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In recent years, Generalized Modules for Membrane Antigens (GMMA) have received increased attention as an innovative vaccine platform against bacterial pathogens, particularly attractive for low- and middle-income countries because of manufacturing simplicity. The assessment of critical quality attributes (CQAs), product-process interactions, identification of appropriate in process analytical methods, and process modeling is part of a robust quality by design (QbD) framework to support further development and control of manufacturing processes. QbD implementation in the context of the GMMA platform will ensure robust manufacturing of batches with desired characteristics, facilitating technical transfer to local manufacturers, regulatory approval, and commercialization of vaccines based on this technology. Here, we summarize the methodology suggested, applied to a first step of GMMA manufacturing process.
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Desarrollo de Vacunas , VacunasRESUMEN
This review reports on an overview of key enablers of acceleration/pandemic and preparedness, covering CMC strategies as well as technical innovations in vaccine development. Considerations are shared on implementation hurdles and opportunities to drive sustained acceleration for vaccine development and considers learnings from the COVID pandemic and direct experience in addressing unmet medical needs. These reflections focus on (i) the importance of a cross-disciplinary framework of technical expectations ranging from target antigen identification to launch and life-cycle management; (ii) the use of prior platform knowledge across similar or products/vaccine types; (iii) the implementation of innovation and digital tools for fast development and innovative control strategies.
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This publication provides some industry reflections on experiences from the Chemistry, Manufacturing, and Controls (CMC) development and manufacture and supply of vaccines and therapies in response to the COVID-19 pandemic. It integrates these experiences with the outcomes from the collaborative work between industry and regulators in recent years on innovative science- and risk-based CMC strategies to the development of new, high-quality products for unmet medical needs. The challenges for rapid development are discussed and various approaches to facilitate accelerated development and global supply are collated for consideration. Relevant regulatory aspects are reviewed, including the role of Emergency Use/Conditional Marketing Authorizations, the dialogue between sponsors and agencies to facilitate early decision-making and alignment, and the value of improving reliance/collaborative assessment and increased collaboration between regulatory authorities to reduce differences in global regulatory requirements. Five areas are highlighted for particular consideration in the implementation of strategies for the quality-related aspects of accelerated development and supply: (1) the substantial need to advance reliance or collaborative assessment; (2) the need for early decision making and streamlined engagement between industry and regulatory authorities on CMC matters; (3) the need to further facilitate 'post-approval' changes; (4) fully exploiting prior and platform knowledge; and (5) review and potential revision of legal frameworks. The recommendations in this publication are intended to contribute to the discussion on approaches that can result in earlier and greater access to high-quality pandemic vaccines and therapies for patients worldwide but could also be useful in general for innovative medicines addressing unmet medical needs.
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COVID-19 , Vacunas , COVID-19/epidemiología , COVID-19/prevención & control , Humanos , Pandemias/prevención & control , Vacunas/uso terapéuticoRESUMEN
Analytical methods are utilized throughout the biopharmaceutical and vaccines industries to conduct research and development, and to help control manufacturing inputs and outputs. These analytical methods should continuously provide quality data to support decisions while managing the remaining of risk and uncertainty. Analytical quality by design (AQbD) can provide a systematic framework to achieve a continuously validated, robust assay as well as life cycle management. AQbD is rooted in ICH guidelines Q8 and Q9 that were translated to the analytical space through several white papers as well as upcoming USP 1220 and ICH Q14. In this white paper, we expand on the previously published concepts of AQbD by providing additional context for implementation in relation to ICH Q14. Using illustrative examples, we describe the AQbD workflow, its relation to traditional approaches, and potential pathways for ongoing, real-time verification. We will also discuss challenges with respect to implementation and regulatory strategies.
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Proyectos de Investigación , Vacunas , Animales , Estadios del Ciclo de VidaRESUMEN
The COVID-19 pandemic has shown itself to be an unprecedented challenge for vaccines which are widely recognized as the most important tool to exit this pandemic. We have witnessed vaccine scientists, developers, manufacturers, and stakeholders deliver several vaccines in just about a year. This is an unprecedented achievement in an environment that was not ready to manage such a global public health crisis. Indeed, the pandemic has highlighted some hurdles that need to be addressed in the system in order to streamline the regulatory processes and be in a situation where life-saving pharmaceutical solutions such as vaccines can be delivered quickly and equitably to people across the globe. More precisely, trade-offs had to be made between the need for regulatory flexibility in the requirements for manufacturing and controls to enable rapid availability of large volumes of vaccines vs the increased stringency and the lack of harmonization in the regulatory environment for vaccines globally. It is also characterized by a high heterogeneity in terms of review and approval processes, limiting equitable and timely access. We review and highlight the challenges relating to several topics, including process validation, comparability, stability, post-approval-changes, release testing, packaging, genetically modified organisms and variants. We see four areas for accelerating access to vaccines which provide solutions for the regulatory concerns, (1) science- and risk-based approaches, (2) global regulatory harmonization, (3) use of reliance, work-sharing, and recognition processes and (4) digitalization. These solutions are not new and have been previously highlighted. In recent months, we have seen some progress at the health authority level, but still much needs to be done. It is now time to reflect on the first lessons learnt from a devastating pandemic to ultimately ensure quick and wide access to medicines and vaccines for the citizens and patients.
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COVID-19 , Vacunas , Humanos , Pandemias/prevención & control , Salud Pública , SARS-CoV-2RESUMEN
Vaccine discovery and vaccination against preventable diseases are one of most important achievements of the human race. While medical, scientific & technological advancements have kept in pace and found their way into treatment options for a vast majority of diseases, vaccines as a prevention tool in the public health realm are found languishing in the gap between such innovations and their easy availability/accessibility to vulnerable populations. This paradox has been best highlighted during the unprecedented crisis of the COVID-19 pandemic. As part of a two series publication on the vaccine industry's view on how to accelerate the availability of vaccines worldwide, this paper offers a deep dive into detailed proposals to enable this objective. These first-of-its-kind technical proposals gleaned from challenges and learnings from the COVID-19 pandemic are applicable to vaccines that are already on the market for routine pathogens as well as for production of new(er) vaccines for emerging pathogens with a public health threat potential. The technical proposals offer feasible and sustainable solutions in pivotal areas such as process validation, comparability, stability, post-approval changes, release testing, packaging, genetically modified organisms and variants, which are linked to manufacturing and quality control of vaccines. Ultimately these proposals aim to ease high regulatory complexity and heterogeneity surrounding the manufacturing & distribution of vaccines, by advocating the use of (1) Science and Risk based approaches, (2) global regulatory harmonization, (3) use of reliance, work-sharing, and recognition processes and (4) digitalization. Capitalizing & collaborating on such new-world advancements into the science of vaccines will eventually benefit the world by turning vaccines into vaccination, ensuring the health of everyone.
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COVID-19 , Vacunas , Humanos , Pandemias , SARS-CoV-2 , VacunaciónRESUMEN
A structured approach to method development can help to ensure an analytical procedure is robust across the lifecycle of its use. The analytical target profile (ATP), which describes the required quality of the reportable value to be produced by the analytical procedure, enables the analytical scientist to select the best analytical technology on which to develop their procedure(s). Once the technology has been identified, screening of potentially fit for purpose analytical procedures should take place. Analytical procedures that have been demonstrated to meet the ATP should be evaluated against business drivers (e.g., operational constraints) to determine the most suitable analytical procedure. Three case studies are covered from across small molecules, vaccines, and biotherapeutics. The case studies cover different aspects of the analytical procedure selection process, such as the use of platform method development processes and procedures, the development of multiattribute analytical procedures, and the use of analytical technologies to provide product characterization knowledge in order to define or redefine the ATP. Challenges associated with method selection are discussed such as where existing pharmacopoeial monographs link acceptance criteria to specific types of analytical technology.
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Proyectos de Investigación , Vacunas , Control de CalidadRESUMEN
Stability assessment of pharmaceuticals in specific storage and shipment conditions is a key requirement to ensure that safe and efficacious products are administered to patients. This is particularly relevant for vaccines, with numerous vaccines strictly requiring cold storage to remain stable. When stability evaluation is exclusively based on real-time data, it may represent a bottleneck for rapid and effective vaccine access. Stability modeling for vaccines represents a key resource to predict stability based on accelerated stability studies; nevertheless, this approach is not fully exploited for these kinds of products. This is likely because of the complexity and diversity of vaccines, as well as the limited availability of dedicated guidelines or illustrative case studies. This article reports a cross-company perspective on stability modeling for vaccines. Several examples, based on the direct experience of the contributors, demonstrate that modeling approaches can be highly valuable to predict vaccines' shelf life and behavior during shipment or manipulation. It is demonstrated that modeling methodologies need to be tailored to the nature of the vaccine, the available prior knowledge, and the monitored attributes. Considering that the well-established strategies reported in ICH or WHO guidelines are not always broadly applicable to vaccines, this article represents an important source of information for vaccine researchers and manufacturers, setting the grounds for further discussion within the vaccine industry and with regulators.
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Quality by design (ICH-Topic Q8) requires a prospective summary of the desired quality characteristics of a drug product. This is known as the Quality Target Product Profile (QTPP), which forms the basis for the design and development of the product. An analogous term has been established for analytical procedures called the Analytical Target Profile (ATP). The ATP, in a similar fashion to the QTPP, prospectively summarizes the requirements associated with a measurement on a quality attribute which needs to be met by an analytical procedure. Criteria defined in the ATP relate to the maximum uncertainty associated with the reportable result that is required to maintain acceptable confidence in the quality decision made from the result. The ATP is used to define and assess the fitness of an analytical procedure in the development phase and during all changes across the analytical lifecycle. One or more analytical procedures can meet the requirements of an ATP. The ATP can be applied to any quality attribute across any pharmaceutical modality where an analytical procedure is used to generate a reportable result, and this paper provides examples from three of these modalities: small molecules, oligonucleotides, and vaccines. Some key performance characteristics will be discussed for each ATP, namely specificity, accuracy, and precision, taking into account the expected range of the analyte. The combination of accuracy and precision into a combined uncertainty characteristic is also discussed as a more holistic approach. The use of the ATP concept will help focus attention on the properties of a method which impact quality decisions rather than method descriptions and may enable greater regulatory flexibility across the lifecycle using established conditions based on method performance criteria as proposed in the Step 2 version of ICHQ12. The revision of ICHQ2(R1) and development of the new ICHQ14 guideline (Analytical Procedure Development) will provide a golden opportunity to harmonize the definition of new QbD concepts such as the ATP.
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Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Oligonucleótidos/análisis , Preparaciones Farmacéuticas/análisis , Polisacáridos/análisis , Control de Calidad , Vacunas/análisisRESUMEN
Bexsero is the first approved vaccine for active immunization of individuals from 2 months of age and older to prevent invasive disease caused by Neisseria meningitidis serogroup B. The active components of the vaccine are Neisseria Heparin Binding Antigen, factor H binding protein, Neisseria adhesin A, produced in Escherichia coli cells by recombinant DNA technology, and Outer Membrane Vesicles (expressing Porin A and Porin B), produced by fermentation of Neisseria meningitidis strain NZ98/254. All the Bexsero active components are adsorbed on aluminum hydroxide and the unadsorbed antigens content is a product critical quality attribute. In this paper the development of a fast, selective and sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of the Bexsero antigens in the vaccine supernatant is presented. For the first time in the literature, the Quality by Design (QbD) principles were applied to the development of an analytical method aimed to the quality control of a vaccine product. The UHPLC method was fully developed within the QbD framework, the new paradigm of quality outlined in International Conference on Harmonisation guidelines. Critical method attributes (CMAs) were identified with the capacity factor of Neisseria Heparin Binding Antigen, antigens resolution and peak areas. After a scouting phase, aimed at selecting a suitable and fast UHPLC operative mode for the vaccine antigens separation, risk assessment tools were employed to define the critical method parameters to be considered in the screening phase. Screening designs were applied for investigating at first the effects of vial type and sample concentration, and then the effects of injection volume, column type, organic phase starting concentration, ramp time and temperature. Response Surface Methodology pointed out the presence of several significant interaction effects, and with the support of Monte-Carlo simulations led to map out the design space, at a selected probability level, for the desired CMAs. The selected working conditions gave a complete separation of the antigens in about 5min. Robustness testing was carried out by a multivariate approach and a control strategy was implemented by defining system suitability tests. The method was qualified for the analysis of the Bexsero vaccine.
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Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Vacunas Meningococicas/análisis , Porinas/análisis , Cromatografía Líquida de Alta Presión , Neisseria meningitidis Serogrupo B , Control de CalidadRESUMEN
This chapter reports an overview of the recent advances in the analysis of mono- and oligosaccharides by capillary electrophoresis (CE); furthermore, relevant reviews and research articles recently published in the field are tabulated. Additionally, pretreatments and procedures applied to uncharged and acidic carbohydrates (i.e., monosaccharides and lower oligosaccharides carrying carboxylate, sulfate, or phosphate groups) are described.Representative examples of such procedures are reported in detail, upon describing robust methodologies for the study of (1) neutral oligosaccharides derivatized by reductive amination and by formation of glycosylamines; (2) sialic acid derivatized with 2-aminoacridone, released from human serum immunoglobulin G; (3) anomeric couples of neutral glycosides separated using borate-based buffers; (4) unsaturated, underivatized oligosaccharides from lyase-treated alginate.
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Carbohidratos/sangre , Electroforesis Capilar/métodos , Monosacáridos/sangre , Oligosacáridos/sangre , Carbohidratos/química , Glicósidos/sangre , Glicósidos/química , Humanos , Monosacáridos/química , Ácido N-Acetilneuramínico/sangre , Ácido N-Acetilneuramínico/química , Oligosacáridos/químicaRESUMEN
CE applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case the degree of substitution was as low as 14 %.The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization.The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50 % galactose substituted HA.Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters ξ.In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.
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Carbohidratos de la Dieta/aislamiento & purificación , Electroforesis Capilar/métodos , Ácido Hialurónico/aislamiento & purificación , Polisacáridos/aislamiento & purificación , Esterificación , Ácido Hialurónico/química , Peso Molecular , Polisacáridos/químicaRESUMEN
Iodinated contrast media (ICM) are persistent and ubiquitous water pollutants. Because of their high water solubility and biochemical stability, their phase-separation and recovery from the aquatic environment is very difficult. Here, borate was chosen as a complexing agent of the two diagnostic aids iomeprol and iopamidol in order to provide them with a negative charge and to fix the resulting adducts on Dowex 1X4 ion exchangers. A systematic characterization study of the complex by means of capillary zone electrophoresis and 11B NMR revealed that iomeprol and iopamidol interact with borate anions in aqueous solutions giving a 1:1 single-charged adduct and that the association constant at 25 °C for both contrast agents is highest at pH 10.5. These findings allowed the proper calibration of experimental parameters for further batch adsorption-desorption trials, where the two ICM were shown to be almost completely removed from the water phase and released from the solid sorbents in mild conditions, enabling the recovery of functional resin.
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Boratos/química , Medios de Contraste/química , Yopamidol/análogos & derivados , Yopamidol/química , Contaminantes Químicos del Agua/química , Adsorción , Resinas de Intercambio Aniónico , Reciclaje , Resinas Sintéticas , Eliminación de Residuos Líquidos/métodos , Rayos XRESUMEN
Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).
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Cicloheximida/análogos & derivados , Colorantes Fluorescentes/química , Biosíntesis de Proteínas , Acetilación , Animales , Cicloheximida/síntesis química , Colorantes Fluorescentes/síntesis química , Ratones , Células 3T3 NIH , Espectrometría de Fluorescencia/métodosRESUMEN
N-linked or O-linked glycans derived from glycoprotein processing carry, an N-acetylglucosamine or an N-acetylgalactosamine respectively, at their reducing termini. The presence of the N-acetylamino group on C-2 of reducing sugar residues has been reported to hamper the derivatization reaction with a chromophore at the anomeric centre. In this paper N-acetyllactosamine, N-acetylglucosamine, N-acetylgalactosamine and several other neutral monosaccharides are coupled to three different dyes (4-aminobenzonitrile, 2-aminopyridine, 2-aminobenzoic acid (2-AA)) by reductive amination and analysed by CE with UV detection. The 2-AA derivatives showed the lowest concentration detection limits, varying approximately in the 2-3 muM range for the saccharides tested including the N-acetamido ones. The possibility to separate and detect with the same sensitivity ten 2-AA-labelled monosaccharides mainly found in mammalian or plant glycoproteins in a single CE run is highlighted. The analysis has been carried out in less than 25 min using the borate-complexation method in CZE mode. The influence of the strength of the acid used as catalyst in the chemical modification of the sugars with 2-AA is also shortly addressed.
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Amino Azúcares/análisis , Cromatografía Capilar Electrocinética Micelar/métodos , Electroforesis Capilar/métodos , Acetilgalactosamina/análisis , Acetilglucosamina/análisis , Aminopiridinas/química , Concentración de Iones de Hidrógeno , Nitrilos/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , ortoaminobenzoatos/químicaRESUMEN
Malignant transformation of epithelial cells is frequently associated with the alteration of glycosylation pathways. Tn is a common tumor-associated carbohydrate antigen present in 90% of human carcinomas and its expression correlates with metastatic potential and poor prognosis. Despite its relevance, the functional role of Tn in tumor biology has not been firmly established probably for the lack of appropriate experimental tools. Our aims were to produce highly reactive monoclonal antibodies against Tn making use of synthetically produced Tn and to test their usefulness for in vivo imaging as well as to define their potential functional activity in tumor cell spread. We immunized mice with Tn clustered on cationized BSA and screened the positive hybridomas with Tn-biotinylated alginate. Enzyme-linked immuno sorbent assay and immunofluorescence assays revealed that the most reactive anti-Tn IgM mAb (2154F12A4) selectively recognized Tn on the MCF7 breast cancer cell line since its binding to the cell membrane was completely abolished by preincubation with purified Tn. Importantly, QDot 800-conjugated mAb injected in MCF7-tumor bearing mice specifically bound to primary tumor lesions as well as to metastases in lymph nodes. In addition, this mAb was able to inhibit cancer cell adhesion to lymphatic endothelium suggesting a novel involvement of Tn in the lymphatic dissemination of cancer cells and hypothesizing future applications in inhibiting lymphatic metastases.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Ganglios Linfáticos/inmunología , Neoplasias/inmunología , Animales , Antígenos de Carbohidratos Asociados a Tumores/química , Adhesión Celular , Línea Celular Tumoral , Endotelio/inmunología , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática/inmunología , Metástasis Linfática/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Molecular , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The physico-chemical characterization of NadADelta(351-405), a recombinant protein discovered by reverse vaccinology, component of a candidate vaccine against Neisseria meningitidis serotype B is presented. Analytical methods like mass spectrometry, electrophoresis, optical spectroscopy and SEC-MALLS have been applied to unveil the structure of NadADelta(351-405), and to evaluate Product-Related Substances. Moreover, analysis of the protein after intentional denaturation has been applied in order to challenge the chosen methods and to determine their appropriateness and specificity. All the obtained results were inserted in a model allowing in-depth understanding of the antigen NadADelta(351-405): it is present in solution as a homo-trimer, retaining a high percentage of alpha-helix secondary structure, and able to reassemble from monomeric subunits after thermal denaturation; this structural organization is consistent with that foreseen for MenB NadA (Neisseria Adhesin A). The analytical data sets produced during process development for clinical phases I-III material confirm product quality and manufacturing consistency.
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Adhesinas Bacterianas/química , Vacunas Meningococicas/química , Neisseria meningitidis Serogrupo B/química , Proteínas Recombinantes/química , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Femenino , Vacunas Meningococicas/inmunología , Ratones , Datos de Secuencia Molecular , Neisseria meningitidis Serogrupo B/inmunología , Mapeo Peptídico , Conformación Proteica , Proteínas Recombinantes/inmunología , Espectrometría de Fluorescencia , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunologíaRESUMEN
This chapter reports an overview of the recent advances in the analysis of neutral sugars by capillary electrophoresis (CE); furthermore, some relevant reviews and research articles in the field are tabulated. Comparison of CE with chromatography is also presented, with special attention to separation efficiency and sensitivity. The main routes aimed at pretreatment and CE analysis of uncharged mono-, oligosaccharides, and glycosides are described. Representative examples of such procedures are reported in detail, upon describing robust methodologies for the study of (1) neutral mono- and oligosaccharides derivatized by reductive amination and by formation of glycosylamines; (2) underivatized mono- and di-saccharides analyzed using highly alkaline buffers; and (3) anomeric couples of glycosides separated using borate-based buffers.
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Electroforesis Capilar/métodos , Glicósidos/análisis , Monosacáridos/análisis , Oligosacáridos/análisis , Boratos , Tampones (Química) , Cromatografía Capilar Electrocinética Micelar , Glicósidos/química , Monosacáridos/química , Oligosacáridos/química , Oxidación-ReducciónRESUMEN
This chapter illustrates the usefulness of capillary electrophoresis (CE) for the analysis of sugar acids, that is, monosaccharides and lower oligosaccharides carrying carboxylate, sulphate or phosphate groups. In order to provide a general description of the main results and challenges in the field, some relevant applications and reviews on CE of such saccharidic compounds are tabulated. Furthermore, some detailed experimental procedures are shown, regarding the CE analysis of sugar acids released upon hydrolysis of acidic polysaccharides and of glycans linked to glycoproteins. In particular, the protocols will deal with the following compounds: (i) unsaturated, underivatized oligosaccharides from lyase-treated alginate; (ii) oligosaccharides derivatized with 4-aminobenzonitrile, arising from chemical hydrolysis of alginate; (iii) sialic acid derivatized with 2-aminoacridone, released from human serum immunoglobulin G.
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Electroforesis Capilar/métodos , Azúcares Ácidos/análisis , Alginatos/química , Cromatografía Capilar Electrocinética Micelar , Humanos , Inmunoglobulina G/química , Ácido N-Acetilneuramínico/análisis , Ácido N-Acetilneuramínico/química , Azúcares Ácidos/químicaRESUMEN
Capillary electrophoresis (CE) applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case, the degree of substitution was as low as 14%. The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization. The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50% galactose-substituted hyaluronic acid (HA). Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters xi. In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.