Recent advances in the capillary electrophoresis of recombinant glycoproteins

Analytica Chimica Acta 383 (1999) 137±156

Review

Recent advances in the capillary electrophoresis of recombinant glycoproteins Anastasia Pantazakia, Myriam Tavernab,*, Claire Vidal-Madjarc a

Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece Laboratoire de Chimie Analytique, Centre d'Etudes Pharmaceutiques, 5 rue Jean-Baptiste CleÂment, 92296 ChaÃtenay-Malabry, France c Laboratoire de Recherche sur les PolymeÁres, CNRS UMR C7581, 2 rue Henry Dunant, 94320 Thiais, France

b

Abstract Highly ef®cient methods are required to analyze recombinant proteins for clinical use. These proteins generally produced from mammalian expression systems are highly glycosylated and consist of a population of glycosylated variants (glycoforms). This review presents the different microscale techniques of capillary electrophoresis (CE) for analyzing the intact recombinant glycoproteins and for monitoring their bioproduction. Because of several advantages such as simplicity, speed and automation, capillary zone electrophoresis (CZE) has been generally employed for the routine analysis of the glycoform populations of intact glycoproteins. Capillary isoelectric focusing (CIEF) is a powerful method for a charge-based separation of the glycoforms. Micellar electrokinetic capillary chromatography (MEKC) represents an alternative method to CZE for the purity control of recombinant glycoproteins, while the sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) with replaceable gel matrices gives an estimation of the glycoform molecular masses. The results from CIEF and SDS-CGE are comparable to those from the corresponding slab gel techniques. The recent advances in the coupling of CZE with mass-spectrometry (MS) offers new perspectives not only for precise molecular mass determinations, but also to better understand the mechanisms involved in the CE separation of glycoforms. # 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Review; Capillary electrophoresis; Recombinant; Glycoprotein; Glycoform separation

1. Introduction A large number of proteins are obtained from the recombinant DNA technology. Some of therapeutic value are glycoproteins. The carbohydrate groups are covalently attached at the polypeptidic chain through the amide nitrogen of the asparagine residue (N-glycans) or via O-linkage to serine, threonine or, in rare cases, to hydroxyproline residues. In addition, Noligosaccharides can be classi®ed on the basis of *Corresponding author.

the nature of the monosaccharide moieties of the glycan chains [1]. 1. High mannose oligosaccharides only consist in mannose and N-acetylglucosamine residues. 2. The molecular structure of complex-type oligosaccharides contains galactose, fucose and neuraminic acid residues in addition to the monosaccharide moieties found in the high mannose glycan type. 3. Hybrid structures are made of elements of both high mannose and complex oligosaccharide structures.

0003-2670/99/$ ± see front matter # 1999 Published by Elsevier Science B.V. All rights reserved. PII: S0003-2670(98)00495-4

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In contrast, O-linked oligosaccharide side chains are generally shorter than the N-linked analogues, but show larger differences with respect to their monosaccharide moieties. They include glucose, xylose, N-acetylgalactosamine, arabinose and other monosaccharide moieties found in N-glycans. One feature of glycosylated proteins is the presence of sites that can be linked to glycan chains of various structures giving rise to extremely heterogeneous glycoprotein populations, named glycoforms. The oligosaccharide structure of recombinant glycoproteins greatly depend on the system used for gene expression and on the culture conditions [2]. For example the recombinant proteins expressed in Escherichia coli are not glycosylated [3]. Differences in the oligosaccharide structures have been found among recombinant glycoproteins expressed in mammalian cells. These differences are related to the presence and activities of the glycosyltransferases and glycosidases. In cells such as those from chinese hamster ovary (CHO) and from baby hamster kidney (BHK) a functional enzyme, -2,6-sialyltransferase is lacking and these cells synthesize exclusively 2,3linked sialic acids, while the recombinant C127 cells generate N-linked oligosaccharides containing only the linkage sialic acid (2,6)-bonded to galactose moiety. Variations in the oligosaccharide chain structure present on glycoproteins can signi®cantly affect many protein properties such as solubility, speci®c activity, circulatory half life, antigenicity, resistance to protease attack and thermal denaturation [2]. Furthermore, a variety of factors in the cell culture environment have been recently implicated in affecting N-linked glycosylation [4]. Ammonium ion concentrations of the culture medium ranging from 0 to 10 mM have been shown to signi®cantly reduce the level of sialylation of granulocyte colony stimulating factor (G-CSF) produced by recombinant CHO cells [5]. In addition, cultivation mode (either adherent or suspended), process time or cell ages may also affect the glycosylation of recombinant glycoproteins. In conclusion, not only the protocol for protein puri®cation may have a strong effect on the distribution of puri®ed glycoforms, but also the reproducibility of glycoform distribution will depend on that of the puri®cation procedure [2,6].

For these reasons, quality control of the ®nal product is compulsory for the delivery of a well de®ned and safe therapeutical agent. To monitor the puri®cation procedure, in-process controlling will also be necessary. The purity of the product has to be assessed, since the complex way of production may result in byproducts which are likely to be biologically active (e.g. host cell proteins, dimers. . .). Moreover, during the puri®cation and product-®lling, proteins may form aggregates, be partially degraded by protease or thermally denatured. For example, in the produced polypeptide misfolded or aggregated forms can be generated. Monitoring of the structural integrity of the DNA-derived product constitutes a signi®cant part in the control of the identity of recombinant proteins. As the glycosylation pattern can vary quite easily with the fermentation and cell culture conditions, the consistency from lot to lot is an important aspect to be considered. Demands from regulatory authorities require increasing ef®ciency in carbohydrate analyses as part of the validation of products or processes [7]. Recombinant glycoproteins, however, form one of the most challenging classes among the biopharmaceutical products to analyze. For these reasons, a wide range of analytical investigation is necessary in order to guarantee the activity, purity, identity and safety of the active product. Several publications have recently reported the contribution of capillary electrophoresis (CE) in carbohydrate or glycoprotein analysis [8±11]. A review on the applications of high-performance CE for glycoprotein analysis [12] has appeared in a special thematic issue. One can also refer to papers published in this volume for detailed informations concerning various aspects of carbohydrate analysis by chromatography and CE. The characterization of glycoproteins is generally achieved by analyzing the peptide and glycopeptide fragments enzymatically releazed from the glycoproteins. As this classical approach was described in detail, the current review will consider the main strategies for analyzing the glycoforms of the intact recombinant glycoproteins by means of the various modes afforded by CE: capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MEKC), capillary isoelectrofocusing (CIEF) and capillary gel electrophoresis (CGE). Recent applications of the coupling of CE with mass spectrometry will be also described. This paper will

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summarize the methods of CE and point out the practical problems encountered in quality control and in the production of recombinant derived glycoproteins. A particular emphasis will be given to the new strategies recently proposed to overcome several problems such as adsorption of proteins on the capillary wall and lack of solubility of some glycoproteins. 2. Analysis using capillary zone electrophoresis (CZE) Many examples of separations of intact recombinant glycoproteins using CE have been already reported. For glycoform separation, CZE is well suited as the variants can be separated even in neutral medium on the basis of their charge differences mainly related to their various degrees of sialylation, sulfatation or phosphorylation, as well as to differences in masses. One of the main limitations of the CZE of glycoproteins and proteins in general is the adsorption of sample components on the capillary walls. Besides sample loss, the consequences are: decrease of separation ef®ciency and poor reproducibility of migration time. Basically three strategies are generally employed to overcome this problem [13]. Electrostatic interactions can be weakened with a decrease of the charge of the capillary wall by selecting a low pH for separations. Alternatively working above the pI of the glycoproteins may induce electrostatic repulsions between protein molecules and capillary surface. Another method is the inclusion of various additives into the separation buffer which compete with the protein molecules for the negatively charged silanols of the capillary wall. The third method of reducing glycoprotein adsorption is chemical modi®cation of the capillary wall in order to give the best shield of silanol groups. Critical conditions such as capillary coating, buffer composition, concentration and pH for separating the microheterogeneous components of glycoproteins by CE have been recently discussed by Chen [14]. 2.1. Glycoform separation by CZE 2.1.1. CZE with untreated fused-silca capillaries The CZE method was used to analyze the glycoform population of recombinant human tissue

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plasminogen activator (rtPA), a glycoprotein which exhibits a certain complexity in its glycosylated structure. The rtPA, with a molecular mass of about 60 000 Da and 527 aminoacids, is a ®brin-speci®c plasminogen activator approved for the treatment of myocardial infarction. Two glycosylation variants exist [15]: type I is glycosylated at asparagine residues 117, 184, 448, whereas type II is glycosylated at asparagine residues 117 and 448. At Asn 117, Nlinked high mannose oligosaccharides are mainly found. At Asn 184 and 448, the N-linked glycosylation sites consist of bi-, tri-, tetra-antennary structures containing sialic acid residues. This heterogeneity in combination with the microheterogeneity at each glycosylation site forms a plurality of glycoforms. The ®rst attempts to separate the glycoforms of rtPA were reported by Wu et al. [16] using a polyacrylamidecoated capillary. The separation was unsuccessful, with a large single broad peak. An incomplete resolution of several glycoforms was achieved by Taverna et al. [17] using fused-silica capillaries in presence of phosphate buffer (pH 3.6). With the uncoated capillaries employed, a strong protein adsorption occurs onto the capillary walls. As shown by Yim [18] a partial resolution of the plethora of the rtPA glycoforms is possible with an ammonium phosphate buffer (pH 4.6) containing 0.01% Triton X-100 and 0.2 M aminocaproic acid (EACA). The resolution is not as good as in the HCIEF mode but still 15 peaks can be observed. The improved separation is ascribed to an increase of the rtPA solubility in presence of EACA. Several papers have been published about the characterization and analysis of human recombinant erythropoietin (rHuEPO) by CZE. Erythropoietin (EPO) is a glycoprotein hormone produced in adult kidney and fetal livers; it regulates the red blood cell production [19]. rHuEPO has been produced using recombinant DNA technology by the cloning and expressing of the human EPO gene and pharmaceutical preparations of rHuEPO are commercially available since 1988. Its molecular mass is 30 400 Da, with a carbohydrate content of about 40% [20]. Several glycoforms exist that differ by the degree of glycosylation and the number of sialic acid residues [21]. The CZE was used to separate rHuEPO into different glycoform populations. Tran et al. [22] studied the effects of various factors such as pH, buffer type and organic additives on glycoform resolution in free

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solution CZE. The critical role of the phosphate ions in improving the separation was demonstrated. The separation into four different major and one minor glycoforms was obtained with a mixed buffer 100 mM acetate±phosphate. A 10 h reequilibration time is needed for reproducible results. Watson and Yao [21] described the CZE separation of the glycoforms of rHuEPO into six well resolved peaks with an uncoated silica-fused capillary using a tricine buffer (pH 6.2) with the addition of 2.5 mM 1,4-diaminobutane (DAB) and 7 M urea (Fig. 1(c)). The rHuEPO has three N-linked oligosaccharides units with a variable number of sialic acid residues. At pH>4, these sialic residues are negatively charged and migrate against the electroosmotic ¯ow (EOF). It is thus considered that the glycoforms elute in the order of increasing number of sialic acid residues. Additional evidence of this elution was given from preparative gel isoelectric focusing (IEF) and from the electrophoretic separation of the rHuEPO in which the sialic acid groups were removed by a treatment with neuramidase. The electropherogram of Fig. 1(a) shows a poor resolution with a single broad peak, when the buffer is used with no additives. A decrease of the EOF is observed by adding 2.5 mM of 1,4-DAB and the resolution is greatly improved (Fig. 1(b)). The optimal results are obtained by the addition of urea (Fig. 1(c)) giving six well resolved peaks. Various simultaneous phenomena can be invoked to explain the ef®ciency of this separation: reduction of the EOF by the addition of the organic cation modi®er, increase in the charge differences between glycoforms, minimization of solute±wall interaction. The improvements due to urea addition were attributed to a deaggregation of the sample and a disruption of hydrophobic and noncovalent interactions, causing a reduction of solute adsorption on the capillary walls and thus an improvement of peak sharpness. Watson and Yao [23] have evaluated free CZE as an alternative technique to separate the glycoforms of recombinant human granulocyte-macrophage colony stimulating factor (rhGM±CSF). The rhGM±CSF produced in CHO cells was selected because this wellcharacterized protein is obtained in a highly puri®ed state. It contains two O-linked carbohydrate moieties having one or two sialic acid residues. The effect of pH on the separation of the rhGM±CSF glycoforms

was evaluated. At pH values of 7±9, the separation gives two peaks of equal size. The best resolution was obtained by adding 2.5 mM of 1,4-DAB to the phosphate±borate buffer. The glycoforms migrate in the order of increasing number of sialic acid residues. The usefulness of DAB and of diaminoalkanes in general to achieve the CZE analysis of proteins and glycoproteins has been reported by other authors. [24±27]. Oda and Landers [28] have investigated the possible mechanisms by which these compounds in¯uence the resolution of ovalbumin glycoforms. This favorable effect is attributed to a reduction of protein adsorption induced not only by the binding of these diamines to silica, with a reduction of the EOF (they behave as divalent cations in the electrolyte) but also to a favorable combination of borate complexation with diaminoalkanes cations [17,26]. Human recombinant blood coagulation factor VII has been obtained from a mammalian expression system and its activated form (rFVIIa) was puri®ed and characterized [29]. The CZE technique was tested to separate the glycoforms of this recombinant glycoprotein having multiple O and N-glycosylated sites [30]. Polyamines and mainly putrescine were used as additives to the 100 mM phosphate buffer with an optimum separation at pH 8. The electropherogram showed more than six distinct glycoforms primarily related to differences in the content of N-acetyl-neuraminic acid. These results suggest that the resolution induced by the addition of putrescine was not only caused by a reduction of the EOF but also by an ion-pairing mechanism between the divalent cations and the glycoforms. Replacement of putrescine by cadaverine resulted in an almost identical glycoform pattern indicating that probably other ,!-diaminoalkanes are potential ion-pair reagents. Further CE and high-performance liquid chromatography (HPLC) experiments were performed to characterize the rFVIIa after hydrazinolysis or neuramidase treatments. A recombinant basic chimeric glycoprotein, FG, was characterized in CZE by using fused-silica capillaries and a dynamical coating with an amphipathic polymer [31]. This dynamic coating layer is useful for the separation of basic proteins as it enables to reverse the EOF to the anode [32]. A net positively charged amine layer is formed on the capillary wall surface that reduces the adsorption of cationic proteins. The

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Fig. 1. Effect of additives on the CZE separation of rHuEPO: Sample: 1 mg/ml; fused-silca capillary (50 cm75 mm i.d.); voltage: 10 kV. Buffers at pH 6.2: (A) 10 mM tricine/10 mM NaCl; (B) 10 mM tricine/10 mM NaCl/2.5 mM 1,4-diaminobutane; (C) 10 mM tricine/10 mM NaCl/2.5 mM 1,4-diaminobutane/7 M urea. UV detection at 214 nm (reprinted from [21] with permission from Academic Press).

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amphipathic polymer-coated column is stable for one electrophoretic run and the column has to be recoated with the reagent before every analysis. The glycoprotein (pI 9.4) is composed of the fusion protein (F) and the receptor protein (G). The chimeric FG glycoprotein is developed as a vaccine for lower respiratory disease in children younger than two years old. With a 50 mM sodium citrate-acetic acid buffer (pH 5.2), a good resolution in two peaks was obtained. It was not possible to separate this highly glycosylated protein into its glycoforms. Poor results were obtained with the C8 or C18 derivatized capillary columns and a single highly skewed elution peak is observed, most probably because of protein adsorption on the capillary walls due to the hydrophobic interaction of protein molecules with the grafted alkyl chains. As described in next sections, hydrophilic coatings are needed to decrease protein adsorption on the silica capillary walls. 2.1.2. CZE with permanently coated capillaries Modi®ed fused-silica capillaries by a covalently bound polymer have several advantages in the CE of proteins: the shielding of the silanol groups of the silica surface will minimize the protein-capillary wall interactions and reduce peak tailings and band broadenings. A decrease and control of the EOF in a wide pH range of the buffer is useful to separate proteins with different pI values. The reproducibility of analyses will be increased by a better stability of the EOF in presence of additives in the buffer. Finally, it may not be necessary to use organic modi®ers in the buffer for improving the resolution. Many different hydrophilic polymeric coatings were developed for the CZE of proteins, as covalently attached methylcellulose and dextran [33], polyethylene glycol (PEG) [34±36], polyvinylalcohol (PVA) [37], polyacrylamide (PAA) [36±38], polyethyleneimine [39] and precoated capillaries are now commercially available. In spite of the recent advances in capillary modi®cation technology to minimize protein-wall interactions, there is still a problem with the adsorption of basic proteins. Moreover, the stability of the surface coatings is limited at high pH values [40]. Wu et al. [16] demonstrated the potential of CE to analyze the charged variants of proteins produced by recombinant-DNA technology. The examples selected in this study range from a non-glycosylated protein,

the recombinant human growth hormone (rhGH), to a glycoprotein of moderate complexity, the soluble form of a T4 receptor protein (rCD4) and to a glycoprotein (rtPA) of large microheterogeneity. To reduce interactions between the proteins of relatively high pI values (pI7±8) and the capillary wall, the separations were carried out on a precoated commercially available capillary. For rtPA, the charge heterogeneity was observed as a single broad peak. For rCD4, a truncated form of human CD4 secreted from transfected CHO cells, the charged variants are well resolved at pH 5.5. Compared to the results with rtPA, the improved resolution observed with rCD4 is explained by a lower degree of sialylation and a less molecular weight heterogeneity of the rCD4 protein. More recently, Thorne et al. [41] evaluated the performances of bare fused-silica capillaries and capillaries covalently modi®ed with a polymeric hydrophilic coating for the CZE analysis of rtPA. With an EACA buffer, the rtPA are greatly adsorbed on the surface of bare silica capillaries and the results of Yim [18] cannot be reproduced. Adsorption of rtPA onto the capillary wall was minimized by using the PAA- and PVA-coated capillaries. The best separation of rtPA glycoforms was observed with the PVA-coated capillary. The effect of a series of !-amino acid buffers was studied. The protein recovery was optimal by adding 0.01% (v/v) of Tween 80 detergent to the EACA buffer. Using precoated capillaries and a simple phosphate buffer (pH 2.5) with no additives, Yim et al. [42] reported the CZE separation into glycoforms of a basic glycoprotein (pI>8.5), the recombinant human bone morphogenic protein-2 (rhBMP-2). It is a disul®delinked homodimeric glycoprotein which induces bone formation in vivo in several animal model systems. All the rhBMP-2 glycoforms could be separated according to their number of mannose residues, but the glycoforms having the same number of mannose residues were not resolved in the CZE system used. A separation of the 15 glycoforms of rhBMP-2 into nine peaks was thus observed (Fig. 2). The glycan and peptide mapping enabled to identify the glycoforms as (rhBMP-2)2-(GlcNAc)4-(Manz), where z varies from 10 to 18. These results were con®rmed with the matrix-assisted laser desorption ionization-time of ¯ight (MALDI-TOF) mass-spectrum of a reduced and alkylated rhBMP-2 sample. As shown in Fig. 2,

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Fig. 2. Overlay of the CZE profiles of intact rhBMP-2 and (1-2)-mannosidase digested rhBMP2. Full line: intact rhBMP-2; dotted line: digested to oligomannose 10, at an enzyme±protein ratio of 50 mU/mg in 1 ml of sodium acetate, pH 5, at 378C for 48 h. Precoated capillary (50 cm50 mm i.d.); voltage: 5±12 kV. 0.1 M phosphate buffer, sample injected by electromigration, UV detection at 200 nm (reprinted from [42] with permission from Elsevier Science).

when rhBMP-2 is digested with (1±2)mannosidase, a single peak is observed that coincide with (rhBMP2)2-(GlcNAc)4-(Man5Man5) peak. As the glycoforms have the same charge, the CZE separation mechanism is interpreted in term of the diffuse double layer model. The zeta potential of the various glycoforms was calculated from their electrophoretic mobilities and it is concluded that the separation is based on the size of the mannose residue which is large enough to shield the charges on the protein molecule and reduce its mobility. The CZE method was shown to be useful for the analysis of natural and recombinant interleukin-2 (rIL-2) [43]. The natural inteleukin-2 (nIL-2) is a polypeptide which is synthesized and secreted by activated T-cells. The smallest nIL-2 component of molecular mass 15 000 Da is non-glycosylated and the species of larger molecular masses (16 500 and 17 000 Da) are glycosylated and sialylated. The rIL2, produced from DNA technology, is derived from Escherichia coli expression and is non-glycosylated. CZE was employed using a precoated capillary and a phosphate buffer at pH 2.5. Three distinct peaks were obtained for the natural product, demonstrating that the method was able to separate the nonglycosylated from the mono- and disialylated forms of IL-2.

2.2. CZE monitoring in formulations or in body fluids Several groups reported the use of CZE for on-line analysis to monitor the bioproduction of pharmaceutical glycoproteins at different stages of the puri®cation process such as cultivation step, downstream process and characterization. Using this technique, it is possible to examine the effect of culture environment on the glycosylation pattern of a recombinant protein. There are important needs in the development of analytical methods for the quanti®cation of rHuEPO in pharmaceutical formulations and for the doping control [44]. A CE method was developed to analyze rHuEPO in ®nal drug preparations. Large amounts of human serum albumin (HSA) were used as a protein excipient. The addition of 1 mM nickel chloride to a 200 mM sodium phosphate buffer lead to a complete separation of the two proteins without affecting the resolution pattern of rHuEPO into several glycoform populations. The effect of metal ions in the electrophoretic buffer was investigated as a way to alter the mobility of one of the protein components present in the sample. It appears that the addition of nickel ions in the buffer, selectively decreases the electrophoretic mobility of HSA. The method allows a quanti®cation

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of rHuEPO in drug formulations, as shown from different tests: range of linearity, limits of detection, precision of the method. To avoid the use of additives in the running buffer [45], CZE experiments were performed using a C8coated capillary column with a low concentration phosphate buffer at pH 7. The limit of detection for rHuEPO is above the natural concentration of EPO in urine. Because of the low sensitivies in UV detections, the method cannot be applied to the analysis of the glycoprotein hormone in urine samples. The CE technique was used by Apffel et al. [46] to control the purity of the sample preparation of an intact recombinant glycoprotein. As an example of an heterogeneous glycoprotein, the Desmodus salivary plasminogen activator (DSPAa1) was selected. The DSPAa1 is a serine protease that has potential applications in several cardio-vascular diseases. The experiments were performed with a fused-silica capillary. The electrolyte consisted of a 100 mM sodium phosphate buffer (pH 2.4) and 100 mM NaCl. The glycoprotein is a large complex molecule, with six sites for potential glycosylation (four O-linked and two N-linked). The charge heterogeneity resulting from the variable sialic acid content gives a relatively broad peak. The presence of a single peak demonstrates the low-level of other contaminating proteins. To assess sample purity CE is used as a complementary method to reversed-phase HPLC and matrixassisted laser desorption ionization-time of ¯ight mass spectrometry (MALDI-TOF-MS). With reversedphase HPLC the purity level is given in terms of hydrophobicity while the separation mechanism is based on charge differences in CE. The informations from MALDI-TOF, based on mass/charge ratio are similar to those from CZE based on size/charge ratio. The main drawback of the CE technique is the poor concentration sensitivity. The characterization of the proteolytic digest of the intact protein was further performed using the above techniques and the on-line combination of HPLC and electrospray ionization mass spectrometry. Pedersen and Biedermann [47] characterized the proteinase A glycoforms secreted by recombinant Saccharomyces cerevisiae. This approach is important for the evaluation not only of product purity but also to investigate the suitability of the host organism. From CZE analysis employing N-glycosidase F digestion

and CNBr cleavage, it was shown that 70% of the protein produced was native proteinase A, glycosylated at Asn 68 and Asn 269 and 30% is a variant glycoform with no carbohydrate group at Asn 269. The mass of the two proteins (40 755 and 38 132 Da) were measured by laser desorption mass spectrometry. The CZE of the proteinase A and the variant glycoform was performed using an untreated silica capillary and a 100 mM acetate±phosphate buffer (pH 3.2). Both proteins are resolved into three peaks that may correspond to the glycosylation variants having two, one or no phosphate substituted on the high-mannosetype sugar chain at the Asn 68 site. Reif and Freitag [48] used the CZE technique for monitoring the production of the recombinant antithrombin III (rAT III). Human antithrombin III is a therapeutically important glycoprotein which inhibits serine proteases. The CZE experiments were performed with an uncoated capillary using a 50 mM phosphate buffer (pH 2.0). Instead of using precoated capillaries, a dynamic dextran coating [33] was obtained by adding 0.1% hydroxypropylmethylcellulose (HPMC) to the acidic buffer. The detection limit is ca. 50 mg/ml with a UV detector at 200 nm. The advantages of using CZE for on-line analysis is the automated instrumentation used, short analysis times and simple sample preparations. 3. Analysis using capillary isoelectric focusing (CIEF) Capillary isoelectric focusing (CIEF) is an important tool for analyzing the charged variants of recombinant glycoproteins as the glycoforms are based on differences in sialic acid content. Important ®elds of applications are genetic engineering, focusing in pI evaluation, structural assessment of recombinant proteins or comparison of the chemico-physical parameters of the biotechnology product with those of the natural product. The traditional technique for separating the various glycoforms is isoelectric focusing (IEF) in slab gel with visualization in discrete bands. Several recent reviews outlined the advantages of CIEF over this laborious technique [49±51] as faster sample analysis, ease of automation and ability to perform quantitative analysis. In the two-step method the CIEF focuses the proteins into near stationary zones before they are

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Fig. 3. Effect of changing the ampholytes ratio from 100% pH 5±8 to 100% pH 3±10 in CIEF of rtPA. Precoated capillary (20 cm50 mm i.d.); reversed polarity at 500 V/cm. UV detection at 280 nm. The number on the left at the bottom of each electropherogram indicate the proportion of the pH 5±8 to pH 3±10 ampholyte proportion (reprinted from [53] with permission from Elsevier Science).

mobilized by a second procedure, chemical or hydrodynamic (pressure or vacuum). With this method, the CIEF is generally performed in coated capillaries to minimize the EOF. More recently, a rapid one step CIEF method in which focusing and mobilization occur simultaneously, was developed [52,53]. In this method, the proteins which focus past the detector window are swept by the EOF in the opposite direction. The CIEF suffers from several drawbacks as a resolving power of about 0.01 pH unit, low UV detection limits due to carrier ampholyte absorbances, problems of precipitation at the pI of the protein [54], increased by the large concentrations needed to reveal the minor glycoforms. 3.1. Glycoform separation by CIEF Yim [18] reported ®rst the application of CIEF to the fractionation of the glycoforms of rtPA. The ampholyte solution used contained 2% of ampholyte (pH 6±8), 2% of 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS) and 6 M of urea. The method was able to discriminate the subtle differences between the two variants of rtPA: type I and type II which differ by the presence or the absence of glycan groups at the Asn 184. The CIEF pattern of the neuraminidase treated rtPA was considerably simpler indicating that the microheterogeneity was mainly due to a variable content in sialic acid residues.

Nevertheless, there was a poor reproducibility in migration times, making the correlation of pI versus migration times very dif®cult to assess. More recently, Moorhouse et al. [53] developed a rapid one step CIEF method for the separation of rtPA glycoforms based on the simultaneous focusing and mobilization of the sample. The separation was performed with precoated ``neutral'' capillaries in presence of hydroxypropylmethylcellulose (HPMC) to reduce the EOF. Focusing was achieved using reverse polarity. To optimize the separation of rtPA glycoforms, 4 M of urea and 0.1% of HPMC were added to a mixture of pH (5±8) and pH (3±10) ampholytes (pharmalytes). The effect of varying the proportion of the ampholytes used from 100% of pH (5±8) to 100% of pH (3±10) is shown in Fig. 3. Thus, a series of 8±10 peaks reveals the large level of charge heterogeneity present in the rtPA recombinant glycoprotein. The best separation was obtained by mixing the wide range ampholyte (pH 3±10) with that covering a narrower pH range. The addition of urea was required to maintain protein solubility during focusing. At pH values near the isoelectric point, the solubility problems would have lead to poor resolution. To validate the use of CIEF as an alternative to the slab gel IEF technique, intact rtPA was analyzed by both methods. Using slab gels, a greater concentration of urea (8 M) was required to avoid precipitation of the protein. The gel visualization detects 10 major bands

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which correspond to the same number of peaks resolved by CIEF. Later, a validation of the CIEF method was performed by the same group [55] in a series of experiments examining accuracy, precision, speci®city and ruggedness of the method. A detailed study examined the effect of different parameters which affect the separation pro®le quality such as capillary age, temperature range, voltage, concentration of the rtPA in the sample. The method showed acceptable recovery, good sensitivity (25 ng of protein could be resolved into constituents) and appeared rugged with respect to the operating conditions. The detection limits were two order of magnitude below that of Coomassie Blue staining with slab gel IEF. As part of the CIEF method validation [56], four commercial ampholytes were tested and compared for their ability to resolve the rtPA glycoforms. For the CIEF system used, the best results were obtained with ``Ampholine'' and ``Pharmalyte'' ampholytes. Thorne et al. [41] applied the two step method, with focusing into narrow zones and pressure mobilization to perform the CIEF separation of the rtPA glycoforms. A precoated capillary was used to minimize the EOF. Ampholytes were added to an urea/polymer solution. A good migration time reproducibility was obtained because of the increased solution viscosity. Three commercially available ampholytes were tested and the best separation of rtPA was achieved with ``Ampholine 3.5±10''. Using a fused-silica capillary and a dynamic coating with either PEG or HPMC, Kubach and Grimm [57] have developed a simple CIEF method with pressure mobilization of the focused zones to separate the glycoforms of both rtPA and rhEPO under denaturing conditions. Using this standard method, six isoforms of rhEPO with pI values ranging from 4.6 to 5.1 could be separated. Good reproducibilities of peak areas and migration times were obtained. As previously described, the CZE technique was used to monitor the puri®cation process of rATIII [48]. Additional CIEF experiments were performed to characterize rATIII. The CIEF method, with pressure mobilization of the focused zones, was used to determine the isoelectric point of the puri®ed products. Precoated PAA and dextran capillaries were tested. A better stability was obtained when using the dextran coating, with reproducible data for at least 50 runs. Addition of the detergent Triton X-100 to the sample

buffer was needed to prevent protein precipitation. N,N,N0 ,N0 -tetramethylenediamine (TEMED) was added to obtain the focusing of the sample components before the detection window. A pI calibration graph was obtained with standard proteins (Fig. 4(a) and (b)). For the r-AT III protein, a pattern of six fractions focused in the pH range 4.7±5.2 was observed (Fig. 4(c)) with three major peaks corresponding to pI values of 4.7, 4.75 and 4.85 and three minor peaks corresponding to pI values of 5.0, 5.1 and 5.3. Similar isoelectric points were determined by conventional IEF. 3.2. CIEF for quality control monitoring The CIEF was used to separate the isoforms of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) [58]. This antibody consists of two light and two heavy chains, mutually attached by disul®de bonds. The constant region of each heavy chain contains an Asn glycosylation site. The exhibited charge heterogeneity is due to the C-terminal clipping and deamidation, but not to sialylation [59]. The CIEF experiments were performed with precoated commercial capillaries and chemical mobilization after focusing. TEMED and HPMC were added to the ampholytes. Five isoforms were separated with pI values in the range 8.2±9. These results agree well with the pIs determined on slab gel IEF. The good precision obtained in terms of migration time, peak area and area per cent in the sample, clearly demonstrates the potentialities of CIEF for routinely monitoring the quality control. A multi-compartment electrolyzer with isoelectric immobiline membranes, was described by Wenisch et al. [60] to purify to homogeneity large amounts of proteins and mainly to remove the contaminants from r-DNA proteins. The system is able to resolve isoforms as close as 0.001 in pI difference. By this technique, the isoforms of human monoclonal antibodies against the gp-41 of AIDS virus and of recombinant superoxide dismutase have been puri®ed. Analytical IEF in immobilized pH gradients (IPG) and CZE techniques were used to monitor the progress of puri®cation. Comparisons between the CZE, IPG and chromatofocusing patterns show that the resolution power of the three methods decreases in the order IPG>CZEchromatofocusing. An extremely high

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resolution is obtained with IPG, but the advantages of CZE over the other techniques are speed and quanti®cation. 4. Analysis using micellar electrokinetic capillary chromatography (MEKC) There are several papers reporting protein separation by MEKC [61]. In this method, surfactants are added to the running electrolyte at concentrations above the critical micellar concentration and the electrophoretic migration of proteins is in¯uenced by protein±micelle interactions. Some problems encountered in the CE analysis of glycoforms may be addressed with MEKC. The highly signi®cant bene®t of the presence of surfactant in the separation buffer is the elimination of protein±wall interactions. Moreover, in contrast to other CE modes, MEKC does not require any special capillary treatment in-between each run, as often needed in CZE for free silanol equilibration and reproducible migration times. Sodium dodecyl sulfate (SDS) is generally used in glycoprotein analyses, as the presence of this anionic surfactant has several advantages. First, it has the capacity to bind to (glyco)proteins leading to anionic protein±SDS complexes which are repelled from the negatively charged capillary wall, limiting thereby their adsorption. Second, SDS induces a protein denaturation that may be necessary to achieve the separation of glycoproteins present as dimers or oligomers. This denaturation exposes the inner core of the protein to the solution environment and may facilitate resolution. When investigation of native proteins is desired, MEKC has the advantage that it can be non-denaturing through the use of zwitterionic or non-ionic surfactants [61]. 4.1. Separation of glycoforms by MEKC Fig. 4. Characterization of the isoelectric points of r-AT III by CIEF: (a) standard proteins; (b) calibration graph; (c) r-AT III. Dextran-coated capillary (20 cm50 mm i.d.), UV detection at 254 nm; focusing voltage 12 kV (2 min); then 8 kV and pressure mobilization; anolyte: 10 mM phosphoric acid; catholyte: 20 mM sodium hydroxide samples suspended in a buffer containing 0.01% HPMC, 0.1% TEMED, 0.001% Triton X-100, 2% Ampholine 4/6, 0.5% pharmalytes 3/10, 0.5% pharmalytes 2.5/5 and 0.5% pharmalytes 4/6.5 in deionized water. (reprinted from [48] with permission from Elsevier Science).

James et al. [62] employed MEKC to resolve r-human interferon-g (rIFN-g) into glycoform populations by using uncoated fused-silica capillaries and a borate buffer containing SDS. The rIFN-g is produced by CHO cells. As the natural human interferon-g, the recombinant glycoprotein exists as heterogenous populations of hydrogen-bonded dimeric glycoforms exhibiting variable site occupancy. This glycoprotein

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has two potential N-glysosylation sites. The N-linked oligosaccharides are found either attached to both Asn 25 and Asn 27 or to Asn 25 only. They may also be entirely absent. NMR studies [63] examining N-linked oligosaccharides of IFN-g expressed in CHO cells, have demonstrated that the N-linked oligosaccharides were mainly of the complex biantennary type and that the microheterogeneity could result from the presence or absence of terminal sialic groups and core fucose residues. The initial experiments using borate alone were unsuccessful with no peak observed, probably because of a strong adsorption of rIFN-g on the capillary wall. Although there was no resolution of the glycoforms, the addition of SDS to the separation buffer prevented protein adsorption. Taking into account that the extent of anionic-borate-sugar diol complexation increases with pH and borate concentration, a high ionic-strength borate buffer was employed to attain maximum resolution. Optimal conditions were obtained with a 400 mM borate, 100 mM SDS, pH 8.5. The glycoform migration times were inversely related to the amount of carbohydrate associated to the protein. This work shows that the increased separation ef®ciency is a result of the synergic action of both the reduced EOF and the increased surfactant concentration as separate functional entities eliminating protein adsorption. It appears that the presence of polar, hydrophilic glycan structures reduces the interaction of the glycoprotein with the SDS micelles. Therefore, it was concluded that the analytes with the shortest migration times had the highest carbohydrate content, i.e. the largest glycan structures. As a modi®cation of an immunoglobulin G (IgG), the BR96 chimeric antibody represents a glycoprotein with an average molecular mass of 150 000 Da, where about 3% of the mass is accounted for by carbohydrate moieties. Four major isoforms of the BR96 antibody were separated by MEKC using a borate buffer containing SDS [64]. The separated species were shown not to be a result of carbohydrate heterogeneity or partial oxidation/deamidation, but rather to different forms of the same primary structure. Heat treatment induced interconversions between species which was easily monitored by a change in the CE pro®le. Kats et al. [65] further applied MEKC to separate structurally similar isoforms and/or conformers of a fusion protein BR 96 sFv-PE40, a single-chain immunotoxin using a

fused-silica capillary and cholic acid in a borate buffer at pH 9.0, as a micelle-forming surfactant. Attempts to separate BR96 sFv-PE40 isoforms by using a SDS micelle-containing buffer were unsuccessful; with these experimental conditions, the protein migrates as a single peak. The addition of denaturants that alter the secondary and tertiary protein structures, such as guanidine hydrochloride or tri¯uoroethanol, modi®es the separation pattern in a concentration-dependent manner. The differences in the electrophoretic mobilities of closely related species may be assigned to the differences in exposure of the hydrophilic and hydrophobic domains of the globular surface of the protein molecules during the formation of protein±micelle association complexes. 4.2. MEKC for quality control monitoring MEKC represents an alternative method for process control and purity testing of recombinant DNAderived (rDNA) proteins. The MEKC method [66] was used to monitor the puri®cation process and test the purity of a highly glycosylated hepatitis C virus (HCV) rDNA protein expressed in CHO cells. The MEKC experiments for production control were performed with a fused-silica capillary using a high concentration of SDS (100 mM, critical micellar concentration 8.1 mM) added to the electrolyte. A high pH borate buffer (pH 9.5) was employed to increase the EOF and to reduce the adsorption of proteins on the capillary walls. Fig. 5 shows the CE pro®les of the HCV protein obtained by two different production methods. The paper indicates for both methods an identical procedure of protein puri®cation, but no further informations about sample preparations. Differences in the elution pattern are displayed, with an additional major peak for the protein prepared by method 2. The MEKC approach is rapid, easy to perform and enables to quantify the purity level in each sample. The results from the MEKC technique correlate well with those from the conventional slab gel electrophoresis technique. MEKC has been also reported as an ef®cient tool to monitor the effect of fermentation conditions on the glycosylation pattern of a recombinant glycoprotein in order to assess the carbohydrate heterogeneity and the stability of fermentation variants [67]. The various fermentation conditions used to cultivate human inter-

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is responsible for the generation of virus variants. For an HIV-1 vaccine, the recombinant glycoprotein (rgp120) is derived from a mammalian system. It is expressed on the external surface of the HIV-1 virus particle. The purity of rgp120 was con®rmed by different techniques: SDS±polyacrylamide gel electrophoresis, MEKC laser desorption mass spectrometry, total amino acid analysis and N-terminal amino acid sequencing. An almost single and symmetrical peak was obtained using a phosphate borate buffer pH 9.4 containing 50 mM SDS indicating that the rgp120 peak was homogeneous and corresponded to 98±99% of the total material absorbing at 200 nm. 5. Analysis using capillary gel electrophoresis

Fig. 5. MEKC control of the production of HCV expressed in CHO cells. Fused-silica capillary (40 cm75 mm i.d.); voltage: 30 kV. 100 mM borate buffer pH 9.5 plus 100 mM SDS. UV detection at 214 nm. The HCV production modes in CHO cells (methods 1 and 2) are not specified in [66] (reprinted from [66] with permission from ISC Technical Publications, Inc.).

feron-w (IFN-w) expressed in CHO cells produce alterations in the glycoform patterns as shown from the MEKC electropherograms, with a 150 mM buffer containing 50 mM SDS. The most signi®cant glycosylation alterations resulted from the change of various parameters such as initial ammonia concentration in the production medium, cultivation mode (adherent versus suspended) or process time. The HIV gp120 molecule is still a strong candidate for incorporation into a recombinant sub-unit HIV vaccine [68]. Hypervariability within the HIV gp120

SDS-polyacrylamide gel electrophoresis (SDSPAGE) is the most widely used analytical tool for routine separations of proteins. A disruption of protein structure with unfolded polypeptide chains result from the SDS binding to proteins. In presence of a denaturant agent, a direct analysis assumes a migration according to the relative masses, on the basis of two hypotheses. First, all SDS±protein complexes have an identical charge/mass ratio in presence of SDS in excess, with an average of 1.4 g of SDS associated to 1 g of protein. Second, the SDS±protein complexes have similar shape and their size varies linearly with the molecular mass. These assumptions are not valid for some classes of proteins because deviations from the predicted charge/mass ratio are often observed with the SDS±protein complex. The differences in the nature of the protein are at the origin of this non-ideal migration behavior. A lower ratio is found for proteins with a high pI, because of the presence of positively charged amino acids, whereas hydrophobic membrane proteins give a larger charge/mass ratio. Among proteins, the glycoproteins are the most numerous that exhibit this non-ideal effect. Responsible of the lower than predicted charge/mass ratio is the presence of carbohydrate moieties leading to a decreased migration and an over-estimation of molecular masses [69]. With the capillary format a Ferguson plot was proposed to minimize the inaccuracies of glycoprotein molecular mass determination in presence of SDS, by using a replaceable sieving matrix [70]. The Ferguson method is based upon the observation that the loga-

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rithm of protein mobility varies linearly as a function of the gel concentration employed [71]. From the slope of the line obtained by plotting the log of protein molecular weight versus polymer concentration, it is possible to calculate the retardation coef®cient Kr of various standard proteins. Then, the calibration curve is obtained by plotting the logarithm of protein molecular mass against the square root of Kr. Linear regression provides the slope and intercept for the calculation of the molecular mass of the protein. The Ferguson analysis yields molecular masses for glycoproteins similar to those obtained from other techniques, the least deviations observed are due to a modi®ed shape of the glycoprotein caused by the presence of the carbohydrate moiety as compared to a non-glycosylated protein of the same molecular mass. In the production and control of therapeutic proteins, SDS-PAGE is often used to evaluate the purity as well as to determine the presence of non-dissociable aggregates of the product. Using capillary gel electrophoresis (CGE), the detection and quantitation are performed on-line. Moreover, shorter analysis times are achieved with analysis in acrylamide gel-®lled capillaries [72]. Despite these advantages, there are several problems in the CGE technique: they include defective gel formation in polymerization, the breakdown of the gels in a high electric ®eld and crosscontamination through the matrix. An alternative is now to use replaceable polymers in solution that still provide a size-based separation medium and that can be easily replaced in-between the analysis [73,74]. The utility of sieving polymer networks for protein separation in capillary electrophoresis received the attention of several workers. Various materials were suggested for CGE such as linear polyacrylamides, hydroxyethylcellulose, dextran, agarose, polyethyleneoxide [75]. 5.1. Estimation of molecular masses by SDS-CGE The CGE was examined as an alternative method to high-performance size exclusion chromatography for the analysis of recombinant bovine somatotropin (rbSt) [76]. The experiments were performed in a coated capillary column ®lled with a SDS non-acrylamide gel solution. The use of a coated capillary is required to eliminate the electroosmosis. A well

resolved separation was observed, with peaks corresponding to the monomer, dimer, trimer and tetramer of rbSt. A linear relationship for the calibration curve was obtained by plotting on a log±log scale, the molecular masses of standard proteins against the electrophoretic mobilities. The difference between the observed and the theoretical value of the molecular mass of rbSt was explained by the non-reduced state of the protein: the protein could not unfold in full length and the migration time was lower than expected. For a precise measurement of the protein molecular masses, a CE-electrospray mass spectrometer interfacing is needed. Reif and Freitag [48] combined the different CE methods to characterize recombinant antithrombin III (rAT III). The molecular mass was determined by CGE, applying removable dextran gels. The experiments were performed in precoated dextran or PAA capillaries, ®lled with dextran gel and the separation buffer was 100 mM tris(hydroxymethyl)aminomethane (Tris)-2-(N-cyclohexylamino)ethanesulfonic acid (CHES) containing 0.1% SDS. The value of the molecular mass correlates well with previously published data obtained with the conventional slab gel technique. The CGE could also detect the af®nity complex between rAT III and thrombin as evidenced by the new peak which appeared in the electropherogram after incubation rAT III with thrombin. The rate of complex formation was increased by addition of catalytic amounts of polysaccharide heparin. CGE is thus an adequate and useful method to study and evaluate biological af®nities such as enzyme-inhibitor reactions. In the series of experiments exploring the feasability of the CE technique for quantitating the two variants of rtPA which differ by the number of sites which are glycosylated (type I and type II), Thorne et al. [41] have demonstrated the usefulness of SDSCGE. A commercial kit was used with a polymer for sieving medium and a precoated capillary. The exposure of plasminogen treated rtPA samples to -mercaptoethanol results in cleavage of the disul®de bond that holds the A chain (Gly 1-Arg 275) and the B chain (Ile 276-Pro 527). However, plasminogen treated rtPA was separated into three polypeptide chains, one B chain and two A chains. The two variants of plasminogen treated rtPA (type I and type II) give different electropherogram pro®les. Since glycosylation is

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HER2 [58]. As shown in Fig. 6(a), seven peaks were detected by SDS-CGE with a non-reduced sample. This result correlates well with the seven bands observed using SDS-PAGE. The peak area percent attributed to the high molecular mass aggregates (peak 7) is consistent with that found using size exclusion chromatography with an SDS-containing mobile phase. The SDS-CGE analysis of this glycoprotein at two different storage temperatures, 58C and 378C, held for 27 days is shown in Fig. 6(b). The authors observed a slight increase in peak area percent for the lower molecular mass peaks (1±5) with the sample stored at 378C. These data were explained by a fragmentation of the molecules due to storage at elevated temperature and the method could therefore be used for stability indicating purposes. 6. Coupling of CE with mass spectrometry

Fig. 6. SDS-CGE of rhuMAbHER2 under nonreducing conditions: (a) Electropherograms of rhuMAbHER2 and of molecular mass markers. (b) Electropherogram of ElrhuMAbHER2 samples stored at 58C and 378C for 27 days. Fused-silica capillary (19.5 cm75 mm i.d.); voltage: 22 kV; SDS buffer. Sample: 1 mg/ml (reprinted from [58] with permission from Elsevier Science).

known to affect the activity of rtPA, the method could be useful to determine the variant composition of rtPA. 5.2. MEKC for quality control monitoring A recombinant humanized monoclonal antibody (rhuMAbHER2) was analyzed by SDS-CGE. The SDS-CGE method was compared to the traditional gel SDS-PAGE method for the evaluation of the purity, consistency and percent distribution of the rhuMAb-

The determination of the molecular masses of the components separated by CE is useful to give structural information about recombinant glycoproteins with complexities arising from variabilities in both the carbohydrate distribution and composition. The advantages of coupling mass spectrometry (MS) detection with CE are well recognized and the recent advances in CE±MS in instrumentation and applications have been published in several reviews [77, 78]. 6.1. Applications of CE±electrospray mass-spectrometry The coupling of CE to electrospray mass-spectrometry (ESI MS) is now increasingly used for the online determination of the molecular mass of the separated compounds. The combination of CE±ESI MS for peptide mapping is useful for protein characterization but only few examples of applications exist as the technique is still considered as a complementary method to HPLC±MS experiments [78,79]. The recent developments in the instrumentation have permitted applications in the CE±ESI MS analysis of proteins [80]. Kelly et al. [81] reported the on-line coupling CE to electrospray mass spectrometry (ESI MS) to analyze glycoproteins in both intact and digested forms.

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Examples of applications are presented for ribonuclease B (RNase B), ovalbumin, horseradish peroxidase and a lectin from Erythrina corallodendron. The separations, compatible with the operation of electrospray ionization, were achieved with capillaries dynamically coated with ``Polybrene''. An anodal EOF enabled to resolve the glycoproteins and glycopeptides according to the number of attached carbohydrate residues. The detection of the oxonium ions generated from in-source fragmentation [82], i.e. those of hexose at m/zˆ163 (Hex‡) and those of Nacetylhexosamine at m/zˆ204 (HexNAc‡) facilitates the identi®cation of the glycopeptides from proteolytic digests or chemical cleavages. Yeung et al. [83] developed a method to analyze by on-line CE±ESI MS high-mannose glycoproteins. The potentialities of the technique was demonstrated by selecting as examples ribonuclease B (RNase B) and recombinant human bone morphogenic protein-2 (rhBMP-2). The recombinant glycoprotein rhBMP-2 gives a total of six dimer isoforms, each of them carrying glycoforms derived from the high-mannose glycans. The term `isoform' describes a protein variant with differences other than those arising from the carbohydrate structure (for example, differences in protein sequence). The CE separation of nine glycoforms was previously described [42] using an acetic phosphate buffer. For the on-line CE±ESI MS experiment, a non-volatile acidic -alanine buffer was employed and a zero EOF was obtained by using a linear PAA-coated capillary. A good resolution with several isoform and glycoform peaks was observed with the rhBMP-2 monomer obtained after reduction, alkylation and desalting. As shown in Fig. 7(b), three isoforms are well separated. The masses detected allow to assign the extended form as peak I, the mature form, with a glutamine residue at N-terminus, as peak II and the pyroglutamic form, with a cyclization of this glutamine residue, as peak III. On the basis of CE±UV and CE±MS observations, the peak IV should be a non-covalent aggregate of either or both the mature and the pyroglutamic forms. Experiments were performed to monitor the oxonium ions (Hex‡, m/zˆ163) and (HexNAc‡, m/zˆ 204) and the two ions arising from losses of water (m/zˆ145 and m/zˆ127) all generated from insource fragmentation of the intact proteins. The

value of the Hex‡/HexNAc‡ ratio is useful to compare the carbohydrate contents of the different glycoforms. 6.2. Applications of CE-MALDI-TOF mass spectrometry The matrix assisted laser desorption±ionization mass spectrometry (MALDI-MS) is an effective technique for the precise mass determination of large biomolecules [84]. In this technique the focused laser pulses are directed on a mixture of a sample plus matrix deposited on a surface and a desorption/ionization plume is formed. The time of ¯ight (TOF) mass spectrometers equipped with MALDI ion sources allow improved mass resolution and excellent sensitivity [85]. The on-line coupling with CE is not easy to achieve and several studies describe the off-line MALDI-TOF-MS method to characterize proteins isolated by CE [86±88]. Combining CE and off-line MALDI-TOF-MS, Chakel et al. [89] presented a protocol for the glycoform analysis of intact glycoproteins. The model proteins selected for this study, ovalbumin and DSPAa1, were both highly heterogeneous glycoproteins. When expressed in CHO cells, the DSPAa1 is heterogeneous with four O-linked and two N-linked sites for potential glycosylation. The number of possible glycoforms is larger than 330 000. An electropherogram of DSPAa1 was obtained at pH 3.0 (phosphate buffer), using a bovine serum albumintreated fused-silica capillary. In Fig. 8 are shown the four fractions that were collected for subsequent MALDI-TOF mass analysis (accuracy of 0.1%). The collected fractions are still a mixture of glycoforms and it is dif®cult to explain the observed differences in mass. The MALDI-TOF-MS is a powerful tool not only for the quality control of recombinant glycoprotein pharmaceuticals but also for a better understanding of the mechanisms involved in the CE separation of glycoforms. 7. Conclusion It has become increasingly accepted that the glycosylation of a therapeutic protein may in¯uence its in

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Fig. 7. On-line CE±electrospray mass spectrometry of rhBMP-2 monomer. (a) CZE±UV and (b) total ion chromatogram of CE±MS analysis. Coated capillary with linear PAA, 50 mm i.d.; length: 50 cm (a) and 65 cm (b). Buffer: 50 ml -alanine at pH 3.5 with acetic acid; sample: 5 mg/ml (reprinted from [83] with permission from American Chemical Society).

vivo ef®cacy. Glycosylation analysis is therefore incorporated into the development and the production processes for therapeutic glycoproteins. Fast analysis of glycoform heterogeneity are of prime importance if a precise understanding of the effect of culture environment on the glycosylation pattern of a recombinant protein is to emerge. The characterization of complex glycoproteins requires the

use of a combination of different analytical methods with orthogonal selectivity. CE with its various operation modes has already a great potential in glycoprotein analysis and may be selected as one alternative for high speed and ef®cient separations. In the future, CE is believed to make signi®cant contributions in the quality control of the biotechnology products. The technique may be employed not only to monitor the

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Fig. 8. Analysis of DSPAa1 using EC coupled with off-line MALDI-TOF-MS: (a) Electropherogram of DSPAa1. (b) MALDI-TOF-MS spectra of the fractions 1±4 were collected from CE. BSA treated capillary (41 cm50 mm i.d.); voltage: 500 V/cm. Sample: 10 mg/ml; 100 mM sodium phosphate buffer (pH 3.0). UV detection at 200 nm (reprinted from [89] with permission from Elsevier Science).

fermentation, cell cultures and puri®cation processes, but also to identify the ®nal product, the consistency of its glycosylation, assess its purity or study its stability.

The improvements in reproducibility and sensitivity will allow broad applications of the CE techniques in routine analytical separations.

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