RESUMO
The cytotoxic activities of several natural and semisynthetic anthracyclines against L1210 leukemia and two human colon tumor cells (Colon 4, HT 29) in vitro were examined after short (1 h) and long (7 days) incubation times and correlated with the water/octanol partition coefficients and the DNA-binding affinity of the compounds. Analysis of equation in which cytotoxicity against L1210 (1-h incubation) was parabolically related to the partition coefficient revealed an almost exclusive correlation (r = 0.80) between the cytotoxicity and the parameters, and this correlation was only slightly improved by addition of DNA-binding affinity (r = 0.85). On the other hand, cytotoxic activities displayed after continuous incubation were partially related to both partition coefficients (parabolic dependence) and DNA-binding affinities (linear dependence). In this case the correlation between the activity and partition coefficient (r = 0.67) was significantly improved by addition of DNA-binding affinity (r = 0.90). Similar results were also obtained for human colon tumor cells although the corresponding correlation coefficients were generally of lower value, indicating that cytotoxic activity of anthracyclines against these primary resistant cells may be influenced by additional factors not yet determined.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/metabolismo , Fenômenos Químicos , Físico-Química , Neoplasias do Colo/tratamento farmacológico , DNA/metabolismo , Humanos , Leucemia L1210/tratamento farmacológico , Estrutura Molecular , Análise de Regressão , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Sera of patients with anti-plasmodial antibodies determined by indirect fluorescence antibody test (IFAT) were used to evaluate an enzyme immunoassay (IgG ELISA) for determining antibodies to Plasmodium falciparum merozoite antigen. The two tests correlated well. Antibodies to species other than P. falciparum, however, were detected by P. falciparum merozoite antigen to a limited extent only.
Assuntos
Anticorpos Antiprotozoários/análise , Malária/imunologia , Animais , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Plasmodium falciparum/imunologiaRESUMO
A new in vitro potency assay for erythropoietin has been developed and compared to the exhypoxic polycythemic mouse bioassay for rhuEPO batch release, required according to Ph. Eur. 2001. Evaluation of 14 batches of EPO has shown that the new assay is highly sensitive and able to reveal subtle changes in N-linked carbohydrates. In combination with regular EPO batch release assays (such as sialic acid determination, isoelectric focusing, RP-HPLC, and peptide mapping), this assay may enable to replace the highly variable exhypoxic mouse bioassay for EPO batch release and stability studies and thus reduce the use of laboratory mice.
Assuntos
Alternativas aos Testes com Animais , Eritropoetina/química , Eritropoetina/uso terapêutico , Sequência de Aminoácidos , Animais , Eritropoetina/genética , Eritropoetina/metabolismo , Glicosilação , Humanos , Focalização Isoelétrica , CamundongosAssuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Experimentais/radioterapia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígeno Carcinoembrionário/imunologia , Humanos , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Ácido Pentético/uso terapêutico , Radioterapia/métodos , Receptores de Droga/biossíntese , Receptores de Droga/imunologia , Radioisótopos de Ítrio/uso terapêuticoRESUMO
The overall isoform number I, calculated from capillary zone electrophoresis (CZE) data, is introduced as a new parameter that enables the assessment of the quality of erythropoietin (EPO) in a simple, yet efficient manner. I is defined as the sum of the products of the individual CZE peak area percent shares (pn) of the EPO isoforms (n = 1-8) and the corresponding individual isoform numbers (n): I=p1x1+p2x2+p3x3+ p4x4+p5x5+p6x6+p7x7+p8x8 The results from an internal collaborative study were used to calculate I-numbers for 2 successive batches of EPO Biological Reference Preparations (BRPs) established by the European Pharmacopoeia (Ph. Eur.) Commission. Based on the results of 6 participating laboratories each, the I-numbers calculated for batch 1 (BRP1) and candidate batch 2 (cBRP2) were I = 518.7 +/- 3.5 (coefficient of variation (CV) = 0.7 %) and I = 542.4 +/- 2.2 (CV = 0.4 %) respectively. Thus, the I-numbers of the 2 EPO preparations clearly indicated the difference between BRP1 and cBRP2 described in the literature, but in a much more apparent and simple manner. Notably, one of the 7 laboratories participating in the study with cBRP2 yielded an outlier value of I = 525.6, pointing towards a suboptimal CZE analysis, and was therefore excluded from the cBRP2 mean value calculation. It is suggested that the overall isoform number I of erythropoietin provides a new and highly valid quality control measure that makes it possible to ensure the batch-to-batch consistency of the active pharmaceutical ingredient of EPO market products.
Assuntos
Eritropoetina/química , Isoformas de Proteínas , Estabilidade de Medicamentos , Eletroforese Capilar , Europa (Continente) , Farmacopeias como Assunto , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The invasion of human red blood cells (RBC) by plosmodiol merozoites is a key event during malaria infection, and the inhibition o f invasion is regarded as a crucial goal of malaria vaccine development. For Plasmodium falciparum it has been suggested that the red cell sialoglycoproteins, glycophorins A, B and C, are receptors for invasion and that O-linked or N-linked carbohydrate structures may be involved as receptor sites(1-3). However, recent evidence suggests that the role o f these sialoglycoproteins and carbohydrates may have been overestimated. In this article, Peter Hermentin discusses the contradictory findings and presents a revised model for the invasion process.
RESUMO
Recent advances in the understanding of the cellular, biochemical and molecular aspects of the highly specific interactions between P. falciparum merozoites and human red blood cells are summarized. Special attention is given to glycophorins A, B and C as receptors of invasion and to the role of carbohydrates and internal domains of glycophorin A, such as the Wrb antigen, as receptor site. Video pictures displaying the liberation of merozoites from schizont-infected red blood cells and the invasion of human erythrocytes by P. falciparum merozoites are also provided.
Assuntos
Eritrócitos/parasitologia , Malária/sangue , Animais , Antígenos , Antígenos de Protozoários/isolamento & purificação , Sítios de Ligação , Carboidratos/sangue , Glicoforinas/imunologia , Glicoforinas/fisiologia , Humanos , Malária/parasitologia , Plasmodium falciparum/fisiologiaRESUMO
The development of monoclonal antibody-drug (anthracycline) conjugates (immunocytostatics) that has emerged during the last decade is briefly reviewed. Stress is layed on the various procedures that have been employed for the chemical attachment of daunorubicin (daunomycin) and doxorubicin (adriamycin) to spacers, high-molecular weight carrier molecules, and immunoglobulins. Anthracycline conjugates that have proved effective in mice with respect to the treatment of cancer are especially evaluated. Also a new method developed for the conjugation of rhodosaminyl anthracyclinones via a hydrolysable spacer, developed in our laboratories, is briefly communicated. Finally, import aspects for further developments of monoclonal antibody-drug conjugates are discussed.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Imunotoxinas/uso terapêutico , Neoplasias/terapia , Fenômenos Químicos , Química , Humanos , Naftacenos/uso terapêuticoRESUMO
Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.
Assuntos
Metabolismo dos Carboidratos , Eritrócitos/parasitologia , Plasmodium falciparum/metabolismo , Animais , Antígenos de Grupos Sanguíneos , Carboidratos/farmacologia , Dissacarídeos/farmacologia , Haptenos , Humanos , Metilglicosídeos/farmacologia , Monossacarídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Trissacarídeos/farmacologiaRESUMO
We have examined the inhibitory potencies of glycophorin A, a mixture of glycophorins B and C, chymotryptic fragments of GpA, desialylated GpA, alkaliborohydride treated GpA, and the O-linked tetrasaccharide isolated from GpA on the invasion of human red blood cells by synchronous Plasmodium falciparum (strain FCB). 50% inhibition of invasion, as measured by 3H-hypoxanthine incorporation into parasites, was achieved at 14 and 155 microM for GpA and GpA-CH1, respectively. We have noticed, however, that isolated GpA exhibits a toxic effect on the intraerythrocytic growth of the parasite whereas the chymotryptic fragment (amino acid residues 1-64 of GpA) does not. Thus the inhibitory potency of isolated GpA during erythrocyte invasion by the merozoite should be regarded as the result of both an inhibitory and a toxic effect. The inhibitory effect should be attributed to the carbohydrate-rich outer portion of GpA carrying clusters of neuraminic acid. The toxic effect should be attributed to the hydrophobic region of GpA which might be capable of inserting into the membrane of free merozoites and/or erythrocytes. Our data suggest that results previously obtained with glycoprotein inhibitors carrying hydrophobic portions may have to be questioned.
Assuntos
Eritrócitos/parasitologia , Glicoforinas/intoxicação , Plasmodium falciparum/efeitos dos fármacos , Sialoglicoproteínas/intoxicação , Glicoforinas/isolamento & purificação , Glicoforinas/toxicidade , Humanos , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Uromodulin was isolated from urine of three pregnant women. Urine of each donor was collected at subsequent stages of their pregnancy and at one month after gestation. Each batch of uromodulin was enzymatically N-deglycosylated and the released N-glycans were isolated, quantified and profiled by high-pH anion-exchange chromatography. In the course of pregnancy no significant changes were detected in the negative charge distribution stemming from sialic acid and sulfate residues on the complex-type carbohydrate chains of uromodulin. Furthermore, no significant changes in the molar ratio between Man6GlcNAc2 and Man7GlcNAc2 were found in the course of pregnancy, only uromodulin from non-pregnant periods showed small differences.
Assuntos
Manose/metabolismo , Mucoproteínas/química , Polissacarídeos/metabolismo , Gravidez/urina , Cromatografia por Troca Iônica , Feminino , Humanos , Concentração de Íons de Hidrogênio , Polissacarídeos/química , UromodulinaRESUMO
Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd approximately 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd approximately 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proportion of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.
Assuntos
Antitrombinas/metabolismo , Heparina/metabolismo , Animais , Células CHO , Células Cultivadas , Cromatografia de Afinidade , Cricetinae , Eletroforese em Gel de Poliacrilamida , Fluorescência , Glicosilação , Humanos , Cinética , Oligossacarídeos/análise , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.
Assuntos
Eritropoetina/análogos & derivados , Eritropoetina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Configuração de Carboidratos , Linhagem Celular , Cricetinae , Glicosilação , Humanos , Rim , Mesocricetus , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Receptores da Eritropoetina/metabolismo , Taxa Secretória , Relação Estrutura-AtividadeRESUMO
We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.
Assuntos
Polissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia por Troca Iônica , Bases de Dados Factuais , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Polissacarídeos/análise , Terminologia como AssuntoRESUMO
The lectin of Dolichos biflorus, a hemagglutinin previously considered to be blood group A specific, is now found to react much more strongly with the terminal disaccharide unit [alpha DGalNAc(1 leads to 3) beta DGalNAc] of the Forssman antigenic determinant. In contrast, the relative reactions of the lectins of Helix pomatia (which also agglutinates A erythrocytes) and Wistaria floribunda (which agglutinates A, B, and O erythrocytes) with the Forssman pentasaccharide were substantially weaker than that of Dolichos biflorus. The combining site of the lectin of Helix pomatia has a broader affinity for terminal 2-acetamido-2-deoxy-alpha-D-galactopyranose (alpha DGalNAc) residues than does that of Dolichos biflorus. The reactions of the lectin with terminal alpha DGalNAc units are strongly dependent on the nature of the aglycon and remain ill defined. The lectin may also react with appropriately presented terminal 2-acetamido-2-deoxy-beta-D-glucopyranose units. The broad affinity of the lectin of Wistaria floribunda which reacts both with a range of blood group specific glycoproteins (A, B, H, Lea, and Leb) and with non blood group glycoproteins [Sugii, S., & Kabat, E.A. (1980) Biochemistry 19, 1192-1199] appears best assigned to a combining site that favors pauci- or multivalent cooperative effects of clustered terminal beta-D-galactopyranose units. An attempt is made to rationalize certain of the inhibition data in terms of topographical features at the surfaces of the carbohydrate structures which are considered compatible for binding within essentially hydrophobic combining sites.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Antígenos Heterófilos , Antígeno de Forssman , Caracois Helix/imunologia , Hemaglutininas , Plantas/imunologia , Animais , Testes de Inibição da Hemaglutinação , Humanos , Monossacarídeos , Oligossacarídeos , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
The production of recombinant glycoprotein therapeutics requires characterization of glycosylation with respect to the lot-to-lot consistency. Here we introduce the ¿hypothetical N-glycan charge Z' as a parameter that allows to characterize the protein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein is deduced from the N-glycan mapping profile obtained via HPAE-PAD. In HPAEC, N-glycans are clearly separated according to their charge, i.e., their number of sialic acid residues, providing distinct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined as the sum of the products of the respective areas (A) in the asialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region, each multiplied by the corresponding charge: [formula: see text] Thus, a glycoprotein with mostly C4-4* structures will provide Z approximately equal to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3* structures will amount to Z approximately equal to 300 (e.g., bovine fetuin, Z = 290), a glycoprotein with mostly C2-2* structures will have Z approximately equal to 200 (e.g., human serum transferrin, Z = 207, or human plasma AT III, Z = 180), and a glycoprotein carrying only high-mannose type or trunkated structures will provide Z approximately equal to 0 (e.g., bovine pancreas ribonuclease B, Z = 15, and hen ovomucoid, Z = 15, respectively). The determination of Z was validated in multiple repetitive experiments and proved to be highly accurate and reliable. Z may therefore be regarded as a new and characteristic parameter for protein N-glycosylation.
Assuntos
Glicoproteínas/química , Polissacarídeos/química , Amidoidrolases/metabolismo , Animais , Bovinos , Glicoproteínas/metabolismo , Glicosilação , Humanos , Hidrazinas/metabolismo , Modelos Químicos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
We find Wr(a + b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wrb directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group O Rh- red blood cells. Despite this, different lines of evidence still indicate that glycophorin A is one of the receptors for erythrocyte invasion by P. falciparum. The Wrb epitope, however, does not appear to represent a distinct receptor site, which is in contrast to previous suggestions.
Assuntos
Eritrócitos/imunologia , Malária/imunologia , Plasmodium falciparum/imunologia , Sistema ABO de Grupos Sanguíneos , Suscetibilidade a Doenças , Humanos , Masculino , Sistema do Grupo Sanguíneo Rh-HrRESUMO
The distribution of the monoclonal antibody (MAb) BW494 in human pancreatic carcinoma biopsies during high dose intravenous immunotherapy was investigated. Using immunohistochemical techniques combined with anti-idiotypic, endothelial cell-specific and bispecific MAbs, it was shown that 3 days after onset of immunotherapy, MAb BW494 was bivalently bound to tumour cells in some highly vascularised areas near capillaries. No binding was observed in other highly vascularised tumour cell areas although the epitope detected by MAb BW494 was present. In contrast to our expectation the majority of the tumour cells were not yet saturated by the antibody, probably due to diffusion barriers in the solid tumour tissue.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias Pancreáticas/metabolismo , Anticorpos Monoclonais/uso terapêutico , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pancreáticas/terapiaRESUMO
The distribution of the monoclonal antibody (MAb) BW494 in human pancreatic carcinoma biopsies during high dose i.v. immunotherapy was investigated. Using immunohistochemical techniques combined with anti-idiotypic, endothelial cell specific and bispecific MAbs it was shown that 3 days after onset of immunotherapy, MAb BW494 was bivalently bound to tumor cells in some highly vascualized areas near capillaries. No binding was observed in other highly vascularized tumor cell areas although the epitope detected by MAb BW494 was present. In contrast to our expectation the majority of the tumor cells was not yet saturated by the antibody, probably due to diffusion barriers in the solid tumor tissue.
Assuntos
Anticorpos Monoclonais , Imunoterapia , Neoplasias Pancreáticas/patologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Humanos , Imuno-Histoquímica , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapiaRESUMO
The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.