Comparative studies on the analysis of glycosylation heterogeneity of sialic acid-containing glycoproteins using capillary electrophoresis

Kinoshita, Mitsuhiro; Murakami, Etsuko; Oda, Yasuo; Funakubo, T.; Kawakami, Daisuke; Kakehi, Kazuaki; Kawasaki, Nana; Morimoto, Kazushige; Hayakawa, Tetsuo

doi:10.1016/s0021-9673(99)01080-8

Cited by 54 publications

(67 citation statements)

References 26 publications

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“…A molécula apresenta estrutura peptídica idêntica ao hormônio natural, mas a composição de carboidratos é heterogênea, a atividade biológica é equivalente (7,8). Diferenças significativas entre a composição de isoformas e propriedades biológicas foram observadas entre as rhEPOs, refletindo, provavelmente, variações nas linhagens de células recombinantes usadas para expressão, condições de cultura ou processos de purificação (4,9,10). A heterogeneidade da rhEPO e da EPO urinária foi pesquisada, e foram separadas 5 a 8 isoformas que são devidas ao grau de glicosilação, bem como aos resíduos de ácido siálico com ou sem Nacetilactosamina (9,11).…”

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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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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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“…In the methodology using CE described in the 2002 European Pharmacopoeia, the separation was performed using a bare fused silica and separation buffer containing putrescine and urea. 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].…”

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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“…In some cases, zwitter ions enhance the resolution of glycoforms due to the formation of ion pairs with glycoproteins. Kinoshita et al (2000) reported partial resolution of recombinant human erythropoietin (rhEPO) in the buffer containing trimethyl-ammonium propansulfonate as the zwitterionic substance using an untreated fused-silica capillary. Further, this mode of separation also made it possible to determine the ingredients in pharmaceutical preparations.…”

Section: Analysis Of Multiple Forms Of Glycoproteins (Glycoform Analymentioning

confidence: 99%

“…Urea was added to inhibit protein aggregation (Watson and Yao, 1993). Kinoshita et al (2000) re-examined this mode of separation in the analysis of rh EPO. A few examples after repetitive analyses of an rhEPO sample separated by this mode are shown in Fig.…”

Section: Analysis Of Multiple Forms Of Glycoproteins (Glycoform Analymentioning

confidence: 99%

“…Typical parameters (linearity and range, limit of detection and limit of quantitation, precision) were evaluated for validation of the nickel-containing capillary electrophoretic method. Kinoshita et al (2000) compared several methods hitherto reported for the glycoform analysis of glycoprotein using rhEPO, fetuin and a1-acid glycoprotein as model samples of glycoprotein. The studies indicated that the resolution of glycoforms was most influenced by sialic acid residues in the protein.…”

Section: Analysis Of Multiple Forms Of Glycoproteins (Glycoform Analymentioning

confidence: 99%

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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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Comparative studies on the analysis of glycosylation heterogeneity of sialic acid-containing glycoproteins using capillary electrophoresis

Cited by 54 publications

References 26 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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