Estimation of the composition of recombinant human erythropoietin mixtures using capillary electrophoresis and multivariate calibration methods

Benavente, Fernando; Giménez, Estela; Olivieri, Alejandro C.; Barbosa, J.; Sanz‐Nebot, Victoria

doi:10.1002/elps.200600132

Cited by 13 publications

(7 citation statements)

References 29 publications

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“…In summary, two trends can be found in literature: (1) Sialoform separation seems to be best accomplished close to the pI of the glycoprotein of interest and can then be applied to determine charge heterogeneity using UV detection, nice examples are given in [116,121]. (2) Sialoform and (partial) glycoform (of like charge) separation relies on strongly acidic conditions for most glycoproteins.…”

Section: Resolution and Information Achieved For Intact Glycoproteinsmentioning

confidence: 96%

“…Applications of intact glycoprotein analysis are comprehensively summarized in [135]. Examples for a broad range of applications are: the determination of protein stability [116], the assessment of the charge heterogeneity of a recombinant protein within quality control [116], the observation of glycosylation changes due to host cell or growth media changes [116], the quantitative comparison between EPO alpha and beta in binary mixtures using PLS [121].…”

Section: Summary and Applicationsmentioning

confidence: 99%

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Protein glycosylation analysis with capillary-based electromigrative separation techniques

Pattky

Hühn

2010

Bioanal Rev

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An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-ofprinciple studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.

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Section: Resolution and Information Achieved For Intact Glycoproteinsmentioning

confidence: 96%

Section: Summary and Applicationsmentioning

confidence: 99%

Protein glycosylation analysis with capillary-based electromigrative separation techniques

Pattky

Hühn

2010

Bioanal Rev

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“…Recently, commercially available polymers (EOTrol and UltraTrol) from Target Discovery (Palo, CA, USA) have been introduced [43] and provided glycoform profiling of erythropoietin [44,45], fetuin or a-acid glycoprotein [46]. Alkylamines as BGE additives have been reported not only to prevent protein adsorption by ion association with the negative charges on the capillary surface [47][48][49], but also to improve the glycoform resolution [50][51][52]. Combination of alkylamine and borate buffer, which is well known to promote glycoform separation through its interaction with sugars, is also a successful strategy [53][54][55].…”

Section: Introductionmentioning

confidence: 99%

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

Alahmad

Tran

Duboeuf³

et al. 2009

Electrophoresis

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The aim of the present work was to develop a simple high-resolution CZE method for glycoform profiling and quality assessment of recombinant human interleukin-7 (rhIL-7) produced in Chinese hamster ovary cells. Classical and isoelectric buffers and several monoamines used as alkaline component of BGE at very low concentration have been investigated in order to optimize the resolution. The best separation was achieved using a fused-silica capillary and a buffer composed of 25 mM citrate/triethanolamine at pH 2.6. Under these acidic conditions, triethanolamine was able to reverse EOF favorably and to limit rhIL-7 adsorption. This method allowed the separation of four groups of peaks ranging from low to high-sialylated glycoforms. An extensive study on inter-run rinsing procedures has been conducted. Rinsing with 50 mM SDS was retained to achieve the optimal repeatability. Excellent intermediate precision was obtained for migration time (RSD < 0.6%), while RSD for intraday studies were only less than 2.9%. Satisfactory inter and intraday repeatabilities were also observed for relative peak area. We finally demonstrated that reliable information could be obtained to address comparability studies and demonstrate batch-to-batch consistency of biomanufactured rhIL-7.

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“…In such cases, the methods traditionally used for the analysis of MS data may be excessively time consuming. Chemometrics-assisted multiway data analysis is an excellent alternative for handling these complex data sets [10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

“…Using three-way data, determinations in the presence of unknown interferences are possible, when the instrumental signals are separated mathematically by achieving the second-order advantage [11]. We have recently demonstrated that a first-order multivariate calibration method with partial least squares (PLS) can be used to characterize mixtures of different erythropoietin samples based on the analysis of the electrophoretic separations of their glycoforms at a single wavelength [12]. However, the use of second-order CE-DAD data for multiway analysis of individual protein isoforms is precluded because the UV spectra of the comigrating isoforms are indistinguishable from each other.…”

Section: Introductionmentioning

confidence: 99%

A multiway approach for classification and characterization of rabbit liver apothioneins by CE‐ESI‐MS

et al. 2008

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We applied a multiway approach to extract information from the analysis of protein isoforms by CE-ESI-MS. Metallothioneins (MT) are low-molecular-weight proteins (6-7 kDa) with a strong affinity for heavy-metal ions. Rabbit liver MT-I and MT-II fractions are purified from MT samples. At low pH, the bound metal ions were released from the amino acid structures, giving rise to apothioneins. MT-I, MT-II and MT apothioneins, which are complex mixtures of protein isoforms, were analyzed by CE-ESI-MS. After data pre-processing, parallel factor analysis (PARAFAC) and multivariate curve resolution-alternating least squares (MCR-ALS) were applied to the data sets. In both cases, the models enabled classification of the protein samples and identification of their characteristic sub-isoforms using a set of three components. MCR-ALS required an initial estimate of the pure mass spectra of the three components. Thus, PARAFAC loadings were used to initialize the MCR-ALS optimization. The classifications obtained with MCR-ALS were slightly better than those obtained with PARAFAC, probably because MCR-ALS was less affected by the small migration time shifts of the preprocessed electropherograms. However, no differences were found between the pure mass spectra of the three components in either model. Finally, MCR-ALS allowed us to obtain an individual electrophoretic profile of each of the three components for each of the samples analyzed, which proved valuable for characterization and quantification purposes.

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Estimation of the composition of recombinant human erythropoietin mixtures using capillary electrophoresis and multivariate calibration methods

Cited by 13 publications

References 29 publications

Protein glycosylation analysis with capillary-based electromigrative separation techniques

Protein glycosylation analysis with capillary-based electromigrative separation techniques

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

A multiway approach for classification and characterization of rabbit liver apothioneins by CE‐ESI‐MS

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