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1.
Anal Chem ; 81(17): 7357-62, 2009 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-19663468

RESUMEN

The productivity of mammalian cell culture expression systems is critically important to the production of biopharmaceuticals. In this study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-X(L) gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomarker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene that is known to have growth regulating properties in human cells. A total of 392 proteins were identified in this study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were downregulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-X(L). The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures.


Asunto(s)
Ovario/citología , Ovario/metabolismo , Proteoma/análisis , Proteoma/genética , Proteómica , Proteína bcl-X/genética , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Femenino , Expresión Génica , Transfección
2.
Anal Chem ; 80(19): 7473-81, 2008 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-18729476

RESUMEN

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon beta-1a (IFN-beta-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-beta1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-beta1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-beta1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.


Asunto(s)
Interferón beta/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Infecciones por Cardiovirus/tratamiento farmacológico , Infecciones por Cardiovirus/virología , Línea Celular Tumoral , Efecto Citopatogénico Viral/efectos de los fármacos , Virus de la Encefalomiocarditis/fisiología , Etilmaleimida/análogos & derivados , Etilmaleimida/química , Humanos , Interferón beta-1a , Interferón beta/análisis , Interferón beta/farmacología , Pulmón/efectos de los fármacos , Pulmón/virología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología
3.
J Chromatogr A ; 1123(2): 189-98, 2006 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-16716331

RESUMEN

Characterization and quantitative analysis of oxidation plays an important role in biopharmaceutical development. This study demonstrates an approach to the assessment of susceptible to oxidation methionine residues in monoclonal antibodies and recombinant proteins. A method for the determination of oxidation levels by peptide mapping with mass spectrometric (MS) detection is described and its advantages compared to the UV detection are presented. Good linearity and reproducibility for determination of oxidation with MS detection are demonstrated (R2 > 0.99; RSDs of 4-9%). Aspects of method transfer to quality control group (QC) are discussed. As well, a quick and easy flow injection/MS method is proposed to substitute for peptide map analysis. Peptide coverage, linearity, reproducibility, robustness, sensitivity and quantitative oxidation results are compared for the flow injection/MS and LC/MS approaches.


Asunto(s)
Espectrometría de Masas/métodos , Metionina/química , Proteínas/química , Control de Calidad , Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión , Peróxido de Hidrógeno/química , Oxidación-Reducción , Mapeo Peptídico , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Serina Endopeptidasas/metabolismo
4.
Carbohydr Res ; 341(3): 410-9, 2006 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-16378604

RESUMEN

A comparison of three techniques for quantitative analysis of galactosylation present on immunoglobulins is described. ESIMS, MALDI-TOF MS, and anion-exchange chromatography with fluorescence detection were evaluated in terms of repeatability, limit of quantitation, selectivity, and linearity. A recombinant monoclonal IgG was enzymatically modified in vitro to produce essentially completely galactosylated and degalactosylated forms of the immunoglobulin. Samples of known galactosylation levels were prepared by mixing the modified forms with the native form of the immunoglobulin. Good repeatability and linearity were demonstrated for all three assays (RSDs<1.0%, correlation coefficients>0.99). Differences in selectivity, sensitivity, and other performance aspects of the three techniques are also discussed in this paper.


Asunto(s)
Anticuerpos Monoclonales/química , Carbohidratos/análisis , Inmunoglobulinas/análisis , Anticuerpos Monoclonales/metabolismo , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Fluorescencia , Glicosilación , Datos de Secuencia Molecular , N-Acetil-Lactosamina Sintasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Streptococcus pneumoniae/enzimología
5.
J Chromatogr A ; 1079(1-2): 254-65, 2005 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-16038312

RESUMEN

A simple high-resolution capillary zone electrophoresis (CZE) method capable of rapidly assessing the micro-heterogeneity of a 24 kDa molecular weight glycoprotein, has been developed. Separation is carried out using a bare silica capillary at a pH of 2.5 in a commercially available electrophoresis buffer system composed of triethanolamine and phosphoric acid. Over 30 peaks were detected within a run time of 15 min using a 27 cm capillary and approximately 60 peaks were detected using a 77 cm capillary. Although most of the peaks arise from differences in the oligosaccharide structures present on the one glycosylation site on this molecule, other forms of micro-heterogeneity due to the presence of the nonglycosylated form of this glycoprotein and various types of chemical degradation, e.g., deamidation, are also responsible for the multitude of peaks observed. Although the exact chemical identity of each peak in the resulting electropherogram of this glycoprotein is not known, useful information can be obtained for assessing comparability, stability, and batch consistency. Factors impacting the resolution, precision, accuracy, and robustness of the assay are also discussed along with inherent advantages and limitations associated with measuring the micro-heterogeneity of intact glycoproteins.


Asunto(s)
Electroforesis Capilar/métodos , Glicoproteínas/análisis , Antiportadores , Tampones (Química) , Etanolaminas/química , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Monitoreo Fisiológico , Neuraminidasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Ácidos Fosfóricos/química , Proteínas Recombinantes/análisis , Reproducibilidad de los Resultados , Factores de Tiempo
6.
Anal Biochem ; 348(1): 24-39, 2006 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-16289440

RESUMEN

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.


Asunto(s)
Anticuerpos Monoclonales/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Humanos , Polisacáridos/análisis , Isoformas de Proteínas/análisis , Proteínas Recombinantes/análisis , Sensibilidad y Especificidad
7.
Electrophoresis ; 25(21-22): 3890-6, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15565674

RESUMEN

A method based on microfluidic technology was developed to support quantitative analysis of recombinant monoclonal immunoglobulin G4 (IgG4) antibody samples. The assay was performed on an Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip Kit and the Protein 200 Plus assay software. Capillary electrophoresis principles have been transferred to a chip format that integrates all separation, staining, virtual destaining, and detection steps. The method is referred to in this paper as chip-based capillary gel electrophoresis (GelChip-CE method). The GelChip-CE method under nonreducing conditions proved to be a quantitative test for half-antibody determination in IgG4 samples. Similar to the traditional nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, the GelChip-CE method includes a denaturing step prior to separation. We showed that denaturing the sample by heating resulted in an artificial increase in the amount of half-antibody detected, which could be prevented by addition of N-ethylmaleimide to the sample buffer. The GelChip-CE method allowed for analysis of IgG4 samples with more accuracy, higher precision, and a faster turnaround time than SDS-PAGE and reversed-phase high-performance liquid chromatography (RP-HPLC).


Asunto(s)
Anticuerpos Monoclonales/análisis , Electroforesis por Microchip/métodos , Inmunoglobulina G/análisis , Electroforesis por Microchip/normas , Etilmaleimida , Desnaturalización Proteica , Juego de Reactivos para Diagnóstico , Proteínas Recombinantes/análisis , Reproducibilidad de los Resultados
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