Process-related Impurities Research Articles

OPTIMIZATION OF ROBUST HPLC APPROACH FOR ASSESSING PROCESS RELATED IMPURITIES OF LEMBOREXANT AND ELUCIDATION OF STRESS-INDUCED DEGRADATION PRODUCTS THROUGH LCMS/MS

This study primarily aimed to establish an uncomplicated yet highly responsive HPLC technique to effectively separate and quantify process-related impurities of Lemborexant. Additionally, it aimed to explore the forced degradation behavior of lemborexant through systematic assessments by utilizing LCMS. The chromatographic separation of drug substance, process related impurities and its degradation products (DPs) was achieved on Kinetex C18 (150×4.6 mm, 5 μm) column at that was maintained at 350C temperature using 10 mM ammonium formate buffer pH 4.2, acetonitrile and methanol in 65:25:10 (v/v) isocratic elution at 0.7 mL/min. Detection wavelength was selected as 265 nm. In the proposed conditions, lemborexant is identified at 4.06 and 6.19, 9.33 min and 1.60 min respectively for impurity 1, 2 and 3 min with acceptable system suitability and specificity. The method produces LOD at 0.009 for impurities with calibration range of 40–280 µg/mL for lemborexant and 0.04 - 0.28 µg/mL for impurities. The remaining validation parameters were observed to fall within acceptable ranges for both lemborexant and its impurities. The compound underwent exposure to various stress conditions (acid, base, peroxide, thermal, and UV light) as outlined in accordance with ICH Q1A (R2) guidelines. The degradation products formed during the stress study were detected and characterized using LCMS/MS in ESI positive mode. This involved a thorough comparison of collision-induced dissociation mass spectrometry data between the degradation products and lemborexant. Consequently, potential structures for five degradation compounds were proposed. The outcomes of supplementary validation investigations were equally satisfactory, confirming their appropriateness for the regular quantification of lemborexant and its related impurities in both bulk drug and pharmaceutical formulations. Furthermore, these findings have the potential to shed light on the mechanism of stress-induced degradation in lemborexant

Assessing the Manufacturability and Critical Quality Attribute Profiles of Anti-IL-8 Immunoglobulin G Mutant Variants.

Early-phase manufacturability assessment of high-concentration therapeutic monoclonal antibodies (mAbs) involves screening of process-related risks impacting their translation into the clinic. Manufacturing a mAb at scale relies on cost-effective and robust approaches to derisk manufacturability parameters, such as viscosity, conformational stability, aggregation, and process-related impurities. Using a panel of model anti-IL-8 IgG1 mutants, we investigate upstream and downstream processability, phase behavior, and process-related impurities. We correlate trends in the biophysical properties of mAbs with their cell growth, expression, filtration flux, solubility, and post-translational modifications. We find significant trends in increased relative free light chain expression with heavy chain mutants and detect a requirement for adjusted operation pH for cation exchange polishing steps with charge-altering variants. Moreover, trends between phase stability and high-concentration viscosity were observed. We also investigated unique correlations between increased glycosylation and biophysical behavior. Further in-depth analysis and modeling are required to elucidate the impact of the mAb sequence on the metabolism of the expression system, solubility limits, and alternative gelation models as future directions.

Synthesis, Isolation and Characterization of Nine Pharmacopeial Impurities Related to the Active Pharmaceutical Ingredient Olsalazine Sodium

AbstractOlsalazine sodium is an anti-inflammatory agent used to treat ulcerative colitis. Both British and European Pharmacopoeia list nine process-related impurities in their monographs. Quantification of the impurities in the final synthesized active pharmaceutical ingredient (API) is required to meet the quality standards set by the International Council for Harmonisation (ICH). Both literature reports and commercial vendors for these impurities are few and far between. The present report describes the synthesis, isolation, purification and characterization of these nine pharmacopeial impurities.

Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review.

Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

Characterization of host cell proteins in the downstream process of plant-Based biologics using LC-MS profiling

Host cell proteins (HCPs) are process-related impurities found in biopharmaceutical products that can impair their safety and efficacy. While ELISA has traditionally been employed to quantify HCPs, LC-MS emerges as a powerful alternative for precise identification of individual HCPs. In this study, we used LC-MS for profiling HCPs from Nicotiana benthamiana-derived biopharmaceuticals. Our approach involved rigorous false discovery rate control to ensure data integrity and reliability. Comprehensive analysis revealed a systematic reduction of HCPs following purification, demonstrating the efficiency of purification processes in removing non-essential proteins. Furthermore, LC-MS enabled the identification of potential contaminants, refining purification strategies and improving product purity and integrity. Our findings highlight the potential of LC-MS as an analytical tool for HCPs analysis in biopharmaceutical development and manufacturing. By providing detailed insights into HCPs profiles and contaminants, LC-MS facilitates informed decision-making in downstream processing steps, benefiting product quality, patient safety, and the biopharmaceutical sector.

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

COVID-19 Modified mRNA “Vaccines”: Lessons Learned from Clinical Trials, Mass Vaccination, and the Bio-Pharmaceutical Complex, Part 2

The COVID-19 modified mRNA (modmRNA) lipid nanoparticle-based “vaccines” are not classical antigen-based vaccines but instead prodrugs informed by gene therapy technology. Of considerable note, these products have been linked to atypical adverse and serious adverse event profiles. As discussed in Part 1, health-related risks and drawbacks were drastically misreported and underreported in the Pfizer and Moderna trial evaluations of these genetic products. Now in Part 2, we examine the main structural and functional aspects of these injectables. The COVID-19 modmRNA injectable products introduce a unique set of biological challenges to the human body with the potential to induce an extensive range of adverse, crippling, and life-threatening effects. Based on the fact that there is no current method to quantify host (cell-based) spike protein production in vivo following injection with these prodrugs, there is no standard “dose”. This is in part due to differences in spike protein production output, which depends on cell metabolism and transfection efficiency. It is therefore difficult to predict adverse event profiles on an individual basis, but considering that millions of adults across the world have reported severe and serious adverse events in the context of these modmRNA COVID-19 products, valid concerns are raised regarding injection of infants and younger age groups for whom COVID-19 poses only minimal risks. We address the process-related genetic impurities inherent in mass production of these products, and the potential risks posed by these contaminants. We then categorize the principal adverse events associated with the modmRNA products with a brief systems-based synopsis of each of the six domains of potential harms: (1) cardiovascular, (2) neurological, (3) hematologic; (4) immunological, (5) oncological, and (6) reproductive. We conclude with a discussion of the primary public health and regulatory issues arising from this evidence-informed synthesis of the literature and reiterate the urgency of imposing a global moratorium on the modmRNA-LNP-based platform.

Stability-Indicating HPLC Method for Quantifying Process-Related Impurities in Fedratinib and Identification of Its Forced Degradation Products Using LC-MS/MS

Background: Pharmaceutical industry is characterized by rigorous quality standards to ensure the safety and efficacy of drugs. Despite stringent manufacturing processes, the presence of impurities or generation of degradation products (DPs) in pharmaceutical products remains a concern. This necessitates a comprehensive and systematic approach to analysis impurities and DPs. Objectives: This study deals with the optimization of the stable HPLC method for quantification of fedratinib impurities and its DPs characterization through LC-MS/MS. Method: Method optimization studies were conducted by analyzing standard solutions in various method parameters. The results noticed in every varied method condition were tabulated for finalizing the appropriate conditions for analyzing fedratinib. The mass spectral response of DPs was interpreted carefully for structural conformation of DPs. Results: The method is optimized as HIQSIL C18 (250mm×4.6mm;5µ) column employing 1.0 mL/min flow of phosphate buffer (pH 5.2) and acetonitrile in 45:65 (v/v) and 257 nm. This method elutes 5.4, 2.6, 9.2 and 3.5 min for fedratinib, impurity 1, 2 and 3 respectively. Method sensitivity was verified to be very sensitive that can evaluate up to 0.003, 0.015 and 0.004 µg/mL for impurity 1, 2 and 3 respectively. Well correlated calibration curve achieved in 50-200 µg/mL for fedratinib and 0.05-0.20 µg/mL for impurities. Various stress studies produce four stress DPs and were identified using LC-MS/MS. The molecular mass (g/mol) and formula of DPs were identified as 426 and C21H25N5O3S, 312 and C17H22N5O, 354 and C17H14N4O3S, 215 and C11H11N4O respectively for DP 1 to 4. Conclusion: The method proposed can successfully be helpful for quantifying the pharmaceutical impurities and DPs of fedratinib in bulk batch samples and formulations.

Reactivity of N terminal histidine of peptides towards excipients/impurity of excipients: A case study of liraglutide excipient compatibility study

The selection of quality excipients is a crucial step in peptide formulation development. Apart from excipient incompatibility, process-related impurities or degradants of an excipient can interact with peptide-active pharmaceutical ingredients, forming the interaction products. The formaldehyde has been reported as an impurity of excipient in polyethylene glycol, glycerol, magnesium stearate, microcrystalline cellulose, mannitol, etc. The peptide contains various amino acids such as histidine, lysine, and arginine having free amine groups. These amine groups act as strong nucleophile and can increase the reactivity of peptides. PLGA is the most widely used biodegradable polymer in sustained-release formulations. The hydrolysis of PLGA generates glycolic acid and lactic acid impurities, which can form the interaction product with the amines of peptides. During the formulation development of Liraglutide, we have found few interaction products. The systematic characterization and mechanistic understanding of these interaction products lead us to imidazopyrimidine, glycolyl, and lactolyl moieties. These interaction products have been characterized thoroughly with the use of LC-HRMS, MS/MS, and hydrogen-deuterium exchange mass studies. The study revealed that the reactivity of N-terminal histidine must be considered for formulation development. Moreover, the quality of excipients with respect to presence of impurities must be considered as critical material attributes.

Single-use chromatographic clarification to eliminate endonuclease treatment in production of recombinant adeno-associated viral vectors

In current manufacturing processes for recombinant adeno-associated viral vectors (rAAV), endonuclease treatment is used prior to depth filtration to break down host cell DNA that is released from cells post-lysis. Without this treatment, traditional depth filters exhibit negligible clearance of host cell DNA, a key process-related impurity. In this work, we evaluate single-use chromatographic clarification using Harvest RCTM filters (3 M) to process rAAV8 and rAAV9 HEK293 cell culture lysate with and without endonuclease pretreatment. We demonstrate endonuclease-free clarification for both serotypes with yield >90 % and up to 3 log reduction of host cell DNA, from >104 ng/mL to <50 ng/mL, matching or exceeding the filtrate quality in traditional depth filtration with 100 U/mL of endonuclease added during lysis. The addition of 100 mM of salt in the harvest buffer is also shown to improve filtration throughput, prevent filter clogging, and limit rAVV binding to the filter device. Finally, a mechanistic cake-fiber fouling model is shown to be a good representation of the Harvest RCTM filtration mechanism. The endonuclease-free clarification approach enables cost savings of USD 100,000 per 500 L batch, equivalent to the cost of cGMP-grade endonuclease at this scale. The benefits of endonuclease-free clarification further include easier raw material sourcing as well as eliminating the need for a residual endonuclease assay in rAAV manufacturing processes.

Isolation, Identification, and Biological Activities of a New Chlorin e6 Derivative.

Chlorin e6 is a well-known photosensitizer used in photodynamic diagnosis and therapy. A method for identifying and purifying a novel process-related impurity during the synthesis of chlorin e6 has been developed. Its structure was elucidated using NMR and HRMS. This new impurity is formed from chlorophyll b rather than chlorophyll a, which is the source of chlorin e6. The intermediates formed during chlorin e6 synthesis were monitored using HPLC-mass spectrometry. This new impurity was identified as rhodin g7 71-ethyl ester, the structure of which remains unknown to date. The cytotoxic effects of this novel compound in both dark and light conditions were studied against five cancer cell lines (HT29, MIA-PaCa-2, PANC-1, AsPC-1, and B16F10) and a normal cell line (RAW264.7) and compared to those of chlorin e6. Upon irradiation using a laser at 0.5 J/cm2, rhodin g7 71-ethyl ester demonstrated higher cytotoxicity (2-fold) compared to chlorin e6 in the majority of the cancer cell lines. Furthermore, this new compound exhibited higher dark cytotoxicity compared to chlorin e6. Studies on singlet oxygen generation, the accumulation in highly vascular liver tissue, and the production of reactive oxygen species in MIA-PaCa-2 cancer cells via rhodin g7 71-ethyl ester correspond to its higher cytotoxicity as a newly developed photosensitizer. Therefore, rhodin g7 71-ethyl ester could be employed as an alternative or complementary agent to chlorin e6 in the photodynamic therapy for treating cancer cells.

A new process related impurity identification, characterization, development of analytical method by HPLC and method validation for Voriconazole API

AbstractDuring the testing of laboratory Voriconazole API batches, one unidentified impurity (IMP-5.312) was detected employing the Pharmeuropa HPLC technique at a level in excess of 0.10%. This IMP-5.312 was synthesized and then characterized as 6-(3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl) butan-2-yl)-5-fluoropyrimidin-4-ol by the corresponding spectral information (MS, 1H-NMR, 13C-NMR, and IR). The IMP-5.312 impurity was effectively quantified using an enhanced HPLC based-technique that was developed as well as validated. The approach made use of a Novapak C18 column with an inner diameter of 3.9 mm and a length of 150 mm (4.0 µm) for chromatographic separation. The analysis of IMP-5.312 was made at 45 °C, with a flow rate (isocratic) of 1.0 mL min−1 and a 256 nm detection wavelength. Acetonitrile, methanol, and 0.1% aqueous trifluoro acetate buffer (pH 4.0) were mixed at a ratio of 15:30:55 (v/v/v) to create the mobile phase for a 20 μL sample injection. The linearity range of 0.25281–1.51690 μg mL−1 had a correlation coefficient more than 0.99942, and the accuracy ranged from 89.3 to 100.3%. It was noted that the established HPLC based-technique was sensitive, specific, and precise. The technique was executed on the current batches of VRC API for IMP-5.312 analysis, and the outcomes were good. For quality control purposes during the manufacturing procedure of VRC, the identification as well as analysis of IMP-5.312 should be helpful. The in silico approach was applied to predict the IMP-5.312 toxicity. The reports indicated that IMP-5.312 in non-mutagenic and categorized as ICH M7 class-5 impurity.

A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

Raman and NIR spectroscopy-based real-time monitoring of the membrane filtration process of a recombinant protein for the diagnosis of SARS-CoV-2

This research shows the detailed comparison of Raman and near-infrared (NIR) spectroscopy as Process Analytical Technology tools for the real-time monitoring of a protein purification process. A comprehensive investigation of the application and model development of Raman and NIR spectroscopy was carried out for the real-time monitoring of a process-related impurity, imidazole, during the tangential flow filtration of Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein. The fast development of Raman and NIR spectroscopy-based calibration models was achieved using offline calibration data, resulting in low calibration and cross-validation errors. Raman model had an RMSEC of 1.53 mM, and an RMSECV of 1.78 mM, and the NIR model had an RMSEC of 1.87 mM and an RMSECV of 2.97 mM. Furthermore, Raman models had good robustness when applied in an inline measurement system, but on the contrary NIR spectroscopy was sensitive to the changes in the measurement environment. By utilizing the developed models, inline Raman and NIR spectroscopy were successfully applied for the real-time monitoring of a process-related impurity during the membrane filtration of a recombinant protein. The results enhance the importance of implementing real-time monitoring approaches for the broader field of diagnostic and therapeutic protein purification and underscore its potential to revolutionize the rapid development of biological products.

Immunogenicity Risk Assessment of Process-Related Impurities in An Engineered T Cell Receptor Cellular Product

Cell therapies such as genetically modified T cells have emerged as a promising and viable treatment for hematologic cancers and are being aggressively pursued for a wide range of diseases and conditions that were previously difficult to treat or had no cure. The process development requires genetic modifications to T cells to express a receptor (engineered T cell receptor (eTCR)) of specific binding qualities to the desired target. Protein reagents utilized during the cell therapy manufacturing process, to facilitate these genetic modifications, are often present as process-related impurities at residual levels in the final drug product and can represent a potential immunogenicity risk upon infusion. This manuscript presents a framework for the qualification of an assay for assessing the immunogenicity risk of AA6 and Cas9 residuals. The same framework applies for other residuals; however, AAV6 and Cas9 were selected as they were residuals from the manufacturing of an engineered T cell receptor cellular product in development. The manuscript: 1) elucidates theoretical risks, 2) summarizes analytical data collected during process development, 3) describes the qualification of an in vitro human PBMC cytokine release assay to assess immunogenicity risk from cellular product associated process residuals; 4) identifies a multiplexed inflammatory innate and adaptive cytokine panel with pre-defined criteria using relevant positive controls; and 5) discusses qualification challenges and potential solutions for establishing meaningful thresholds. The assessment is not only relevant to establishing safe exposure levels of these residuals but also in guiding risk assessment and CMC strategy during the conduct of clinical trials.

LC-MS/MS characterization of stress degradation products of Gilteritinib and establishment of HPLC method for analysis of process related impurities of Gilteritinib

Background: The current investigation entails the characterization of seven degradation products (DPs) formed in different stress conditions of gilteritinib employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methodology: This study developed a stability-indicating reversed-phase high-performance liquid chromatographic (HPLC) method for precisely determining gilteritinib in the presence of its process-related impurities in bulk drug and formulation samples. To explore the stability profile of gilteritinib, it was exposed to forced degradation experiments conducted under various conditions, including acidic, basic, oxidative, photolytic, and thermal stress. These experiments revealed the degradation of gilteritinib under basic, acidic, and photolytic conditions, forming seven distinct DPs. Result: The chromatographic resolution of gilteritinib and its impurities along with DPs was effectively achieved using a Waters Symmetry C18 (250 mm × 4.6 mm, 5 μm) column using equal volumes of solvent A and B (pH 4.5 phosphate buffer and acetonitrile in 25:75 (v/v) as solvent A, acetonitrile and methanol in 75: 25 (v/v) as solvent B) pumped isocratically at 0.7 mL/min and 230 nm wavelength. The method produces an accurate fit calibration curve in 25-175 μg/mL for gilteritinib and LOQ (0.025 μg/mL) – 0.175 μg/mL for its impurities with acceptable precision, accuracy, and recovery. Conclusion: The efficacy of this method was validated through LC-MS/MS, which allowed for the verification of the chemical structures of newly generated degradation products of gilteritinib. Hence, this method is appropriate for the resolution and evaluation of process-related impurities of gilteritinib and can also be applied for evaluating stress degradation products.

Assessment and Control of Organic Impurities in Medicinal Products: A Review

INTRODUCTION. The determination of impurities is a key requirement for the quality assessment of medicines because impurities can significantly impact the quality and therapeutic effectiveness. Pharmacopoeias are the most important scientific and methodological guidelines for manufacturers developing medicinal product specifications and regulators assessing these specifications as part of registration dossiers. Therefore, it is essential to harmonise national and international approaches to impurities.AIM. This study aimed to analyse and summarise pharmacopoeial requirements for and methodological approaches to the control, evaluation, and identification of organic impurities in medicines.DISCUSSION. The authors compared requirements for the control of organic impurities in small-molecule medicines set forth in national and international pharmacopoeias and guidelines of the International Council for Harmonisation of Technical Requirements for Medicinal Products for Medical Use (ICH) and the Eurasian Economic Union (EAEU). This comparison highlighted the differences in current approaches that require further harmonisation of the existing regulatory documentation. Additionally, this study analysed the popularity, advantages, and disadvantages of different options for determining impurities in two-component combination products (i.e. identified and/or unidentified impurities in one or each of the active substances). The analysis demonstrated the need to control nitrosamines and genotoxic impurities and to use selective and highly sensitive chromatographic methods.CONCLUSIONS. When drafting pharmacopoeial monographs for medicinal products, experts should consider the general approach set forth in the EAEU pharmacopoeia and regulations and in the ICH Q3B guideline. This approach recommends that process-related impurities of active substances should not be controlled at the medicinal product level. Therefore, pharmacopoeial monographs for active substances should distinguish degradation products from process-related impurities. Impurities should be determined for each active substance to ensure the quality and safety of fixed combination medicinal products. Priorities for improving the methodological approach to the control of organic impurities include using reference standards for impurities and acknowledging the necessity of impurity quantification.

Development of sensitive stability indicating HPLC method for quantification of process related impurities of Fluphenazine; LC–MS/MS elucidation of their degradation products and degradation pathway

This study focused on the development of a simple and sensitive HPLC method for the resolution and quantification of process related impurities of fluphenazine and further assessment of the forced degradation behavior of fluphenazine. Chromatographic separation was achieved on phenomenex™ Gemini C18 column (250×4.6mm, 5.0μm) at room temperature using acetonitrile, methanol and TFA in 65:30:05 (v/v) at pH 4.2 as the mobile phase was pumped isocratically at a 0.9 mL/min flow rate. The detection wavelength was selected as 255 nm. In the proposed conditions, the retention time of the analytes was noticed to be 6.23 min for fluphenazine and 4.57, 5.30, 9.01 and 8.29 min for impurity 1-4 respectively. The method produces sensitive detection limits of 0.003 μg/mL, 0.013 μg/mL, 0.018 μg/mL and 0.022 μg/mL for impurity 1 to 4 respectively with calibration range of 75-450 μg/mL for fluphenazine and 0.075 - 0.45 μg/mL for impurities. The other validation parameters were noticed to be within the acceptable levels for fluphenazine and its impurities. The drug was exposed to different stress conditions (acid, base, peroxide, thermal and UV light) according to the ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in the ESI positive mode.

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Host cell protein networks as a novel co-elution mechanism during protein A chromatography.

Host cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.