| 1. |
- Nilsson Broberg, Malin
(författare)
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Metabolite Profiling of Drugs using Mass Spectrometry : Identification of analytical targets for doping control and improvements of the metabolite search process
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Doping is defined as the use of prohibited substances or methods by the World Anti-Doping Agency and the aim with doping control analysis is to detect the use of these illicit substances or methods. Substances that are prohibited in human or equine sports have either a positive or negative impact on the performance. Since administered drugs generally are metabolized to a varying degree and thereby not only excreted in their original form, their metabolite profiles are of high interest because drug metabolites may be present in the body for a longer time than the administered drug itself. Thereby detection of metabolites can improve the window of detection. Unfortunately, the metabolite profiles of non-approved drugs that are mainly available on the Internet, such as Selective Androgen Receptor Modulators (SARMs) are often unknown. This thesis consists of four papers that all encompass drug metabolite profiling either in vivo, in vitro or in a combination, utilizing separation with liquid chromatography and detection with high resolution mass spectrometry. In paper I and II, the equine in vivo metabolite profiles of the two SARMs ACP-105 and LGD-3303 were investigated and the results showed that using drug metabolites as analytical targets can prolong the detection time. For ACP-105, the in vivo metabolite profile was compared with different incubation models such as liver microsomes, S9 fractions and the fungus Cunninghamella elegans. The in vivo and in vitro metabolite profiles showed an interesting overlap for several metabolites, demonstrating the importance and usefulness for in vitro methods in doping control, especially since microsome incubates are allowed as reference material. An optimization of microsome incubation conditions utilizing experimental design was presented in paper III and IV, showing that the optimized conditions greatly impacted the yield of drug metabolites, but also that the optimal conditions are substance dependent. In paper III, a multivariate data analysis search tool utilizing OPLS-DA was presented, which greatly simplified the in vitro drug metabolite identification process of ACP-105 and the results showed relevance in comparison with human in vivo metabolites.In conclusion, several new analytical targets with improved detectability for equine and human doping control have been presented, where the drug metabolite profile showed to be of great importance. All together, these new analytical targets, the optimized microsome incubation conditions for improved metabolite yield and the search tool that aids the metabolite investigation through multivariate data analysis, have made a positive contribution to the doping control area.
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| 2. |
- Nilsson, Gunnel, 1950-
(författare)
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Zopiclone degradation in biological samples : Characteristics and consequences in forensic toxicology
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bio-analytical results are influenced by in vivo factors such as genetics, pharmacological and physiological conditions and in vitro factors such as specimen composition, sample additives and storage conditions. Zopiclone (ZOP) is a short-acting hypnotic drug (Imovane®) used for treatment of insomnia. ZOP is metabolized by three major pathways; oxidation to the active zopiclone N-oxide (ZOPNO), demethylation to the inactive N-desmethylzopiclone (NDZOP) and oxidative decarboxylation to other inactive metabolites. ZOP is increasingly being encountered in forensic cases and is a common finding in samples from drug-impaired drivers, users of illicit recreational drugs, victims of drug facilitated sexual assaults and forensic autopsy cases. ZOP is a notoriously unstable analyte in biological matrices and analytical results depend on pre-analytical factors, such as storage time and temperature. The overall aim of this thesis was to investigate the stability of ZOP and the factors of importance for degradation during storage in biological samples and to identify consequences for interpretation of results in forensic toxicology.In paper I, different stability tests in spiked samples were performed including short-term, longterm, freeze-thaw and processed stability. Analyses of ZOP were performed by gas chromatography with nitrogen phosphorous detection and ZOP concentrations were measured at selected time intervals. The degradation product 2-amino-5-chloropyridine (ACP) was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The stability investigations showed a very poor short-term storage stability of ZOP.Therefore, in paper II, the influence of pre-analytical conditions was further investigated in dosed subjects. Whole blood from volunteers was obtained before and after oral administration of Imovane®. In this study, the influence from physiological factors such as drug interactions, matrix composition and plasma protein levels were minimized. The results showed that ZOP was stable in whole blood for only one day at room temperature, one week in a refrigerator and at least three months frozen in authentic as well as in spiked whole blood. The rapid degradation of ZOP at ambient temperature can cause an underestimation of the true concentration and consequently flaw the interpretation. However, by also analyzing the degradation product ACP the original concentration of ZOP may be estimated.In papers III and IV, two LC-MS-MS methods were validated for the quantitation of ACP, ZOP and NDZOP in blood and ACP, ZOP, NDZOP and ZOPNO in urine. These methods were used in a controlled pharmacokinetic study where whole blood and urine were obtained after oral administration of Imovane®. Samples of blood and urine were aliquoted, analyzed and stored under different conditions and the formation of ACP was monitored. Additionally, at each studied time point the pH of the blood and urine samples was measured using i-STAT® system. The results showed that ACP was formed in equimolar amounts to the degradation of ZOP and its metabolites.In urine samples, the formation of ACP occurred at elevated pH or temperature and mirrored the degradation of ZOP, NDZOP and ZOPNO. The high concentrations of metabolites, which also degraded to ACP, made it impossible to estimate the original ZOP concentration.The results from analysis of blood samples containing ACP were also used to develop mathematical models to estimate the original ZOP concentration. Both models showed strong correlation to the original ZOP concentration (r=0.960 and r=0.955) with p<0.01. This study showed that the equimolar degradation of ZOP and NDZOP to ACP could be used to estimate the original concentration of ZOP in blood samples.Absence of ACP in the blood or urine samples analysed strongly suggests that degradation has not occurred and that the measured concentration of ZOP is reliable. For proper interpretation in forensic cases, it is strongly recommended that ZOP and its metabolites as well as ACP are included in the analysis.
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| 3. |
- Pirttilä, Kristian, 1986-
(författare)
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Development of analytical methods for the determination of the small molecule component of complex biological systems
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The research field of untargeted metabolomics aims to determine the relative abundance of all small metabolites in a biological system in order to find biomarkers or make biological inference with regards to the internal or external stimuli. This is no trivial aim, as the small metabolites are both vast in numbers and extremely diverse in their chemical properties. As such, no single analytical method exist that is able to capture the entire metabolome on its own. In addition, the data generated from such experiments is both immense in volume and very complex. This forces researchers to use algorithmic data processing methods to extract the informative part of this data. Such algorithms are, however, both difficult to parametrize and designed to be highly inclusive, the combination of which often leads to errors. One such algorithm is the peak picking procedures used to find chromatographic peaks in liquid chromatography-mass spectrometry (LC-MS) data.In this thesis, four papers are included that focus both on the development of new methods for sample analysis and data processing as well as the application of such, and other, methods in two interdisciplinary research projects. The first paper describes the development and application of a protocol for LC-MS based untargeted analysis of guinea pig perilymph. The focus of the study was to investigate the biochemical processes underlying the protective effect of hydrogen gas on noise-induced hearing loss (NIHL) in guinea pigs exposed to impulse noise. This study sparked two research projects based on limitations observed during the analytical work. The first limitation was that of limited chemical coverage in the analysis when sample volumes are highly limited. The second paper describes the design and validation of a novel separation method for the sequential analysis of both hydrophilic and lipophilic compounds in biological samples. The second limitation observed was the abundance of false peaks reported by peak picking software. These have a negative effect on both downstream data processing as well as data analysis and metabolite identification. The third paper describes the development of a new algorithm for comprehensive peak characterization in untargeted analytical data with the purpose of filtering such false peaks. Both methods presented in the second and third paper were applied to the analysis of guinea pigs perilymph samples in a follow-up study on the attenuating effect of hydrogen gas on NIHL in guinea pigs exposed to broad band continuous noise.
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| 4. |
- Erngren, Ida, 1989-
(författare)
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Analytical method development in liquid chromatography- mass spectrometry based metabolomics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Metabolomics is the analytical field which aims at analyzing all small molecules, metabolites, in a biological system simultaneously. Currently no analytical methods are able to capture the entire metabolome, therefore, the analytical methods are often developed to be as general as possible. However, as research within the metabolomics field is generally driven by biological questions method development is often overlooked. Moreover, method development in metabolomics is very challenging, as evaluation of the methods are difficult since they are not developed for any particular metabolites. Method development is very important though, data quality and accuracy of relative quantitations is paramount if metabolomics is to be used to answer the biological questions at hand.The articles included in the thesis focus around both analytical method development and applications of metabolomics. In the first paper, head and neck cancer cell lines with different sensitivity to ionizing radiation was investigated using LC-MS based metabolomics. A theory on how the radiation resistant (UM-SCC-74B) cell line could alter its metabolism to handle redox status, DNA repair and DNA methylation was formulated. In the second article the sampling of sponge samples (Geodia barretti) was investigated with regard to its effects on detected metabolite profiles and data quality. It was found that freezing the samples directly was the best alternative which allowed for analysis of most metabolite classes. Storing the samples in solvent lead to a substantial extraction of metabolites to the solvent. For metabolomics, the solvents were more useful than the actual sponge samples that had been stored in solvent. In article three the problems caused by high concentrations of inorganic ions in biological samples in HILIC-ESI-MS analyses was described. The inorganic ions can affect relative quantitation and lead to erroneous results and overly complicated datasets inflated by the extra signals caused by cluster formation. To mitigate the problems caused by the inorganic ions a sample preparation method was developed in article four. The method used cation exchange SPE to trap alkali metal ions which, resulted in less ion-suppression, higher signal intensities of relevant metabolites as well as reduced adduct and cluster formation.In conclusion, this thesis have described projects where metabolomics have been applied to answer biological questions as well as analytical method development in LC-MS based metabolomics. Limitations with current methods was described and possible solutions to improve the methods has been presented.
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| 5. |
- Hansson, Annelie
(författare)
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Structural Determination of Drug Metabolites from Doping Classed Compounds Using Mass Spectrometry
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Doping control in equine sports is important for a fair competition, but also to ensure the integrity of the betting system, as well as for animal welfare reasons. To detect the use of illicit compounds, screening for the parent compound is common. However, by using a metabolite as the analytical target instead, the detection time can be prolonged. For some compounds, the use of a metabolite is a necessity since the parent drug may not be detected at all.The metabolites of the selective androgen receptor modulators (SARM) S1, S4 and S22 were investigated in horse urine and plasma. The unchanged parent compounds had the longest detection time in plasma, but were not detected at all in urine. Instead, the longest detection time was measured for the metabolites 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate (SARMs S1 and S4) and 2-amino-5-cyano-4-(trifluoromethyl)phenyl hydrogen sulfate (SARM S22). These metabolites were thus suggested as analytical targets for doping control in urine while the parent compounds were suggested for plasma samples. 2-amino-5-nitro-4-(trifluoromethyl)phenyl hydrogen sulfate could also be produced in large quantities by the fungus Cunninghamella elegans to potentially be used as reference compound.The horse metabolites of the SARM LGD-4033 were also studied in urine and plasma. The formate adduct of LGD-4033 had the longest detection time in plasma and in urine after hydrolysis with β-glucuronidase. In non-hydrolyzed urine, the glucuronidated LGD-4033 was detected instead.Different in vitro models were used to predict in vivo metabolites of roxadustat, a hypoxia-inducible factor stabilizer. Cunninghamella elegans was successful in producing more metabolites compared to human and equine liver microsomes and human hepatocytes.The metabolite detection and identification in all experiments were accomplished using a UHPLC-Q-TOF MS instrument, where the high-resolution MS data was vital in determining which metabolites were formed.The thesis shows the benefits of investigating the metabolites of doping substances to allow for a successful doping control method in horse urine and plasma by prolonging the detection time. It also highlights the usefulness of Cunninghamella elegans as an alternative to the more commonly used in vitro models for both predicting and producing metabolites.
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| 6. |
- Tevell Åberg, Annica, 1978-
(författare)
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Detection and Structure Elucidation of Drug Metabolites in Biological Samples using HPLC-MS/MS Techniques
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the structure elucidation of drug metabolites in biological samples by the use of high performance liquid chromatography (HPLC) atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). Due to their different advantages, various mass analyzers have been used in the different experiments. The metabolism of clemastine, flutamide, and meloxicam were studied in vitro and/or in vivo in different species such as humans, dogs, and horses. Accurate mass measurements with the quadrupole-time of flight mass spectrometer and MSn data supplied by the ion trap instrument were useful in the structural investigation of the product ions of the drugs and their metabolites. Different scan modes of the triple quadrupole mass spectrometer resulted in great flexibility, selectivity, and sensitivity in the qualitative and semi-quantitative studies. Additionally, hydrogen/deuterium exchange and experiments with atmospheric pressure chemical ionization were conducted, and the fungus Cunninghamella elegans was utilized to produce amounts of drug metabolites sufficient for structural investigation. Six isomers of oxidized clemastine were detected and characterized in C. elegans incubations and their retention times and mass spectral data were compared to the metabolites detected in urine samples. Two of the metabolites were concluded to be diastereomeric N-oxides. In urine from horses treated with meloxicam, the peak of 5'-hydroxymethylmeloxicam resulted in much higher intensity than the parent drug or the other metabolites, and it was detectable for at least 14 days after the last dose in some of the horses. That is useful information in the development of analytical methods for the detection of prohibited use of meloxicam. A mercapturic acid conjugate of hydroxyflutamide was detected in urine from cancer patients, which indicated that a reactive metabolite was formed. This metabolite could be responsible for the adverse events reported for flutamide. The results from the four papers included in the thesis clearly demonstrate the usefulness and the flexibility of the HPLC-API-MS/MS technique.
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| 7. |
- Carlsson, Andreas
(författare)
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Synthesis and spectroscopic characterization of emerging synthetic cannabinoids and cathinones
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The application of different analytical techniques is fundamental in forensic drug analysis. In the wake of the occurrence of large numbers of new psychoactive substances possessing similar chemical structures as already known ones, focus has been placed on applied criteria for their univocal identification. These criteria vary, obviously, depending on the applied technique and analytical approach. However, when two or more substances are proven to have similar analytical properties, these criteria no longer apply, which imply that complementary techniques have to be used in their differentiation.This work describes the synthesis of some structural analogues to synthetic cannabinoids and cathinones based on the evolving patterns in the illicit drug market. Six synthetic cannabinoids and six synthetic cathinones were synthesized, that, at the time for this study, were not as yet found in drug seizures. Further, a selection of their spectroscopic data is compared to those of already existing analogues; mainly isomers and homologues. The applied techniques were mass spectrometry (MS), Fourier transformed infrared (FTIR, gas phase) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. In total, 59 different compounds were analyzed with the selected techniques.The results from comparison of spectroscopic data showed that isomeric substances may in some cases be difficult to unambiguously identify based only on their GC-MS EI spectra. On the other hand, GC-FTIR demonstrated more distinguishable spectra. The spectra for the homologous compounds showed however, that the GC-FTIR technique was less successful compared to GC-MS. Also a pronounced fragmentation pattern for some of the cathinones was found.In conclusion, this thesis highlights the importance of using complementary techniques for the univocal identification of synthetic cannabinoids and cathinones. By increasing the number of analogues investigated, the more may be learnt about the capabilities of different techniques for structural differentiations, and thereby providing important identification criteria leading to trustworthy forensic evidence.
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| 8. |
- Stenholm, Åke, 1954-
(författare)
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Investigation of degradation of toxic substances in fungal cultures by mass spectrometric techniques
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Micropollutants in water are biological or chemical contaminants that are present in ground and surface waters in trace quantities. They are a result of human activity and include pharmaceutically active compounds (PhACs) and endocrine disrupting compounds (EDCs). They are eluted to urban wastewater treatment plants (UWWTPs) from households, industries and hospitals. Some of the compounds are recalcitrant (persistent) which means that they enter the aquatic environments in intact forms. In this thesis, some selected micropollutants in water of environmental concern have been chosen to be investigated whether they are suited for biodegradation using filamentous fungi in non-sterile environments. The compounds of key interest were the non-steroidal anti-inflammatory drug (NSAID) diclofenac, nonylphenol polyethoxylates (NPEOs) and finally the aminoglycoside antibiotic neomycin.The white rot fungus (WRF) Trametes versicolor was chosen as the main fungal species candidate in the project. It was used in batchwise and in small scale bioreactor experiments. Mycelia of the species were immobilized on polyurethane foam (PUF) and it was shown that PUF could be used as adsorbent for diclofenac and NPEOs. Furthermore, the species could biodegrade both compounds under co-metabolic conditions (presence of external nutrients). Using UHPLC-Q-TOF MS, with reversed phase chromatography, it was possible to measure the concentration levels of these two target compounds and to tentatively identify previously known and unknown biodegradation products.A screening of 42 fungal species was performed to investigate their ability to survive and grow in a matrix containing toxic nitrogen containing industrial chemicals. Based on this investigation, it was concluded that there are species that are compatible with these harsh conditions which also contained high salt levels. From this study, the mycorrhizal fungal species Rhizoscyphus ericae was selected to be further investigated whether it can biodegrade neomycin.It was concluded that PUF immobilized Trametes versicolor is able to remove a majority of neomycin in co-metabolically performed experiments. In vitro experiments (excluding mycelia), were also performed including a laccase redox mediator system. It was feasible to tentatively determine biodegradation mechanisms that was plausible for both these experimental designs. By varying the levels of nutrients and neomycin and introducing Rhizoscyphus ericae, it was shown that this fungal species is able to use neomycin as nutrient in contrary to Trametes versicolor which only biodegrades neomycin under co-metabolic conditions.
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| 9. |
- Elmongy, Hatem, 1987-
(författare)
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Analytical Methods For Sports Drugs: Challenges and Approaches
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Drugs used to enhance human performance in sport competitions are prohibited by the world anti-doping association (WADA). Biological samples from athletes are continuously tested for adverse analytical findings regarding the identity and/or quantity of the banned substances. The current thesis deals with the development of new analytical methods to determine the concentrations of certain drugs used by athletes and even by regular users for therapeutic purposes. The developed methods aim to analyze the contents of these drugs in the biological matrices; plasma, serum and saliva to provide a successful approach towards either doping detection or therapeutic monitoring. β-adrenergic blockers such as propranolol and metoprolol are used in sports to relief stress and as therapeutic agents in the treatment of hypertension. Both drugs are in chiral forms and available only as racemic mixtures. The different pharmacology of each enantiomer necessitates the monitoring of each enantiomer by stereoselective analytical technique such as chiral liquid chromatography for separation and mass spectrometry for selective detection. The Endogenous anabolic androgenic steroids (EAAS) on the other hand are only notoriously used in sports to increase muscle mass and strength. A method utilizing high-resolution mass spectrometry (HRMS) coupled to ultra-high performance liquid chromatography (UHPLC) was developed for the simultaneous determination of EAAS and their conjugated metabolites to provide a better insight into the steroidal module of the athlete biological passport (ABP). Moreover, the steroidal profile was assessed in serum using the proposed method after the administration of Growth hormone injection as an approach toward the implementation of a new endocrinological module based on steroids biomarkers to hormone doping. Biological samples contain many components that may interfere with the analytical measurements. Therefore, sample preparation methods were developed using solid phase extraction (SPE) and miniaturized techniques such as microextraction by packed sorbents (MEPS) for the purification and pre-concentration of analytes prior to LC/MS analysis.
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| 10. |
- Geurink, Lars
(författare)
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Analytical Quality by Design Method Development for Vaccine Characterization
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Vaccines that are safe, efficacious, and can be rapidly developed are needed to prevent and to react to emerging global infectious disease threats such as influenza, Polio, and Coronavirus diseases. Fast and reliable analytical methods are required without delay to support vaccine process and product development, characterization, and quality control testing. The traditional analytical methods for vaccines are laborious and often lack analytical power, causing slow, expensive, or sometimes failing vaccine development. Capillary electrophoresis (CE) is a technique that has great potential for biopharmaceutical analysis, although there has been limited application in vaccine development.Several novel CE methods were explored, developed, and applied for viral vaccine analysis, making use of the analytical quality by design (AQbD) process and tools. AQbD is a framework of science- and risk-based decision making to achieve in-depth method understanding and to set up fit-for-purpose and in-control analytical methods. Commercial kits for capillary gel electrophoresis (CGE) and imaging capillary isoelectric focusing (icIEF) for antibodies analysis were applied and improved for vaccine analysis. Analytical mechanisms were studied, such as the effect of gel buffer composition on separation, and an AQbD CGE method development strategy was established. The strategy was successfully applied to develop CGE methods for the analysis of seasonal and universal influenza, and sabin inactivated polio vaccine proteins. An icIEF method was also developed, validated, and applied for the universal influenza vaccine protein. A capillary zone electrophoresis (CZE) method development for intact adenovirus concentration determination started with background electrolyte (BGE) and capillary design and screening. An BGE with tris and tricine and a neutral capillary resulted in optimal and robust separation and limited adsorption. The CZE method was validated for seed release, in-process control, product release, and stability testing. The precise, accurate, fast, and robust CZE method was applied for all process intermediates and used at different locations. Process impurities and product degradation could also be characterized.Additionally, CZE methods for chloride and bromide analysis in complex matrices, and a CGE method for host cell DNA characterization were developed for characterization as well as to support process development.Development of CE methods using AQbD reduced lead times and costs. The developed CE methods were easier to use, were more accurate and precise, and were more selective for product and process impurities compared to the previously used analytical methods for vaccines. The use of CE and AQbD helped improve on vaccine safety, efficacy, and quality.
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