regular analysis of penfluridol and its process-related impurities in bulk drug and pharmaceutical dosage forms and can also applicable for evaluation of stress degradation mechanism of penfluridol.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of invivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

A two-step purification platform for efficient removal of Fab-related impurities: A case study for Ranibizumab

Antibodies (mAbs) and antibody fragments (Fabs) constitute one of the largest and most rapidly expanding groups of protein pharmaceuticals. In particular, antibody fragments have certain advantages over mAbs in some therapeutic settings. However, due to their greater chemical diversity, they are more challenging to purify for large-scale production using a standard purification platform. Besides, the removal of Fab-related byproducts poses a difficult purification challenge. Alternative Fab purification platforms could expedite their commercialization and reduce the cost and time invested. Accordingly, we employed a strong cation exchanger using a pH-based, highly linear gradient elution mode following Protein L affinity purification and developed a robust two-step purification platform for an antibody fragment. The optimized pH gradient elution conditions were determined on the basis of purity level, yield, and the abundance of Fab-related impurities, particularly free light chain. The purified Fab molecule Ranibizumab possessed a high degree of similarity to its originator Lucentis. The developed purification platform highly intensified the process and provided successful clearance of formulated Fab- and process-related impurities (∼98 %) with an overall process recovery of 50 % and, thus, might be a new option for Fab purification for both academic and industrial purposes.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

Host-cell proteins (HCPs)are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCPinteractions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.

Identification and Synthesis of Process-Related Impurities in Lisdexamfetamine Dimesylate

Abstract: During the process optimization of Lisdexamfetamine dimesylate, two process impurities were observed. Methods: These impurities remained a concern for commercialization and were isolated, on characterization, were found to be H-Lys-ε-Lys-d-amphetamine and other being its regio- isomer H-Lys-α-Lysd- amphetamine. The main target of this study was to synthesize reference compounds for HPLC analysis of these impurities produced in Lisdexamfetamine. Result: To the best of our knowledge, these impurities are not commercially available and no synthetic methods have been reported. Both of these potential impurities were synthesized, and their structures were established by various spectral techniques. Conclusion: Furthermore, herein we also report the synthesis of two process impurities Boc- Lys(Boc)-ε-Lys(α-Boc)-O-Nitrophenol and Boc-Lys(Boc)-α-Lys(ε-Boc)-O-Nitrophenol and their fate for the formation of impurities H-Lys-ε-Lys-d-amphetamine and H-Lys-α-Lys-d-amphetamine.