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1.
J Pharm Biomed Anal ; 243: 116124, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38520959

RESUMEN

Peptide mapping is the key method for characterization of primary structure of biotherapeutic proteins. This method relies on digestion of proteins into peptides that are then analyzed for amino acid sequence and post-translational modifications. Owing to its high activity and cleavage specificity, trypsin is the protease of choice for peptide mapping. In this study, we investigated critical requirements of peptide mapping and how trypsin affects these requirements. We found that the commonly used MS-grade trypsins contained non-specific, chymotryptic-like cleavage activity causing generation of semi-tryptic peptides and degradation of tryptic-specific peptides. Furthermore, MS-grade trypsins contained pre-existing autoproteolytic peptides and, moreover, additional autoproteolytic peptides were resulting from prominent autoproteolysis during digestion. In our long-standing quest to improve trypsin performance, we developed novel recombinant trypsin and evaluated whether it could address major trypsin drawbacks in peptide mapping. The study showed that the novel trypsin was free of detectable non-specific cleavage activity, had negligible level of autoproteolysis and maintained high activity over the course of digestion reaction. Taking advantage of the novel trypsin advanced properties, especially high cleavage specificity, we established the application for use of large trypsin quantities to digest proteolytically resistant protein sites without negative side effects. We also tested trypsin/Lys-C mix comprising the novel trypsin and showed elimination of non-specific cleavages observed in the digests with the commonly used trypsins. In addition, the improved features of the novel trypsin allowed us to establish the method for accurate and efficient non-enzymatic PTM analysis in biotherapeutic proteins.


Asunto(s)
Fragmentos de Péptidos , Proteínas , Mapeo Peptídico/métodos , Tripsina/química , Fragmentos de Péptidos/química , Péptidos/análisis
2.
Molecules ; 28(6)2023 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-36985827

RESUMEN

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Monoclonales/química , Espectrometría de Masas/métodos , Cromatografía Liquida/métodos , Glicosilación , Mapeo Peptídico/métodos
3.
Analyst ; 147(6): 1086-1098, 2022 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-35174378

RESUMEN

Almost 60% of commercialized pharmaceutical proteins are glycosylated. Glycosylation is considered a critical quality attribute, as it affects the stability, bioactivity and safety of proteins. Hence, the development of analytical methods to characterise the composition and structure of glycoproteins is crucial. Currently, existing methods are time-consuming, expensive, and require significant sample preparation steps, which can alter the robustness of the analyses. In this work, we suggest the use of a fast, direct, and simple Fourier transform infrared spectroscopy (FT-IR) combined with a chemometric strategy to address this challenge. In this context, a database of FT-IR spectra of glycoproteins was built, and the glycoproteins were characterised by reference methods (MALDI-TOF, LC-ESI-QTOF and LC-FLR-MS) to estimate the mass ratio between carbohydrates and proteins and determine the composition in monosaccharides. The FT-IR spectra were processed first by Partial Least Squares Regression (PLSR), one of the most used regression algorithms in spectroscopy and secondly by Support Vector Regression (SVR). SVR has emerged in recent years and is now considered a powerful alternative to PLSR, thanks to its ability to flexibly model nonlinear relationships. The results provide clear evidence of the efficiency of the combination of FT-IR spectroscopy, and SVR modelling to characterise glycosylation in therapeutic proteins. The SVR models showed better predictive performances than the PLSR models in terms of RMSECV, RMSEP, R2CV, R2Pred and RPD. This tool offers several potential applications, such as comparing the glycosylation of a biosimilar and the original molecule, monitoring batch-to-batch homogeneity, and in-process control.


Asunto(s)
Algoritmos , Glicosilación , Análisis de los Mínimos Cuadrados , Preparaciones Farmacéuticas , Espectroscopía Infrarroja por Transformada de Fourier/métodos
4.
Methods Mol Biol ; 2271: 1-21, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33907996

RESUMEN

Glycosylation is a common posttranslational modification of therapeutic proteins. The glycosylation pattern is dependent on many parameters such as the host cell line or the culture conditions. N- and O-linked glycans usually play a great role on the stability, safety, and efficacy of the drug. For this reason, glycosylation is considered as a critical quality attribute of therapeutic glycoproteins, and a thorough characterization should be performed, as well as a systematic control for each batch produced. This chapter gives a short presentation of the structure of glycans commonly found on recombinant therapeutic proteins, and their role on the properties of the drug, in terms of stability, pharmacokinetics, safety, and efficacy. Lastly, the use of mass spectrometry for the analysis of glycoproteins is briefly described.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Productos Biológicos/uso terapéutico , Glicoproteínas/uso terapéutico , Procesamiento Proteico-Postraduccional , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/farmacocinética , Productos Biológicos/efectos adversos , Productos Biológicos/farmacocinética , Conformación de Carbohidratos , Estabilidad de Medicamentos , Glicoproteínas/efectos adversos , Glicoproteínas/farmacocinética , Glicosilación , Humanos , Espectrometría de Masas , Conformación Proteica , Proteínas Recombinantes/uso terapéutico
5.
Methods Mol Biol ; 2271: 73-83, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33908000

RESUMEN

Middle-up LC-MS antibody characterization workflows using reduction or IdeS digestion for a focused assessment of N-glycan profiling of three representative glycoengineered monoclonal antibodies (mAbs), namely, obinutuzumab (GlycomAb technology, Glycart/Roche), benralizumab (Potelligent Technology, BioWa, Kyowa Kirin) and mAb B (kifunensine) and compared to mAb A, produced in a common CHO cell line. In addition, EndoS or EndoS2 enzyme are used for quantitative determination of Fc-glycan core afucosylation and high mannose for these antibodies, as requested by health authorities for Fc-competent therapeutics mAbs critical quality attributes (CQAs).


Asunto(s)
Alcaloides/análisis , Anticuerpos Monoclonales Humanizados/análisis , Ingeniería de Proteínas , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray , Alcaloides/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Células CHO , Cromatografía Liquida , Cricetulus , Glicosilación , Proyectos de Investigación , Flujo de Trabajo
6.
Methods Mol Biol ; 2271: 179-188, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33908008

RESUMEN

N-glycans are described to have a large influence on the properties of therapeutic proteins, including safety and efficacy. For this reason, the extent and type of glycosylation is a characterization parameter for the analysis of antibodies and other therapeutic proteins. The method described here is a fast and high-throughput method for identification and semiquantification of N-glycans by HILIC-FLR-ESI-MS. Sample preparation has been optimized and simultaneous preparation of a large number of samples can be achieved within a day. The use of MS coupled to fluorescence detection is an additional tool for identifying the N-glycan type.


Asunto(s)
Anticuerpos Monoclonales/análisis , Cromatografía Liquida , Colorantes Fluorescentes/química , Glicoproteínas/análisis , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray , ortoaminobenzoatos/química , Fluorometría , Glicosilación , Ensayos Analíticos de Alto Rendimiento , Proyectos de Investigación , Flujo de Trabajo
7.
Methods Mol Biol ; 2271: 237-247, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33908012

RESUMEN

The presence of sialic acids is one characteristic of glycosylated therapeutic proteins. The presence of these charged monosaccharides is critical for the immunogenicity properties and structural properties of the proteins. Profiling of the N-glycans and their charge state is a requisite for complete protein characterization. Two analytical methods developed on released N-glycans are described in this chapter, allowing for the determination of the sialoglycosylation with different levels of details. In the first method (AEX-HILIC/FLR), N-glycans are separated based on their charge and the average charge state can be determined from the fluorescence profile. In the second method (AEX-RP-FLR-MS), N-glycans are also separated based on their charge and the sialylation level is determined based on the fluorescence signal. In addition, in this method, the N-glycans are also separated by type and identified with the hyphenated MS. For both methods, an optimized protocol with fast and high-throughput sample preparation and purification is presented.


Asunto(s)
Cromatografía Liquida , Colorantes Fluorescentes/química , Glicoproteínas/análisis , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray , ortoaminobenzoatos/química , Fluorometría , Glicosilación , Ensayos Analíticos de Alto Rendimiento , Proyectos de Investigación , Flujo de Trabajo
8.
J Pharm Biomed Anal ; 184: 113179, 2020 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-32092633

RESUMEN

A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phosphorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both are considered as critical quality attributes and should be determined using robust validated methods. The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur) and optionally determination of sodium (or any other metal) as a counter ion. The performance of the method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model substance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision.


Asunto(s)
Organofosfatos/química , Oligonucleótidos Fosforotioatos/química , Espectrometría de Masas en Tándem/métodos , Fosfatos/química , Fósforo/química , Azufre/química , Tionucleótidos/química
9.
Nat Commun ; 9(1): 4483, 2018 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-30367166

RESUMEN

Targeting the expansion of pathogenic memory immune cells is a promising therapeutic strategy to prevent chronic autoimmune attacks. Here we investigate the therapeutic efficacy and mechanism of new anti-human IL-7Rα monoclonal antibodies (mAb) in non-human primates and show that, depending on the target epitope, a single injection of antagonistic anti-IL-7Rα mAbs induces a long-term control of skin inflammation despite repeated antigen challenges in presensitized monkeys. No modification in T cell numbers, phenotype, function or metabolism is observed in the peripheral blood or in response to polyclonal stimulation ex vivo. However, long-term in vivo hyporesponsiveness is associated with a significant decrease in the frequency of antigen-specific T cells producing IFN-γ upon antigen restimulation ex vivo. These findings indicate that chronic antigen-specific memory T cell responses can be controlled by anti-IL-7Rα mAbs, promoting and maintaining remission in T-cell mediated chronic inflammatory diseases.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Memoria Inmunológica/efectos de los fármacos , Inflamación/tratamiento farmacológico , Receptores de Interleucina-7/antagonistas & inhibidores , Linfocitos T/inmunología , Animales , Enfermedad Crónica , Supresión Clonal/inmunología , Modelos Animales de Enfermedad , Humanos , Memoria Inmunológica/inmunología , Inflamación/inmunología , Interferón gamma/inmunología , Papio , Receptores de Interleucina-7/agonistas , Receptores de Interleucina-7/inmunología , Transducción de Señal/efectos de los fármacos , Piel/inmunología , Piel/patología
10.
J Chromatogr A ; 1498: 128-146, 2017 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-28372839

RESUMEN

Proteins are increasingly used as therapeutics. Their characterization is challenging due to their size and inherent heterogeneity notably caused by post-translational modifications, among which glycosylation is probably the most prominent. The glycosylation profile of therapeutic proteins must therefore be thoroughly analyzed. Here, we illustrate how the use of a combination of various cutting-edge LC or LC/MS(/MS) methods, and operating at different levels of analysis allows the comprehensive characterization of both the N- and O-glycosylations of therapeutic proteins without the need for other approaches (capillary electrophoresis, MALDI-TOF). This workflow does not call for the use of highly specialized/custom hardware and software nor an extensive knowledge of glycan analysis. Most notably, we present the point of view of a contract research organization, with the constraints associated to the work in a regulated environment (GxP). Two salient points of this work are i) the use of mixed-mode chromatography as a fast and straightforward mean of profiling N-glycans sialylation as well as an orthogonal method to separate N-glycans co-eluting in the HILIC mode; and ii) the use of widepore HILIC/MS to analyze challenging N/O-glycosylation profiles at both the peptide and subunit levels. A particular attention was given to the sample preparations in terms of duration, specificity, versatility, and robustness, as well as the ease of data processing.


Asunto(s)
Cromatografía Líquida de Alta Presión , Glicoproteínas/metabolismo , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adalimumab/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Secuencia de Carbohidratos , Cetuximab/metabolismo , Electroforesis Capilar , Etanercept/metabolismo , Glicopéptidos/análisis , Glicopéptidos/aislamiento & purificación , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Polisacáridos/aislamiento & purificación
11.
Methods Mol Biol ; 988: 115-43, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23475717

RESUMEN

Glycosylation of the Fc moiety of a monoclonal antibody is a heterogeneous posttranslational process considered as a critical quality attribute of the purified drug substance due to its major impact on safety and efficacy (i.e., immunogenicity, CDC or ADCC effector functions, etc.). Glycosylation should thus be addressed for batch-to-batch comparability and for drug substance characterization, in terms of identity and/or purity testing. We present below a set of efficient, performing and complementary analytical tests that can be used alone or in combination, depending on the information needed and available laboratory instrumentation. The results obtained using these techniques for "global" glycosylation profile, N-glycans profiling, monosaccharides, and sialic acids determination are presented for the Trastuzumab (Herceptin)-humanized mAb produced in CHO.


Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Glicopéptidos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Glucosa/química , Glucosa/aislamiento & purificación , Glicopéptidos/aislamiento & purificación , Glicosilación , Humanos , Datos de Secuencia Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Estándares de Referencia , Ácidos Siálicos/química , Ácidos Siálicos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Ionización de Electrospray/normas , Coloración y Etiquetado , Trastuzumab
12.
J Mass Spectrom ; 41(1): 50-8, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16287034

RESUMEN

Globotriaosylceramides (Gb(3)) are biological compounds implicated in Fabry disease, a lysosomal storage disease due to the deficient activity of alpha-D-galactosidase A, which results in an accumulation of Gb(3) in many organs. The naturally occurring samples are composed of mixtures of several molecular species differing by the structure of the alkyl chains and the nature of the sphingoid base. Atmospheric pressure photoionization mass spectrometry (APPI-MS) proved to be an efficient method for the analysis of globotriaosylceramide molecular species, both in direct injection and by coupling with liquid chromatography (LC). In the positive ion mode, in-source fragmentations yield very precious information that can be used to determine the structure of the alkyl chains. In the negative ion mode, the chloroform solvent participates to the analyte ionization by forming an adduct with chloride ions generated in situ. Combination of LC on a Porous Graphitic Carbon stationary phase and APPI-MS allowed the detection of a great number of species from biological samples isolated from Fabry patients. This method could be an interesting analytical tool for the biochemical investigation of (sphingo) lipid metabolism.


Asunto(s)
Enfermedad de Fabry/orina , Trihexosilceramidas/orina , Biomarcadores , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Grafito , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fotoquímica
13.
Rapid Commun Mass Spectrom ; 19(22): 3379-88, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16259044

RESUMEN

The small chloroplast protein CP12 plays the role of a protein linker in the assembly process of a PRK/GAPDH/CP12 complex that is involved in CO2 assimilation in photosynthetic organisms. The redox state of CP12 regulates its role as a protein linker. Only the oxidized protein, with two disulfide bonds, is active in complex formation. Several observations indicating that CP12 might bind a metal ion led us to screen the binding of different metal ions on oxidized or reduced CP12 using non-covalent electrospray ionization mass spectrometry (ESI-MS) experiments. The oxidized protein bound specifically Cu2+ and Ni2+ (Kd of 26+/-1 microM and 11+/-1 microM, respectively); other cations such as Fe2+ and Zn2+ did not bind, while cations such as Cd2+ formed non-specific adducts to CP12. Similar results were obtained for metal ions on screening with the reduced CP12. Interestingly, the present results suggest that Cu2+ catalyzes the re-formation of the disulfide bonds of the reduced CP12, leading to recovery of the fully oxidized CP12 that is then able to bind a Cu2+ ion. Finally the high similarity between CP12 and copper chaperones from Arabidopsis thaliana, as judged by hydrophobic cluster analysis, provides additional evidence for the relevance of metal binding for the in vivo situation. The findings that CP12 is able to bind a metal ion, and that Cu2+ catalyzes the oxidation of the thiol groups of CP12, are new characteristics of this protein that may prove to be important in the regulation of the assembly process of the PRK/GAPDH/CP12 complex.


Asunto(s)
Cloroplastos/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Metales/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Chlamydomonas reinhardtii , Iones/química , Complejos Multiproteicos/metabolismo , Mutación Puntual , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
14.
Eur J Mass Spectrom (Chichester) ; 11(4): 409-17, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16204808

RESUMEN

The potential of atmospheric pressure photoionization was investigated for the structural analysis of phosphatidylcholine lipids (PCs). [M+H]+ ions of high abundance were obtained, along with several fragment ions. Three of these dissociation products corresponded to quite unusual fragmentation pathways but allowed the determination of both the nature and the position on the glycerol backbone (sn-1 or sn-2) of the fatty acyl chains. The loss of a methyl group from the choline head was also observed. These results suggest a complex ionization mechanism in APPI. However, this method proved to be very powerful for the rapid structural analysis of PC species without using MS/MS experiments.


Asunto(s)
Fosfatidilcolinas/química , Fotoquímica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Presión Atmosférica , Estructura Molecular
15.
Anal Chem ; 75(21): 5961-8, 2003 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-14588038

RESUMEN

The use of photoionization at atmospheric pressure shows great potential for the mass analysis of large apolar or hydrophobic peptides. Mass spectra that were obtained using this technique showed mainly singly charged ions. While polar peptides spectra do not produce fragment ions, others lead to B-type or C-type in-source fragmentation. These dissociation reactions, which could involve electron capture dissociation processes in the case of the C-type ions, are observed for hydrophobic peptides. Both the compatibility of this ionization mode with reversed- or normal-phase liquid chromatographic separation and its sensitivity allow liquid chromatography coupling to both mass spectrometry and tandem mass spectrometry for the analyses of hydrophobic peptide mixtures. Atmospheric pressure photoionization seems to be an interesting alternative method to study hydrophobic peptides that are not easily ionizable by more classical ionization techniques such as electrospray ionization and matrix-assisted laser desorption/ionization.


Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas/métodos , Péptidos/química , Fotólisis , Secuencia de Aminoácidos , Angiotensina II/química , Presión Atmosférica , Bradiquinina/química , Cromatografía Liquida , Gramicidina/química , Iones/química , Datos de Secuencia Molecular , Fotoquímica , Fotones , Polietilenglicoles/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , Rayos Ultravioleta
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