Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

Cifuentes, Alejandro; Moreno‐Arribas, M. Victoria; Frutos, Mercedes de; Diez-Masa, Jose Carlos

doi:10.1016/s0021-9673(98)00875-9

Cited by 73 publications

(70 citation statements)

References 23 publications

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“…Por esta razão, o grau de sialilação de cadeias polissacarídicas influencia fortemente a mobilidade eletroforética, o ponto isoelétrico (pI) da molécula e a bioatividade. Os perfis isoelétricos das rhEPOs alfa e beta são bastante similares (pI na faixa de 4,42 a 5,11), mas a EPO-beta tem uma banda extra na região menos ácida (12)(13)(14).…”

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Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Schmidt

Ramos

Silva

et al. 2003

Arq Bras Endocrinol Metab

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RESUMORealizou-se a identificação de eritropoietina humana recombinante em produtos farmacêuticos comerciais por eletroforese em gel de poliacrilamida e detecção com anticorpos específicos, demonstrando-se típi-ca banda larga, semelhante ao padrão de rhEPO da Farmacopéia Européia. Igualmente por focalização isoelétrica e imunodetecção, observaram-se 5-6 isoformas características de acordo com o laboratório produtor. A avaliação de potência efetuada através de ensaio biológico em camundongos normocitêmicos forneceu valores entre 67,6% e 119,4% em relação à declarada. A precisão dos ensaios combinados, calculada pela ponderação, variou de 200 a 389. Os testes de endotoxinas bacterianas, toxicidade e pH apresentaram valores variáveis conforme o lote. Concluiu-se destacando a importância dos testes e ensaios de controle para assegurar a qualidade lote a lote e garantir a eficácia terapêutica dos produtos farmacêuticos. The identification of rhEPO in pharmaceutical products was carried out by polyacrylamide gel electrophoresis and detection with specific antibodies that revealed a typical single broad band similar to that obtained with the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The isoelectric focusing with immunodetection revealed extensive heterogeneity with 5-6 isoforms, characteristic according to the manufacturer. The potency was assessed by the normocythaemic mouse assay with values within 67.6% and 119.4% calculated against the stated potency. The precision evaluated by the weight was within 200 and 389, for the combined assays. The toxicity, bacterial endotoxins, and pH gave variable results according to the batch. In conclusion, we emphasize the importance of the batch-to-batch tests and assays that would guarantee the quality and therapeutic efficacy of the pharmaceutical products.

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unclassified

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Schmidt

Ramos

Silva

et al. 2003

Arq Bras Endocrinol Metab

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“…Although excellent resolution among seven glycoforms is observed in the first analysis, we often encounter difficulty in obtaining reproducible separation [26]. The glycoform resolution becomes worse irreversibly after repeating several analyses [25,[27][28][29][30]. Kinoshita et al [26] successfully separated rhuEPO glycoforms using commercially available surface-modified capillaries for capillary GC in the running buffer containing hydroxypropylmethylcellulose (HPMC) with good reproducibility [26].…”

Section: Glycoform Analysismentioning

confidence: 99%

Capillary electrophoresis for the analysis of glycoprotein pharmaceuticals

Kamoda

Kakehi

2006

Electrophoresis

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Carbohydrate chains in glycoprotein pharmaceuticals play important roles for the expression of their biological activities, but the structure and compositions of carbohydrate chains are dependent on the conditions for their production. Therefore, evaluation of the carbohydrate chains is quite important for productive process development, characterization of product for approval application, and routine quality control. The oligosaccharides themselves have complex structure including blanching and various glycosidic linkages, and oligosaccharides in one glycoprotein pharmaceutical generally have high heterogeneity, and characterization of oligosaccharide moiety in glycoprotein has been a challenging target. In these situations, CE has been realized as a powerful tool for oligosaccharide analysis due to its high resolution and automatic operating system. This review focuses on the application of CE to the glycoform analysis of glycoproteins and profiling of the N-linked glycans released from glycoprotein pharmaceuticals. Current applications for structure analysis using CE-MS(n) technique and glycan profiling method for therapeutic antibody are also described.

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“…Shimura et al (2001) have developed a method to determine the isoelectric points using synthetic peptides as the isoelectric point markers. Recently, improved operation conditions were reported for the separation of glycoforms of rhEPO by CIEF (Cifuentes et al, 1999). Better resolution was achieved using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one separately.…”

Section: Analysis Of Multiple Forms Of Glycoproteins (Glycoform Analymentioning

confidence: 99%

Analysis of glycoproteins and the oligosaccharides thereof by high‐performance capillary electrophoresis—significance in regulatory studies on biopharmaceutical products

Kakehi

Kinoshita

Nakano

2002

Biomedical Chromatography

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This review describes the recent development in the analysis of glycoproteins using capillary electrophoresis with various separation techniques, and focuses especially on the analysis of recombinant glycoprotein pharmaceuticals. We include the analysis of glycoprotein multiforms (ie glycoform) as well as glycan analysis. The relationship between glycoprotein multiforms and oligosaccharide distributions in a glycoprotein sample is also discussed. Further, recent development in capillary electrophoresis-mass spectrometry is described.

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Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

Cited by 73 publications

References 23 publications

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Capillary electrophoresis for the analysis of glycoprotein pharmaceuticals

Analysis of glycoproteins and the oligosaccharides thereof by high‐performance capillary electrophoresis—significance in regulatory studies on biopharmaceutical products

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