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1.
Methods Mol Biol ; 2571: 83-94, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152152

RESUMO

Capillary electrophoresis-mass spectrometry (CE-MS) is an ideal method for analyzing various metabolites in biological samples. CE-MS can simultaneously identify and quantify hundreds of charged metabolites using only two acquisition methods for positively and negatively charged metabolites. Furthermore, CE-MS is commonly used for analyzing biological samples to understand the pathology of diseases at the metabolic level and biofluid samples, such as blood and urine, to explore biomarkers. Here, we introduce a protocol that delineates the handling of clinical samples to ensure that the CE-MS analysis yields reproducible quantified data. We have focused on sample collection, storage, processing, and measurement. Although the implementation of rigorous standard operating protocols is preferred for enhancing the quality of the samples, various limitations in an actual clinical setting make it difficult to adhere to strict rules. Therefore, the effect of each process on the quantified metabolites needs to be evaluated to design a protocol with acceptable tolerances. Furthermore, quality controls and assessments to handle clinical samples are introduced.


Assuntos
Eletroforese Capilar , Saliva , Biomarcadores , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos
2.
Methods Mol Biol ; 2571: 95-103, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152153

RESUMO

Capillary electrophoresis-mass spectrometry (CE-MS) is gaining interest for metabolomics studies because of its high separation efficiency, selectivity, and versatility. The ability to inject nanoliters from only a few microliters of sample in the injection vial makes this approach very suited for volume-limited applications. However, the low injection volumes could compromise the detection sensitivity of CE-MS, thereby potentially limiting its scope in metabolomics. To overcome this issue, online sample preconcentration methods have been developed to increase sample-loading volumes without hampering the intrinsic high separation efficiency of CE. In this protocol, online preconcentration with sample stacking based on pH junction was assessed for the direct profiling of endogenous metabolites in rat brain microdialysates. Sample stacking was realized by a pre-injection of ammonium hydroxide, followed by a large sample injection (i.e., about 17% of the total capillary volume). It is shown that this relatively simple and fast preconcentration procedure is fully compatible with the high-salt concentration in microdialysates and significantly improves the detection sensitivity of the CE-MS method.


Assuntos
Eletroforese Capilar , Metabolômica , Hidróxido de Amônia , Animais , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Ratos
3.
Methods Mol Biol ; 2571: 105-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152154

RESUMO

The simultaneous analysis of cationic and anionic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) has been considered challenging, as often two different analytical methods are required. Although CE-MS methods for cationic metabolite profiling have already shown good performance metrics, the profiling of anionic metabolites often results in relatively low sensitivity and poor repeatability caused by problems related to unstable electrospray and corona discharge when using reversed CE polarity and detection by MS in negative ionization mode. In this protocol, we describe a chemical derivatization procedure that provides a permanent positive charge to acidic metabolites, thereby allowing us to profile anionic metabolites by CE-MS using exactly the same separation conditions as employed for the analysis of basic metabolites. The utility of the overall approach is demonstrated for the analysis of energy metabolism-related metabolites in low numbers of HepG2 cells.


Assuntos
Eletroforese Capilar , Espectrometria de Massas por Ionização por Electrospray , Animais , Ânions , Cátions , Eletroforese Capilar/métodos , Mamíferos , Espectrometria de Massas por Ionização por Electrospray/métodos
4.
J Pharm Biomed Anal ; 222: 115089, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36279846

RESUMO

This review provides a comprehensive overview of methodological advances and applications of CE in the analysis and characterization of recombinant therapeutic and diagnostic proteins over the past two decades. The first part of the review discusses various aspects of biotechnological protein production and the related effects on the final product. This covers upstream processes, e.g., selection and transfection of host cells, up-scaling of cell cultures and cultivation conditions, as well as downstream processing and a discussion of future trends in biotechnological manufacturing. This part is essential for relating biotechnological production to analytical challenges and requirements in order to provide a holistic insight. In this context, the influence of manufacturing steps on the quality of the final drug substance/product is discussed in terms of related post-translational modifications of the target molecule with a major focus on glycosylation pattern and conformational effects. Particular attention is given to host cell specific and non-human modifications affecting the efficacy and safety of recombinant products. Endowed with this propaedeutic knowledge, the major part of the review discusses the manifold contributions of different CE techniques to the development and optimization of the manufacturing process, to the evaluation and characterization of the final drug product and their role in quality control. Different CE techniques, such as CZE, capillary gel electrophoresis (CGE), (imaged) capillary isoelectric focusing ((i)CIEF), µChipCE, CE-Western blot, affinity CE (ACE), and CE-MS are discussed including a brief introduction in the respective separation and hyphenation principle as well as their applications in the analysis of different recombinant biologics together with recent strategies. The addressed analyte portfolio comprises a vast variety of recombinant proteins with molecular masses from 4.1 kDa up to 20.3 MDa (for recombinant virus-like particles), and a pI range from 2.0 to 11.2. Antibodies are not explicitly covered in the survey. The review is complemented by compiling validation aspects and proposed suitability tests in order to assure the feasibility of methods to industrial and pharmaceutical needs.


Assuntos
Produtos Biológicos , Eletroforese Capilar , Espectrometria de Massas/métodos , Focalização Isoelétrica/métodos , Eletroforese Capilar/métodos , Proteínas Recombinantes
5.
J Pharm Biomed Anal ; 222: 115117, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36306637

RESUMO

Silodosin is a single isomer selective α1-adrenoreceptor antagonist used for the treatment of benign prostatic hyperplasia. In order to control the enantiomeric purity of the drug a capillary electrophoresis method was developed that is applicable to the analysis of drug substance as well as pharmaceutical formulations. Method development followed a quality by design strategy. After selection of carboxymethyl-ß-cyclodextrin as suitable chiral selector and the starting conditions in the scouting phase, a two-level full factorial design was applied to identify the critical process parameters. The final method optimization was performed using a face-centered central composite design resulting in the conditions 100 mM sodium phosphate buffer, pH 2.9, containing 40 mg/mL car-boxymethyl-ß-cyclodextrin, a capillary temperature of 17 °C and an applied voltage of 28 kV. Robustness testing employing a Plackett-Burman design revealed the importance of careful pH adjustment in order to achieve suitable peak shape and resolution. The method was validated according to the guideline Q2(R1) of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and applied to the analysis of a commercial capsule formulation.


Assuntos
Ciclodextrinas , Eletroforese Capilar , Humanos , Eletroforese Capilar/métodos , Estereoisomerismo , Indóis , Ciclodextrinas/química , Concentração de Íons de Hidrogênio
6.
Talanta ; 252: 123780, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35988299

RESUMO

CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). This work describes for the first time the development of ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9. The potential of using a low-resolution MS detector, especially for a GMP environment, as a low-cost and simple method to identify SpyFi Cas9 is discussed.


Assuntos
Sistemas CRISPR-Cas , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Eletroforese Capilar
7.
Anal Chem ; 94(45): 15546-15552, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36342126

RESUMO

A new analytical methodology, using capillary electrophoresis in indirect UV absorbance mode, is developed for the quantification of analytes in the absence of reference materials. The methodology allows the quantification of organic molecules and/or small ions, anionic or cationic, absorbing or not in the UV range, carrying either one or two electric charges. Two methods of data processing were compared. The first is based on the use of a dynamic simulator of electromigration, and the second uses the Kohlrausch regulating function combined with the electroneutrality equation. The experimental conditions presented in this work allow a precise quantification of anions having electrophoretic mobilities (µep) between -22.71 and -36.92 × 10-9 m2 V-1 s-1 and cations with µep between +30.59 and +63.60 × 10-9 m2 V-1 s-1 with percent relative errors lower than -5.52%. The effect of the integration errors on the reliability of the results is discussed in detail.


Assuntos
Eletricidade , Eletroforese Capilar , Reprodutibilidade dos Testes , Ânions/análise , Cátions , Eletroforese Capilar/métodos
8.
Int J Mol Sci ; 23(21)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36362139

RESUMO

The discovery of new antigens specific to multiple myeloma that could be targeted by novel immunotherapeutic approaches is currently of great interest. To this end, it is important to increase the number of proteins identified in the sample by combining different separation strategies. A capillary zone electrophoresis (CZE) method, coupled with drift tube ion mobility (DTIMS) and quadrupole time-of-flight mass spectrometry (QTOF), was developed for antigen discovery using the human myeloma cell line LP-1. This method was first optimized to obtain a maximum number of identifications. Then, its performance in terms of uniqueness of identifications was compared to data acquired by a microfluidic reverse phase liquid chromatography (RPLC) method. The orthogonality of these two approaches and the physicochemical properties of the entities identified by CZE and RPLC were evaluated. In addition, the contribution of DTIMS to CZE was investigated in terms of orthogonality as well as the ability to provide unique information. In conclusion, we believe that the combination of CZE-DTIMS-QTOF and microfluidic RPLC provides unique information in the context of antigen discovery.


Assuntos
Cromatografia de Fase Reversa , Mieloma Múltiplo , Humanos , Espectrometria de Massas em Tandem/métodos , Microfluídica , Linhagem Celular Tumoral , Eletroforese Capilar/métodos
9.
Clin Lab ; 68(11)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36378005

RESUMO

BACKGROUND: CYP2C19 gene polymorphisms have been described to have an important influence on the drug metabolism observed in human populations. A series of PCR-based molecule detection methods are applied to identify CYP2C19 genotype. The aim of the study is to validate the novel CYP2C19 genotyping approach with other methods and reveal the allele frequency distribution of CYP2C19 in Chinese Han population. METHODS: We applied a novel genotyping approach for CYP2C19 gene which was combining direct PCR and capillary electrophoresis (CE) technique. A series of fluorescent labeled primers were designed to amplify the particular DNA fragments which indicated the wild type of CYP2C19 genotype. The variants consist of CYP2C19*2, CYP2C19*3 and CYP2C19*17 alleles. Both the novel PCR-based CE method and real-time quantitative PCR (RT-qPCR) method were used to identify the CYP2C19 genotypes in 324 whole blood samples originated from Chinese Han population. According to the different criterions for judgement of two methods, we can obtain the CYP2C19 alleles and genotypes of the same participants. Kappa statistics was used to evaluate the consistency of the two results and the frequencies of CYP2C19 alleles. The genotypes in Chinese Han population were calculated using EXCEL. Furthermore, to ensure the accuracy and reliability of the CYP2C19 genotypes obtained by using the novel approach, Sanger sequencing was conducted to validate the CYP2C19 genotypes *1/*17 and *2/*3. RESULTS: Among the 324 specimens, 111 were *1/*1, 141 were *1/*2, 10 were *1/*3, 4 were *1/*17, 46 were *2/*2, 10 were *2*/3, 1 was *2/*17, and 1 was *3/*17. Allele distributions for CYP2C19 were *1, *2, *3, and *17 at 58.18%, 37.65%, 3.24%, and 0.93%, respectively. Both PCR-based CE method and RT-qPCR methods had good consistency in the genotypes of CYP2C19 polymorphism (Kappa value = 1.000, p < 0.05). The DNA sequences of CYP2C19 genotype *1/*17 were composed of c.681 G/G, c.636 G/G, and c.-806 C>T. In the same way, the DNA sequences of CYP2C19 genotype *2/*3 were composed of c.681 G>A, c.636 G>A, and c.-806 C/C. CONCLUSIONS: The variants including the CYP2C19*2 allele were the most common mutations in Chinese Han unrelated individuals. Both PCR-based CE method and RT-qPCR method had good consistency in the genotypes of CYP2C19 polymorphism. Nevertheless, because of more convenience and higher throughput, the novel PCR-based capillary electrophoresis approach showed to be more suitable for clinical gene screening.


Assuntos
Eletroforese Capilar , Polimorfismo de Nucleotídeo Único , Humanos , Citocromo P-450 CYP2C19/genética , Genótipo , Reprodutibilidade dos Testes , Frequência do Gene , Alelos , China
10.
Anal Chem ; 94(46): 16151-16159, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36343965

RESUMO

Neuraminidase inhibitors modulate infections that involve sialic acids, making quantitative analyses of this inhibitory effect important for selecting and designing potential therapeutics. An automated nanogel capillary electrophoresis system is developed that integrates a 5 nL enzyme inhibition reaction in line with a 5 min separation-based assay of the enzymatic product to quantify inhibition as the half maximal inhibitory concentration (IC50) and inhibitor constant (Ki). A neuraminidase enzyme from Clostridium perfringens is non-covalently immobilized in a thermally tunable nanogel positioned in the thermally controlled region of the capillary by increasing the capillary temperature to 37 °C. Aqueous inhibitor solutions are loaded into the capillary during the nanogel patterning step to surround the enzyme zone. The capillary electrophoresis separation provides a means to distinguish the de-sialylated product, enabling the use of sialyllactose which contains the trisaccharide motif observed on serine/threonine-linked (O-linked) glycans. A universal nanogel patterning scheme is developed that does not require pre-mixing of enzymes with inhibitors when an automated capillary electrophoresis instrument is used, thus reducing the consumption of enzymes and enabling adaption of the method to different inhibitors. The universal approach is successfully applied to two classical neuraminidase inhibitors with different electrophoretic mobilities. The IC50 and Ki values obtained for N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA) are 13 ± 3 and 5.0 ± 0.9 µM, respectively, and 28 ± 3 and 11 ± 1 µM, respectively, for Siastatin B. These values agree with literature reports and reflect the weaker inhibition anticipated for Siastatin B in comparison to DANA.


Assuntos
Eletroforese Capilar , Neuraminidase , Nanogéis , Eletroforese Capilar/métodos , Polietilenoimina , Inibidores Enzimáticos
11.
Molecules ; 27(21)2022 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-36364432

RESUMO

Advances in the treatment of HR+/HER2- breast cancer phenotype have been made with the introduction of abemaciclib, ribociclib, and palbociclib, inhibitors of cyclin D dependent kinases 4 and 6 (CDK4/6). Here, a novel, fast, cheap, and green CE method for the simultaneous determination of these three CDK4/6 inhibitors in less than 4 min is proposed for the first time. Separation was achieved by capillary zone electrophoresis in an acidic medium, in accordance with the structures of the analytes and their pKa values. The optimal pH of the running buffer was found to be 2.9. The optimal method conditions were 27.5 kV separation voltage, 30 °C, 5 s injection time under 50 mbar pressure, and 50 mM phosphate background buffer with benzimidazole as an internal standard. The developed method was validated with respect to robustness, selectivity, accuracy, precision, linearity, and limits of detection. The method was shown to be linear in the range of 10 to 100 µg mL-1 with correlation coefficients higher than 0.9981. A greenness assessment of the proposed method was performed, and the method was shown to be green. The validated method was successfully applied to pharmaceutical dosage forms of all CDK4/6 inhibitors.


Assuntos
Aminopiridinas , Inibidores de Proteínas Quinases , Quinase 4 Dependente de Ciclina , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Eletroforese Capilar/métodos , Preparações Farmacêuticas
12.
Viruses ; 14(11)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36423148

RESUMO

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.


Assuntos
Eletroforese Capilar , Vírion , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio , Eletroforese em Gel de Poliacrilamida
13.
J Pharm Biomed Anal ; 221: 115059, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36191459

RESUMO

Acquiring accurate and reliable results are crucial in pharmaceutical and biomedical analyses. There is a demand for the ongoing development and validation of advanced analytical and bioanalytical methods. Mass spectrometry (MS) has become an extremely powerful tool for the identification, quantification, and characterization of small and macro-(bio)molecules. Capillary electrophoresis (CE) presents fast and high-resolution separation and in combination with MS allows sensitive and selective identification and detailed characterization. To date, CE-MS has been used to analyze a wide range of molecules, including pharmaceuticals, biopharmaceuticals, metabolites, peptides, and proteins. This review provides an update on recent applications and approaches of CE-MS relevant to biomedical and (bio)pharmaceuticals between January 2018 and May 2022. Furthermore, the latest developments on the hyphenation of CE with MS, as well as different CE modes including capillary isotachophoresis, capillary zone electrophoresis, and micellar electrokinetic capillary chromatography along with on-capillary on-line analyte stacking methods such as field-amplified sample injection, transient isotachophoresis, dynamic pH junction, and solid-phase extraction are discussed.


Assuntos
Produtos Biológicos , Eletroforese Capilar , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Peptídeos , Preparações Farmacêuticas
14.
Food Res Int ; 161: 111780, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36192876

RESUMO

Sulfite is widely used to prevent enzymatic browning in shrimp and lobster processing. However, sulfite may cause allergy in sensitive consumers. Thus, regulatory agencies set limits for its use. Sulfite is usually controlled by the normalized Monier-Williams (MW) titrimetric method that allows a limited number of samples to be analyzed. This manuscript consolidates an innovative method for sulfite inspection in seafood by capillary zone electrophoresis with diode array detector (CZE-DAD). A simple, fast, and simultaneous extraction and derivatization method was developed to provide high throughput for analytical routine. The high instability of the sulfite was suppressed by its derivatization with formaldehyde producing hydroxymethylsulfonate. The evaluation of its analytical performance yielded excellent results in compliance with the strict parameters required for metrological accreditation. The CZE-DAD method was selective and specific when submitted to confirmatory evaluations by liquid chromatography coupled to mass spectrometry. The limit of detection (3.50 mg kg-1), limit of quantitation (11.7 mg kg-1) and recoveries (99-103%) were adequate for sample analysis. The measurement uncertainty was estimated by the propagation of errors and experimental standard uncertainties (precision, accuracy, and analytical curves) and type B uncertainties from traceable measurement instruments. The low relative uncertainty (10%) and the adequate reproducibility demonstrated method suitability. The CZE-DAD results were compared to the MW method through the respective expanded standard uncertainties and normalized error. This new method is promising to be used in seafood inspection and continuous laboratory evaluations using instrumentation not very expensive to acquire and maintain.


Assuntos
Eletroforese Capilar , Sulfitos , Eletroforese Capilar/métodos , Formaldeído , Reprodutibilidade dos Testes , Alimentos Marinhos/análise
15.
Molecules ; 27(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36234709

RESUMO

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved 18O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 µM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the 18O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the 18O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations.


Assuntos
Amidas , Peptídeos , Amidas/química , Asparagina/química , Eletroforese Capilar , Marcação por Isótopo , Espectrometria de Massas/métodos , Peptídeos/química , Proteínas
16.
J Chromatogr A ; 1684: 463560, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36288623

RESUMO

Critical quality attributes (CQAs) of recombinant monoclonal antibody therapeutics are constantly monitored throughout the life cycle of drug development and manufacturing. In the past few decades, numerous analytical techniques have been developed for the characterization of CQAs. In this regard, non-reduced and reduced capillary electrophoresis - sodium dodecyl sulfate (CE-SDS) methods have been widely adopted by the biopharmaceutical industry for the evaluation of size-related heterogeneities in biologics. In this work we demonstrate that, with recent development of capillary electrophoresis - mass spectrometry (CE-MS) technologies, a clipping variant of bevacizumab may be identified directly by both capillary zone electrophoresis - mass spectrometry (CZE-MS) and capillary isoelectric focusing - mass spectrometry (cIEF-MS) approaches, providing a powerful addition to the traditional CE-SDS analysis workflow. In this novel workflow, linear regression between the mobility and molecular weight first results in an approximate size range of this variant. The intact masses of all species in the bevacizumab are then obtained, after deconvolution of all features identified in the CZE-MS analysis. Subsequent  CZE-MS analysis of the subunits of bevacizumab leads to the confirmation of a clipped heavy chain. Furthermore, cIEF-MS of the intact bevacizumab confirms the existence of this clipping variant. The cross-validation between CE-SDS, CZE-MS, and cIEF-MS, creates a comprehensive roadmap for monoclonal antibody size variants profiling. These CE-based analytical techniques are complementary to each other, leading to orthogonal verification for size heterogeneity characterization.


Assuntos
Anticorpos Monoclonais , Produtos Biológicos , Focalização Isoelétrica/métodos , Dodecilsulfato de Sódio , Anticorpos Monoclonais/análise , Bevacizumab , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Proteínas Recombinantes
17.
Molecules ; 27(20)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36296580

RESUMO

In this study, a sensitive capillary electrophoresis (CE) method based on molecularly imprinted solid-phase extraction (MISPE) was proposed to determine histamine in foods. A molecularly imprinted polymer (MIP) synthesized by bulk polymerization was used as the MISPE adsorbent for the selective extraction of histamine. Under the optimal conditions, the MISPE-CE method possessed good linearity for histamine detection in the concentration range of 0.1-100.0 µg/L. The limit of detection and limit of quantification of the method were calculated to be 0.087 µg/L and 0.29 µg/L, respectively. The histamine in spiked rice vinegar and liquor samples were detected by the developed method with recoveries of 92.63-111.00%. The histamine contents in fish, prawn, pork, chicken breast and soy sauce samples were determined using the developed method and a high-performance liquid chromatography method, with no significant difference found between the two methods.


Assuntos
Impressão Molecular , Animais , Impressão Molecular/métodos , Histamina , Polímeros Molecularmente Impressos , Ácido Acético , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos
18.
Molecules ; 27(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36296650

RESUMO

Capillary electrophoresis (CE) is a potent method for analyzing chiral substances and is commonly used in the enantioseparation and chiral purity control of pharmaceuticals from different matrices. The adoption of Quality by Design (QbD) concepts in analytical method development, optimization and validation is a widespread trend observed in various analytical approaches including chiral CE. The application of Analytical QbD (AQbD) leads to the development of analytical methods based on sound science combined with risk management, and to a well understood process clarifying the influence of method parameters on the analytical output. The Design of Experiments (DoE) method employing chemometric tools is an essential part of QbD-based method development, allowing for the simultaneous evaluation of experimental parameters as well as their interaction. In 2022 the International Council for Harmonization (ICH) released two draft guidelines (ICH Q14 and ICH Q2(R2)) that are intended to encourage more robust analytical procedures. The ICH Q14 guideline intends to harmonize the scientific approaches for analytical procedures' development, while the Q2(R2) document covers the validation principles for the use of analytical procedures including the recent applications that require multivariate statistical analyses. The aim of this review is to provide an overview of the new prospects for chiral CE method development applied for the enantiomeric purity control of pharmaceuticals using AQbD principles. The review also provides an overview of recent research (2012-2022) on the applicability of CE methods in chiral drug impurity profiling.


Assuntos
Contaminação de Medicamentos , Eletroforese Capilar , Estereoisomerismo , Eletroforese Capilar/métodos , Controle de Qualidade , Preparações Farmacêuticas
19.
Monoclon Antib Immunodiagn Immunother ; 41(5): 260-274, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36306517

RESUMO

In past few years many rituximab (RTX) biosimilars have been launched in India. Biosimilars are products that are similar in terms of quality, safety, and efficacy to its innovator product and are expected to offer improved affordability. The less clinical examination is a significant source of reduction in the cost of development of a biosimilar. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. Therefore, the role of National Control Laboratory become very important to ensure the quality of these drugs by carrying out analytical characterization at the point of drug product release level as when referred by National Regulatory Authority for quality evaluation. To assess the similarity between innovator and biosimilars, different physicochemical and biological quality attributes were assessed. A multitude of state-of-the-art analysis of N = 3 RTX biosimilars marketed in India revealed that the impurity profiles of these biosimilars measured by charge variant analysis (cation exchange chromatography-high performance liquid chromatography [HPLC], capillary zone electrophoresis, and capillary isoelectric focusing), aggregates profiling (size exclusion chromatography-HPLC), fragments analysis (capillary electrophoresis-sodium dodecyl sulfate) were found to be significantly varying as compared with the innovator product. There were significant variations in acidic variants (p = 0.023) and basic variants (p = 0.0005), isoelectric point value (p < 0.0001), aggregates (p = 0.0231), and fragments (p < 0.0001) of biosimilars were found as that of innovator product. However, these differences were not affecting the biological activity in the cell-based potency analysis by complement-dependent cytotoxicity (CDC) assay (p = 0.1026), antibody-dependent cell-mediated cytotoxicity (ADCC) (p = 0.3736), and binding assay by flow cytometer fluorescence-activated cell sorting (p = 0.4005) of these biosimilars as compared with the innovator product.


Assuntos
Medicamentos Biossimilares , Medicamentos Biossimilares/química , Rituximab/química , Rituximab/metabolismo , Anticorpos Monoclonais , Eletroforese Capilar/métodos , Citotoxicidade Celular Dependente de Anticorpos
20.
Anal Chem ; 94(44): 15415-15422, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36301587

RESUMO

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant (Kd) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of Kd value for a protein-aptamer complex. We conclude that "accurate constant via transient incomplete separation" (ACTIS) can serve as a reference method for affinity characterization of protein-aptamer binding in solution.


Assuntos
Eletroforese Capilar , Oligonucleotídeos , Eletroforese Capilar/métodos , Simulação por Computador , Ligação Proteica , Oligonucleotídeos/química , Entropia
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