Application of capillary zone electrophoresis and reversed-phase high-performance liquid chromatography in the biopharmaceutical industry for the quantitative analysis of the monosaccharides released from a highly glycosylated therapeutic protein

Racaityte, K.; Kiessig, Steffen; Kálmán, Franka

doi:10.1016/j.chroma.2005.03.080

Cited by 42 publications

(23 citation statements)

References 39 publications

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“…50 l of labeling reagent was added to the hydrolyzed protein solution, and the mixture was incubated for 45 min at 80°C. The samples were diluted to 5 ml in HPLC eluent A (0.3% 1-aminobutane, 0.5% phosphoric acid, and 1% THF in H 2 O), and 90 l were analyzed by HPLC according to separation system I (18). The analysis was performed in three technical replicates.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli

Naegeli

Neupert

Fan

et al. 2014

Journal of Biological Chemistry

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Background: Actinobacillus pleuropneumoniae N-glycosyltransferase is a cytoplasmic glycosyltransferase catalyzing N-glycosylation of polypeptides. Results: In depth analysis of a reconstituted A. pleuropneumoniae glycosylation system in Escherichia coli showed a surprisingly relaxed peptide substrate specificity of N-glycosyltransferase. Conclusion: N-Glycosyltransferase constitutes a general glycosylation system with a preference for autotransporters. Significance: Our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria.

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Section: Methodsmentioning

confidence: 99%

“…Analysis of Monosaccharides on AtaC 1866 -2428 -Analysis was essentially performed as described previously (18). In short, 250 g of protein was precipitated by 25% TCA, and the pellet was washed in acetone and hydrolyzed for 5 h at 95°C in 2.5 M TFA.…”

Section: Methodsmentioning

confidence: 99%

Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli

Naegeli

Neupert

Fan

et al. 2014

Journal of Biological Chemistry

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“…GC-I hydrolysate was hydrolyzed by 2 mol/L trifluoroacetic acid at 100°C for 4 h as previously described (Račaitytė, Kiessig, & Kálmán, 2005), cooled to ambient temperature, neutralized into pH 7.0 using 2 mol/L NaOH, and then detected for galactose content as per the study (Wang & Zhao, 2017) using the galactose assay kit and procedure provided by the kit producer. Lactose content (g/kg protein) was thereby calculated chemically.…”

Section: Chemical Analysesmentioning

confidence: 99%

In vitro immuno-modulatory ability of tryptic caseinate hydrolysate affected by prior caseinate glycation using the Maillard reaction or transglutaminase

Song

Zhao

2017

Food and Agricultural Immunology

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Caseinate was glycated by lactose and oligochitosan via the Maillard reaction and transglutaminase catalysis to yield two glycated caseinates (GC-I and GC-II), followed by trypsin hydrolysis to generate GC-I hydrolysate and GC-II hydrolysate, respectively. In comparison with caseinate hydrolysate, only GC-I hydrolysate showed decreased His, Lys, Tyr, and Val contents. All three hydrolysates in dose-dependent manner exerted in vitro immunomodulatory abilities via activating three immune cells in terms of lymphocytes, macrophages, and natural killer (NK) cells, enhancing the secretion of seven cytokines in lymphocytes and macrophages, but inhibiting the secretion of one cytokine (interleukine-4) in splenocytes. However, GC-I hydrolysate and GC-II hydrolysate showed respective lower and higher immuno-modulatory abilities than caseinate hydrolysate in the most cases. Amino acid losses of GC-I hydrolysate made partially contribution to its impaired immuno-modulatory ability. It is concluded that lactose glycation and oligochitosan glycation of caseinate could lessen and enhance immuno-modulation of tryptic caseinate hydrolysate, respectively. ARTICLE HISTORY

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“…According to previous studies [14,18,19], GlcN and GalN (and other monosaccharides) are derivatized under optimum conditions with AA in methanol-acetate-borate reaction medium to produce derivatives capable of strong absorbance at UV 214 nm. After a very short time (1 h) required for the derivatization process, hexosamines are separated by CE in approximately 10 min (Fig.…”

Section: Resultsmentioning

confidence: 99%

Capillary Electrophoresis

Compton¹,

Brownlee²

2005

Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine

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a b s t r a c tA simple, accurate, and robust quantitative capillary electrophoresis (CE) method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced (i.e., GlcN from Hep and GalN from OSCS) were derivatized with anthranilic acid (AA) and separated by means of CE in approximately 10 min with high sensitivity detection at 214 nm (limit of detection [LOD] of $200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity, allowing direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination given that chondroitin ABC lyase is unable to act on this semisynthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD, and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS, whereas a formulated contaminated Hep was calculated to have 39.7% OSCS.

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Application of capillary zone electrophoresis and reversed-phase high-performance liquid chromatography in the biopharmaceutical industry for the quantitative analysis of the monosaccharides released from a highly glycosylated therapeutic protein

Cited by 42 publications

References 39 publications

Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli

Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli

In vitro immuno-modulatory ability of tryptic caseinate hydrolysate affected by prior caseinate glycation using the Maillard reaction or transglutaminase

Capillary Electrophoresis

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