Nguyet Thuy Tran

Recent innovations in protein separation on microchips by electrophoretic methods: an update.

Lab‐on‐a‐chip electrophoresis is becoming increasingly useful for protein analysis, thanks to recent developments in this field. This review is an update of the review we published at the start of 2008 [Peng, Y., Pallandre, A., Tran, N. T., Taverna, M., Electrophoresis 2008, 29, 156–177]. The superiority of polymers for the manufacture of analytical microchips has been confirmed. This trend implies several modifications to the processes previously used with glass/silicon chips and requires a better understanding of the interfacial phenomena of these materials. Significant progress in chip‐based techniques for protein analysis has been made in the last 2 years. In addition to advances in traditional electrokinetic modes, counter‐flow gradient focusing techniques have emerged as useful methods not only for separation, but also for the online preconcentration of samples. This review, with more than 175 references, presents recent advances and novel strategies for EOF measurement, surface treatment, sample pretreatment, detection and innovations relating to the different modes of separation.

A multi-mode chromatographic method for the comparison of the N-glycosylation of a recombinant HIV envelope glycoprotein (gp160s-MN/LAI) purified by two different processes.

The glycosylation pattern of a recombinant gp160s-MN/LAI variant of human immunodeficiency virus type 1 (HIV-1) was studied in relation to two alternative purification techniques one of which involves an immunoprecipitation step. For analysis a multi-mode high performance liquid chromatography (HPLC) method which combines gel permeation chromatography on the RAAM 2000 GlycoSequencer, weak anion exchange chromatography and normal phase chromatography was developed and profiles were obtained for the fluorescently-labelled glycans released from the two gp160s-MN/LAI preparations. Charged glycans accounted for 77 and 80% of the total glycans for the IAP- and SP-purified samples, respectively. The acidic character of these glycans was mainly due to the presence of sialic acids. However, following sialidase treatment, residual charged glycans were still found. No differences were found in the glycan distributions of the two gp160s-MN/LAI preparations either in their degree of sialylation or in their relative proportion of each separated structure. Although this did not reach statistical significance, a lower proportion of large glycan structures regardless of their charge status was found on the gp160s-MN/LAI prepared by the procedure which involved an immunoaffinity chromatography step.

A validated capillary electrophoresis method to check for batch-to-batch consistency during recombinant human glycosylated interleukin-7 production campaigns

This work reports the validation of a simple CZE method to be used in quality control of recombinant human glycosylated interleukin-7 (rhIL-7) batches produced in Chinese Hamster Ovary (CHO) cells. The separation buffer was a 25mM sodium borate at pH 10 containing 12mM diaminobutane (DAB) used as a dynamic coating agent of the capillary. This method allowed the separation of seven peaks ranging from low to high sialylated glycoforms. An extensive study on conditioning methods of the capillary has been conducted to yield repeatable results. Excellent RSD of EOF mobility (less than 0.6%) was obtained when conditioning included capillary equilibration under virtual analyses and storage in 0.1M NaOH overnight. Method specificity has been demonstrated to be able to discriminate different rhIL-7 glycoforms produced in CHO from formulation matrix. Linearity was demonstrated between 0.5 and 4mg/mL. LOQ was 0.5mg/mL. Repeatability (RSD<1.4 and 3.3% for t(m) and A%, respectively), intermediate precision of inter-day (RSD<2.1 and 4.5), inter-analyst (RSD<2.0 and 3.0) and inter-equipment (RSD<3.8 and 3.7 for electrophoretic mobility and A%, respectively) were all very satisfactory. Evaluation of robustness revealed that pH and DAB concentration are critical parameters in the method while slight alteration of ionic strength of electrolyte or change of capillary source did not affect the results. Finally the method was shown to provide reliable informations to address comparability studies and batch-to-batch consistency of biomanufactured rhIL-7.

In-line coupling SPE and CE for DNA preconcentration and separation.

An in‐line SPE method coupled to CE was developed for the analysis of DNA. The amino silica monolith was prepared in situ by polymerization of tetraethoxysilane and N‐(β‐aminoethyl)‐γ‐aminopropyltriethoxysilane in ethanol aqueous solution at the inlet end of a 100 μm id fused‐silica capillary, and the remaining part of the capillary was used as separation channel. The procedure for this in‐line SPE‐CE method was constructed on the basis of investigation on operational conditions such as the introduction mode of sieving matrix, the composition of elution solvent and the elution time. Twenty millimolar ammonium hydroxide was demonstrated to be effective for DNA desorption from the monolith, and linear poly(N‐isopropylacrylamide) was used as the separation matrix. The proposed method could achieve limits of detection of 0.065–0.123 ng/mL for six DNA fragments ranging 100–2000 bp. Compared with conventional CE, preconcentration factors of over 100 times were obtained. The applicability of the in‐line SPE‐CE method was further demonstrated by analyzing plasmid DNA from Escherichia coli crude lysate.

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

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.

A capillary zone electrophoresis method to detect conformers and dimers of antithrombin in therapeutic preparations.

Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti‐inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide‐coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.

Chapter 20 Analysis of glycoproteins and their glycopeptide and glycan fragments by electrophoresis and capillary electrophoresis

Publisher Summary This chapter discusses the analysis of glycoproteins and their glycopeptide and glycan fragments by electrophoresis and capillary electrophoresis. Glycoproteins are composed of a population of closely related glycosylation variants that contain the same polypeptide chain but vary by their carbohydrate moiety. This carbohydrate-mediated heterogeneity is often called as the “microheterogeneity of glycoproteins,” and the different variants are referred to as “glycoforms.” Carbohydrate groups are covalently attached to the polypeptidic backbone through the amide nitrogen of an asparagine residue ( N -glycosylation) or through an O -linkage with a serine, threonine, and, sometimes, hydroxyproline residue ( O -glycosylation). O -linked oligosaccharides are generally smaller and less complex than N -linked glycans. Glycosylation is often a critical determinant in many properties of glycoproteins; this is particularly important in the case of therapeutic glycoproteins as biological activity, clearance rate, immunogenicity, solubility, and stability may significantly be affected by the type of oligosaccharide attached to a protein.

Capillary Electrophoresis of Proteins and Peptides

Capillary electrophoresis (CE) coupled to fl uorescence detection is an invaluable technique for the quantitative analysis of proteins of interest in the fi eld of clinical diagnosis and quality control of novel biotechnology products. The various chemical and instrumental approaches that have been reported to carry out such sensitive analysis are described in this paper. To illustrate the contribution of CE to the analysis of therapeutic proteins, a detailed protocol for impurities profi ling of a recombinant antibody sample using CE-LEDIF is given.

Characterization of Chemical and Physical Modifications of Human Serum Albumin by Capillary Zone Electrophoresis

Therapeutic proteins can easily undergo chemical or physical changes during their manufacturing, purification, and storage. These modifications might change or reduce their biological activity. Therefore, it is important to have analytical methodologies that are able to reliably detect, characterize, and quantify degradation products in formulations. Capillary Zone Electrophoresis (CZE) is very well suited for the analysis of proteins due to its relatively easiness of implementation, separation efficiency, and resolving power. We describe here a CZE method that allows separating more than nine forms in therapeutic albumin, including oxidized, glycated, and truncated forms. This method uses a polyethylene oxide (PEO) coating and a buffer composed of HEPES and SDS at physiological pH. The method is reproducible (RSD < 0.5 and 4 % for migration times and peak areas, respectively) and allows quantitation of albumin forms in pharmaceutical preparations.