The conjugation of serotype 31 pneumococcal polysaccharide and CRM197 in N,N-dimethylformamide

array:22 ["pii" => "S1576988723000213" "issn" => "15769887" "doi" => "10.1016/j.vacun.2023.02.002" "estado" => "S300" "fechaPublicacion" => "2023-07-01" "aid" => "285" "copyrightAnyo" => "2023" "documento" => "article" "crossmark" => 1 "subdocumento" => "fla" "cita" => "Vacunas. 2023;24:248-54" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "itemSiguiente" => array:18 [ "pii" => "S157698872200200X" "issn" => "15769887" "doi" => "10.1016/j.vacun.2022.12.006" "estado" => "S300" "fechaPublicacion" => "2023-07-01" "aid" => "277" "copyright" => "Elsevier España, S.L.U." "documento" => "simple-article" "crossmark" => 1 "subdocumento" => "cor" "cita" => "Vacunas. 2023;24:255-7" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "en" => array:11 [ "idiomaDefecto" => true "cabecera" => "Letter to the editor" "titulo" => "Interactions between human microbiota and vaccines; Current perspectives" "tienePdf" => "en" "tieneTextoCompleto" => "en" "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "255" "paginaFinal" => "257" ] ] "titulosAlternativos" => array:1 [ "en" => array:1 [ "titulo" => "Interacciones entre la microbiota humana y las vacunas; Perspectivas actuales" ] ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:8 [ "identificador" => "f0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1124 "Ancho" => 2043 "Tamanyo" => 259476 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0005" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "The main mechanism of gut microbiota actions regarding boosting the vaccine-induced immune response against respiratory infections." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Masoud Keikha, Mehdi Zandhaghighi, Shahram Shahraki Zahedani" "autores" => array:3 [ 0 => array:2 [ "nombre" => "Masoud" "apellidos" => "Keikha" ] 1 => array:2 [ "nombre" => "Mehdi" "apellidos" => "Zandhaghighi" ] 2 => array:2 [ "nombre" => "Shahram Shahraki" "apellidos" => "Zahedani" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S157698872200200X?idApp=UINPBA00004N" "url" => "/15769887/0000002400000003/v1_202307141223/S157698872200200X/v1_202307141223/en/main.assets" ] "itemAnterior" => array:18 [ "pii" => "S1576988723000316" "issn" => "15769887" "doi" => "10.1016/j.vacun.2023.04.003" "estado" => "S300" "fechaPublicacion" => "2023-07-01" "aid" => "291" "copyright" => "Elsevier España, S.L.U." "documento" => "article" "crossmark" => 1 "subdocumento" => "rev" "cita" => "Vacunas. 2023;24:218-47" "abierto" => array:3 [ "ES" => false "ES2" => false "LATM" => false ] "gratuito" => false "lecturas" => array:1 [ "total" => 0 ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "Review article" "titulo" => "The status of COVID-19 vaccines in India: A review" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "218" "paginaFinal" => "247" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "El estado de las vacunas COVID-19 en India: Una revisión" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:8 [ "identificador" => "f0065" "etiqueta" => "Fig. 13" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr13.jpeg" "Alto" => 1276 "Ancho" => 2165 "Tamanyo" => 184800 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0065" "detalle" => "Fig. 1" "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "The figure represents the publication trend of COVID-19 as well as vaccine articles in the year 2019, 2020, 2021 and 2022. The data was obtained from NCBI and PubMed using SARS-CoV-2, COVID-19, Vaccine, Coronavirus, SARS-CoV-2 vaccine and COVID-19 vaccine as keywords." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Deepak Kumar Jha, Kumar Pranay, Samiksha, Amit Kumar, Niti Yashvardhini" "autores" => array:5 [ 0 => array:2 [ "nombre" => "Deepak Kumar" "apellidos" => "Jha" ] 1 => array:2 [ "nombre" => "Kumar" "apellidos" => "Pranay" ] 2 => array:1 [ "apellidos" => "Samiksha" ] 3 => array:2 [ "nombre" => "Amit" "apellidos" => "Kumar" ] 4 => array:2 [ "nombre" => "Niti" "apellidos" => "Yashvardhini" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1576988723000316?idApp=UINPBA00004N" "url" => "/15769887/0000002400000003/v1_202307141223/S1576988723000316/v1_202307141223/en/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "Desarrollo en Vacunas" "titulo" => "The conjugation of serotype 31 pneumococcal polysaccharide and CRM197 in N,N-dimethylformamide" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "248" "paginaFinal" => "254" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Chengli Zong, Hongzhao Mao, Huiting Li, Shiyan Mai" "autores" => array:4 [ 0 => array:4 [ "nombre" => "Chengli" "apellidos" => "Zong" "email" => array:1 [ 0 => "chengli.zong@hainanu.edu.cn" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "*" "identificador" => "cr0005" ] ] ] 1 => array:2 [ "nombre" => "Hongzhao" "apellidos" => "Mao" ] 2 => array:2 [ "nombre" => "Huiting" "apellidos" => "Li" ] 3 => array:2 [ "nombre" => "Shiyan" "apellidos" => "Mai" ] ] "afiliaciones" => array:1 [ 0 => array:2 [ "entidad" => "School of Pharmaceutical Sciences, Hainan Universi, Hainan University School of Pharmaceutical Sciences, China" "identificador" => "af0005" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cr0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "La conjugación del polisacárido bacteriano neumocócico del serotipo 31 y CRM197 en N,N-Dimetilformamida" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:8 [ "identificador" => "f0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2389 "Ancho" => 1896 "Tamanyo" => 193343 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0005" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "1H (A), TOCSY (B) NMR of activated native s-31-ps. Calculation of activation level: Unit D 6-methyl 1H NMR peak moved due to activation. The activation level (%) can be obtained by comparing the integration of shifted peak and the total D-6 integration. Activation =100%*(integration of 1.17 ppm + 1.15 ppm)/sum of integration(1.22 ppm + 1.17 ppm + 1.15 ppm) =100%*(0.38 + 0.68)/3.00 = 35%." ] ] ] "textoCompleto" => "1IntroductionStreptococcus pneumonia (Spn) caused invasive pneumococcal disease (IPD) is one of the major death causes for children under 5 years old<a class="elsevierStyleCrossRef" href="#bb0005">1</a>,<a class="elsevierStyleCrossRef" href="#bb0010">2</a>. Polysaccharide conjugate vaccine (PCV), including 7 serotypes (PCV7, Prevnar 7), was approved by FDA in 2000 and recommended for all children. In this vaccine, purified capsular pneumococcal polysaccharide was conjugated to CRM197 to activate T-cell-dependent immune responses<a class="elsevierStyleCrossRef" href="#bb0015">3</a>. Two doses are enough to provide good protection for children and people at greater risk of complications for IPD. PCVs, including 15 and 20 serotypes (PCV15- VAXNEUVANCE and PCV20- Prevnar 20) were approved by FDA in 2021.Serotype 31 is a non-vaccine serotype and has increased in prevalence. By using Nuclear Magnetic Resonance (NMR) as the primary tool, we report the revised serotype 31 polysaccharide (s-31-ps) structure as [→3)-β-D-Galf-(5/6-OAc)-(1 → 3)-β-D-Galp-(1 → 3)-β-L-Rhap-(2-OAc)-(1 → 2)-α-L-Rhap-(1 → 4)-β-D-GlcpA-(1→]n. Furthermore, the reductive amination-conjugate of serotype 31 polysaccharide and cross reacting material (CRM197) protein was prepared in organic solvent (N,N-dimethylformamide, DMF) instead of water. The reaction is faster, and the DMF conjugate elicited comparable immune responses with the aqueous conjugate. S-31-ps conjugate vaccine has the potential of being included in the next-generation PCV vaccines.The conjugation between a large polysaccharide (molecular weight up to a few million) and CRM197 carrier protein (58 kDa) is very complex, making PCV one of the most complicated drugs to manufacture. Most pharmaceutical companies have been employing NaCNBH3 mediated reductive amination in water as the conjugation method. It is very slow and may take days, jeopardizing the protein stability<a class="elsevierStyleCrossRef" href="#bb0020">4</a>. The process must be carefully monitored for a good quality control, which is a significant financial and labor burden for companies. Here we report the detailed procedure (<a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>) for the fast conjugation of s-31-ps and CRM197 by reductive amination in an organic solvent (DMF), using an organic solvent-soluble reducing reagent: sodium triacetoxyborohydride (STAB).<elsevierMultimedia ident="sch0005"></elsevierMultimedia>2Materials and methods2.1ActivationVortex the solution of s-31-ps powder (5 mg, 1 eq (based on the pentasaccharide repeating unit)) in water (500 μL) in a 2 mL microcentrifuge tube for 10 min. If the solution has un-dissolved material, vortex the solution for 15 min. If the solution is still not clear, leave the solution on the bench-top overnight. Usually, the solution will be clear and pipettable. Then 3.0 M sodium acetate solution (pH 5) was added to the solution to achieve a final concentration of 100 mM sodium acetate, followed by the addition of 100 mM NaIO4 stock solution (1 eq, 44 μL). The mixture was stirred by a magnetic bar for 5 h at room temperature. Then the reaction mixture was transferred to a pre-rinsed 15 mL 30 kD mwco Amicon filter, filled up with water, centrifuged at 4500 rpm at 20 °C until the final volume reached ∼100 μL. The filtrate was discarded.The residue may appear very viscous. For the following filtrations, after filling the filter up with water, pipette up and down to mix. Otherwise, the polysaccharide may wrap the unwanted molecules and stick to the bottom of the filter. The filtration step was repeated two more times. Finally, the residue was pipetted to a 2 mL vial. Two portions of 200 μL of water were used to rinse the filter. The aqueous solutions were combined and freeze-dried.2.2FormulationPolysaccharide formulation: Add 250 μL water to 2.5 mg of free-dried polysaccharide. The solution is very viscous at the beginning. Extended stirring did not alleviate the situation. Then 100 μL 30% w/v freshly prepared sucrose solution and 650 μL water to reach a 2.5 mg/mL polysaccharide, 5% sucrose solution. After stirring overnight, the solution became clear and homogeneous. The well-mixed solution was lyophilized to give a white cake.CRM197 formulation: Commercially available CRM197 (1.25 mg in solution) was spin filtered three times by a 10 k Amcon filter. The 100 μL residue was pipetted out and reconstituted to 1 mL 5 mM sodium phosphate pH 7.2 and 5% w/v sucrose solution. The mixture was freeze-dried to give a white cake.2.3Conjugation and purificationSodium triacetoxyborohydride (STAB) preparation: acetic acid (11.8 μL) was added to the mixture of NaBH4 (2.22 mg) in DMF (1 mL). Bubbles were formed upon acid addition. The solution was stirred for 20 min before use.Polysaccharide and CRM197 dissolution in DMF: 1 mL DMF was added individually to the lyophilized polysaccharide and CRM197 cakes at room temperature. The solutions were vortexed at low speed for 2 min. Do not store the solutions for a longer time.Conjugation: The prepared solutions were combined in a 20 mL tube. The freshly prepared STAB solution was added. The mixture was incubated at room temperature at 500 rpm for 3 h. Then 1 mg NaBH4 in 1 mL water was added to the mixture to quench any unreacted aldehyde.Purification: The solution was diluted by adding 40 mL 0.1% w/v PS-20 in water to dilute the DMF percentage to less than 5%, which is the maximum DMF tolerance level for water-compatible Amicon filters. Then the reaction mixture was transferred proportionally to a pre-rinsed 15 mL 100 kD mwco Amicon filter, centrifuged at 4500 rpm at 20 °C until the final volume reached ∼100 μL. Then water was added to fill up the filter and centrifuged until the final volume reached 100 μL. The process was repeated two more times. Finally, the residue was pipetted to a 2 mL vial, two portions of 200 μL of D2O were used to rinse the filter, and the aqueous solutions were combined and analyzed by using NMR and HPLC-RI.3Results and discussionThe structure of s-31-ps has been reported in our previous work<a class="elsevierStyleCrossRef" href="#bb0025">5</a>. The polysaccharide features a pentasaccharide repeating unit, one furanose, and three O-acetate groups with a molecular weight of 910kD and a poly-dispersity index (PDI) of 1.42. The following steps are followed to prepare s-31-ps and CRM197 conjugate: the polysaccharide was activated by NaIO4, followed by formulation with sucrose in water, lyophilization, dissolution in DMF, and finally conjugation with CRM197 by using freshly prepared STAB (<a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>).3.1ActivationS-31-ps powder was firstly dissolved in water at a concentration of 10 mg/mL. It should be noted that the water solution of pneumococcal bacterial polysaccharide with O-acetate groups is usually viscous, probably due to the hydrogen bond between an acetyl-oxygen atom and hydroxyl proton. Longer vortexing or storage time is required for proper dissolution. Various methods are available for polysaccharide activation. Here, sodium periodate (NaIO4) was used to break the vicinal-hydroxyls under slightly acid condition to form the aldehyde conjugation sites. The aqueous solution of the activated product is even more viscous.As indicated by the blue circle in <a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>, there are two activation sites (vicinal-hydroxyl groups). To pinpoint the exact activation sites, the ratio between those sites and the activation level is challenging due to the structural complexity of pneumococcal polysaccharide. It is a mixture of the desired capsular polysaccharide, cell wall polysaccharide, residue amount of protein and other residuals from process. Also, the generated aldehyde group may hydrate or form other active intermediates<a class="elsevierStyleCrossRef" href="#bb0020">4</a>. We attempted to estimate these information by using NMR. The activation level (%) was roughly estimated as 35% by comparing the integration of shifted methyl peaks (1.17 and 1.15 ppm) and the total D-6 integration (<a class="elsevierStyleCrossRef" href="#f0005">Fig. 1</a>) based on the assumption that the activation of ring D and E can both impact the chemical shift of D-6 proton. This assumption may not be accurate. Other derivation methods may be explored to more accurately quantify the activation.<elsevierMultimedia ident="f0005"></elsevierMultimedia>There are two vicinal-hydroxyls activation sites marked in blue circles (<a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>, unit D 3/4 and unit E 2/3). As shown in <a class="elsevierStyleCrossRef" href="#f0005">Fig. 1</a>A, a few new peaks appeared in the anomeric region, partially attributed to the hydrated aldehyde proton. Among those peaks, the highest at 5.01 ppm correlates with the shifted unit D methyl peak at 1.15 ppm, suggesting unit D might be the major activation site (<a class="elsevierStyleCrossRef" href="#f0005">Fig. 1</a>B).3.2FormulationPneumococcal bacterial polysaccharide like s-31-ps does not dissolve in an organic solvent, such as DMF. S-31-ps will be soluble in DMF when properly formulated with sucrose, which is soluble in water and DMF. First, a mixture of s-31-ps (2.5 mg/mL) and 5% w/v sucrose was prepared and freeze-dried. Similarly, CRM197 was formulated with sucrose and pH 7.2 5 mM sodium phosphate to stabilize the protein.3.3ConjugationWith the lyophilized cakes in hand, they were individually dissolved in DMF, combined, followed by the addition of freshly prepared STAB. The mixture was stirred for 3 h. A longer mixing time will lead to precipitation, probably due to over-conjugation forming macromolecules. The unreacted aldehyde was quenched by adding NaBH4 in water. Then the solution was diluted with water and purified by spin filtration using a 100 k mwco Amicon filter to afford the s-31-ps-CRM197 conjugate. Un-reacted CRM197 will be filtered off.3.4Conjugate characterizationThe S-31-ps and CRM197 conjugate was characterized by HPSEC-IR (<a class="elsevierStyleCrossRef" href="#f0010">Fig. 2</a>) and NMR (<a class="elsevierStyleCrossRef" href="#f0015">Fig. 3</a>). The HPSEC-IR data indicated the conjugate was eluted within 20 min, while native s-31-ps was eluted at around 22 min5, indicating a size increase and the success of the conjugation. The NMR spectrum of the conjugate (<a class="elsevierStyleCrossRef" href="#f0015">Fig. 3</a>C) has been stacked with that of native (<a class="elsevierStyleCrossRef" href="#f0015">Fig. 3</a>A) and activated (<a class="elsevierStyleCrossRef" href="#f0015">Fig. 3</a>B) s-31-ps. As can be seen from the spectrum, the conjugate indicated protein aromatic and polysaccharide signals. The O-acetate signals can still be observed at around 2 ppm, indicating the conjugation condition did not touch the O-acetate groups, which may impact the polysaccharide immunogenicity.<elsevierMultimedia ident="f0010"></elsevierMultimedia><elsevierMultimedia ident="f0015"></elsevierMultimedia>Preparation of polysaccharide samples: 3 mg/mL polysaccharide, glucan molecular weight of 70 kD, 150 kD, 200 kD, 500 kD, and 1000 kD standards were dissolved in 1 mL water individually for further analysis.The HPLC chromatographic conditions: Waters e2695 coupled with RI detector (2414) was used for the analysis. Separation was carried out using UltrahydrogelTM-120 column (7.8 × 300 mm) and UltrahydrogelTM-500 column (7.8 × 300 mm) using isocratic elution of 0.05% sodium azide in an aqueous solution at 0.6 mL/min. Column and detector temperature was kept at 40 °C. The total run time was 15 min, and the sample injection volume was 10 μL. Data were processed by GraphPad Prism 8.0.1.NMR data were acquired on JOEL 600 MHz or Brucker 500 MHz instruments. 600 μL of the samples dissolved in deuterated solvents was usually used. Lock, shim, tune was performed before data acquisition. NMR data were processed by MestReNova version 14.2.0. The spectrum was either referenced to DMSO internal standard or CPS choline methyl signal. Water suppression was performed for serotype 31 polysaccharide-CRM197 DMF conjugate.4ConclusionsThe conjugation of polysaccharide and carrier protein is the most challenging step for PCVs. The reaction is usually in water using NaCNBH3 and may take up to several days. Here we report the reductive amination of s-31-ps and CRM197 in DMF using STAB. To assist the polysaccharide and CRM197 dissolution in DMF, they were individually formulated with sucrose as the solubilizer. The target conjugate can be successfully obtained within hours. The current method provides a new polysaccharide and carrier protein conjugation strategy." "textoCompletoSecciones" => array:1 [ "secciones" => array:9 [ 0 => array:3 [ "identificador" => "xres1930703" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "as0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1664178" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres1930704" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "as0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec1664179" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "s0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "s0010" "titulo" => "Materials and methods" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "s0015" "titulo" => "Activation" ] 1 => array:2 [ "identificador" => "s0020" "titulo" => "Formulation" ] 2 => array:2 [ "identificador" => "s0025" "titulo" => "Conjugation and purification" ] ] ] 6 => array:3 [ "identificador" => "s0030" "titulo" => "Results and discussion" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "s0035" "titulo" => "Activation" ] 1 => array:2 [ "identificador" => "s0040" "titulo" => "Formulation" ] 2 => array:2 [ "identificador" => "s0045" "titulo" => "Conjugation" ] 3 => array:2 [ "identificador" => "s0050" "titulo" => "Conjugate characterization" ] ] ] 7 => array:2 [ "identificador" => "s0055" "titulo" => "Conclusions" ] 8 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2022-09-12" "fechaAceptado" => "2023-02-15" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1664178" "palabras" => array:3 [ 0 => "Serotype 31 pneumococcal polysaccharide" 1 => "Polysaccharide conjugate vaccine" 2 => "CRM197 conjugation" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec1664179" "palabras" => array:3 [ 0 => "Polisacárido del serotipo neumocócico 31" 1 => "vacuna polisacárida conjugada" 2 => "conjugación de CRM197" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "Traditionally, PCV (pneumococcal conjugate vaccine) was prepared by the coupling of aldehyde-activated polysaccharide, with a carrier protein via NaCNBH3 (sodium cyanoborohydride) mediated reductive amination in water. However, the reaction is very slow and may take up to several days, which is a significant burden for pharmaceutical companies. Here we report the detailed reaction process of the reductive amination of structurally reassigned serotype 31 polysaccharide and cross reacting material (CRM197) in an organic solvent (N,N-dimethylformamide, DMF) by using STAB (sodium triacetoxyborohydride). The product has been characterized by size exclusion chromatography, nuclear magnetic resonance and transmission electron microscopy. Compared with the traditional method, the reaction can finish within hours and elicited a comparable immune response. The new strategy has the potential of being applied in the preparation of next-generation polysaccharide conjugate vaccines." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "Tradicionalmente, la PCV (vacuna neumocócica conjugada) se ha preparado mediante acoplamiento de un polisacárido activado por aldehído y una proteína portadora a través de la aminación reductora en agua mediada por NaCNBH3 (sodium cianoborohidruro de sodio). Sin embargo, la reacción es muy lenta, pudiendo prolongarse varios días, lo cual supone una carga significativa para las compañías farmacéuticas. Reportamos aquí el proceso detallado de la reacción de aminación reductora del polisacárido del serotipo 31 estructuralmente reasignado y el material de reacción cruzada (CRM197) en un disolvente orgánico (N,N-dimetilformamida, DMF) utilizando STAB (triacetoxiborohidruro de sodio). El producto ha sido caracterizado mediante cromatografía de exclusión de tamaño, resonancia magnética nuclear y microscopio electrónico de transmisión. En comparación con el método tradicional, la reacción puede finalizar en horas, suscitando una respuesta inmunológica comparable. La nueva estrategia tiene el potencial de aplicarse en la preparación de vacunas polisacáridas conjugadas de nueva generación." ] ] "multimedia" => array:4 [ 0 => array:8 [ "identificador" => "f0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2389 "Ancho" => 1896 "Tamanyo" => 193343 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0005" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "1H (A), TOCSY (B) NMR of activated native s-31-ps. Calculation of activation level: Unit D 6-methyl 1H NMR peak moved due to activation. The activation level (%) can be obtained by comparing the integration of shifted peak and the total D-6 integration. Activation =100%*(integration of 1.17 ppm + 1.15 ppm)/sum of integration(1.22 ppm + 1.17 ppm + 1.15 ppm) =100%*(0.38 + 0.68)/3.00 = 35%." ] ] 1 => array:8 [ "identificador" => "f0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 833 "Ancho" => 1587 "Tamanyo" => 78063 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0010" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "HPSEC-RI of serotype 31 polysaccharide (blue line) and the DMSO conjugate (black line). (For interpretation of the references to color in this fig. legend, the reader is referred to the web version of this article.)" ] ] 2 => array:8 [ "identificador" => "f0015" "etiqueta" => "Fig. 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1944 "Ancho" => 3348 "Tamanyo" => 431558 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0015" "detalle" => "Fig. " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "1D 1H NMR of native (A), aldehyde activated (B) and CRM197 conjugate (C) of serotype 31 polysaccharide." ] ] 3 => array:8 [ "identificador" => "sch0005" "etiqueta" => "Scheme 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "sc1.jpeg" "Alto" => 1771 "Ancho" => 3548 "Tamanyo" => 416281 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "al0020" "detalle" => "Scheme " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "The preparation of serotype 31-polysaccharide and CRM197 conjugate in DMF." ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bs0005" "bibliografiaReferencia" => array:5 [ 0 => array:3 [ "identificador" => "bb0005" "etiqueta" => "1." "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Triplex direct quantitative polymerase chain reaction for the identification of streptococcus pneumoniae serotypes" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:1 [ 0 => "M. Ouattara" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1093/infdis/jiab056" "Revista" => array:6 [ "tituloSerie" => "J Infect Dis" "fecha" => "2021" "volumen" => "224" "paginaInicial" => "S204" "paginaFinal" => "S208" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/34469558" "web" => "Medline" ] ] ] ] ] ] ] ] 1 => array:3 [ "identificador" => "bb0010" "etiqueta" => "2." "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Antibodies reactive to commensal Streptococcus mitis show cross-reactivity with virulent Streptococcus pneumoniae serotypes" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:1 [ 0 => "S. Shekhar" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:3 [ "tituloSerie" => "Front Immunol" "fecha" => "2018" "volumen" => "9" ] ] ] ] ] ] 2 => array:3 [ "identificador" => "bb0015" "etiqueta" => "3." "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:4 [ 0 => "F.Y. Avci" 1 => "X. Li" 2 => "M. Tsuji" 3 => "D.L. Kasper" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1038/nm.2535" "Revista" => array:6 [ "tituloSerie" => "Nat Med" "fecha" => "2011" "volumen" => "17" "paginaInicial" => "1602" "paginaFinal" => "1609" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22101769" "web" => "Medline" ] ] ] ] ] ] ] ] 3 => array:3 [ "identificador" => "bb0020" "etiqueta" => "4." "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "A review on the use of sodium triacetoxyborohydride in the reductive amination of ketones and aldehydes" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:2 [ 0 => "A.F. Abdel-Magid" 1 => "S.J. Mehrman" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:3 [ "tituloSerie" => "ChemInform" "fecha" => "2007" "volumen" => "38" ] ] ] ] ] ] 4 => array:3 [ "identificador" => "bb0025" "etiqueta" => "5." "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Conjugate of structurally reassigned pneumococcal serotype 31 polysaccharide with CRM197 elicited potent immune response" "autores" => array:1 [ 0 => array:2 [ "etal" => true "autores" => array:1 [ 0 => "Tiantian Sun" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1016/j.carbpol.2022.119414" "Revista" => array:5 [ "tituloSerie" => "Carbohydrate polymers" "fecha" => "2022" "volumen" => "289" "paginaInicial" => "119414" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/35483835" "web" => "Medline" ] ] ] ] ] ] ] ] ] ] ] ] ] "idiomaDefecto" => "en" "url" => "/15769887/0000002400000003/v1_202307141223/S1576988723000213/v1_202307141223/en/main.assets" "Apartado" => array:4 [ "identificador" => "95988" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Desarrollo en vacunas" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/15769887/0000002400000003/v1_202307141223/S1576988723000213/v1_202307141223/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1576988723000213?idApp=UINPBA00004N"]

Statistics

Desarrollo en Vacunas

The conjugation of serotype 31 pneumococcal polysaccharide and CRM197 in N,N-dimethylformamide

La conjugación del polisacárido bacteriano neumocócico del serotipo 31 y CRM197 en N,N-Dimetilformamida

Chengli Zong

Corresponding author

chengli.zong@hainanu.edu.cn

Corresponding author.

, Hongzhao Mao, Huiting Li, Shiyan Mai

School of Pharmaceutical Sciences, Hainan Universi, Hainan University School of Pharmaceutical Sciences, China

Read

272

Times
was read the article

Total PDF

193

Total HTML

Share statistics

 array:22 [
  "pii" => "S1576988723000213"
  "issn" => "15769887"
  "doi" => "10.1016/j.vacun.2023.02.002"
  "estado" => "S300"
  "fechaPublicacion" => "2023-07-01"
  "aid" => "285"
  "copyrightAnyo" => "2023"
  "documento" => "article"
  "crossmark" => 1
  "subdocumento" => "fla"
  "cita" => "Vacunas. 2023;24:248-54"
  "abierto" => array:3 [
    "ES" => false
    "ES2" => false
    "LATM" => false
  ]
  "gratuito" => false
  "lecturas" => array:1 [
    "total" => 0
  ]
  "itemSiguiente" => array:18 [
    "pii" => "S157698872200200X"
    "issn" => "15769887"
    "doi" => "10.1016/j.vacun.2022.12.006"
    "estado" => "S300"
    "fechaPublicacion" => "2023-07-01"
    "aid" => "277"
    "copyright" => "Elsevier Espa&#241;a&#44; S&#46;L&#46;U&#46;"
    "documento" => "simple-article"
    "crossmark" => 1
    "subdocumento" => "cor"
    "cita" => "Vacunas. 2023;24:255-7"
    "abierto" => array:3 [
      "ES" => false
      "ES2" => false
      "LATM" => false
    ]
    "gratuito" => false
    "lecturas" => array:1 [
      "total" => 0
    ]
    "en" => array:11 [
      "idiomaDefecto" => true
      "cabecera" => "<span class="elsevierStyleTextfn">Letter to the editor</span>"
      "titulo" => "Interactions between human microbiota and vaccines&#59; Current perspectives"
      "tienePdf" => "en"
      "tieneTextoCompleto" => "en"
      "paginas" => array:1 [
        0 => array:2 [
          "paginaInicial" => "255"
          "paginaFinal" => "257"
        ]
      ]
      "titulosAlternativos" => array:1 [
        "en" => array:1 [
          "titulo" => "Interacciones entre la microbiota humana y las vacunas&#59; Perspectivas actuales"
        ]
      ]
      "contieneTextoCompleto" => array:1 [
        "en" => true
      ]
      "contienePdf" => array:1 [
        "en" => true
      ]
      "resumenGrafico" => array:2 [
        "original" => 0
        "multimedia" => array:8 [
          "identificador" => "f0005"
          "etiqueta" => "Fig&#46; 1"
          "tipo" => "MULTIMEDIAFIGURA"
          "mostrarFloat" => true
          "mostrarDisplay" => false
          "figura" => array:1 [
            0 => array:4 [
              "imagen" => "gr1.jpeg"
              "Alto" => 1124
              "Ancho" => 2043
              "Tamanyo" => 259476
            ]
          ]
          "detalles" => array:1 [
            0 => array:3 [
              "identificador" => "al0005"
              "detalle" => "Fig&#46; "
              "rol" => "short"
            ]
          ]
          "descripcion" => array:1 [
            "en" => "<p id="sp4000" class="elsevierStyleSimplePara elsevierViewall">The main mechanism of gut microbiota actions regarding boosting the vaccine-induced immune response against respiratory infections&#46;</p>"
          ]
        ]
      ]
      "autores" => array:1 [
        0 => array:2 [
          "autoresLista" => "Masoud Keikha, Mehdi Zandhaghighi, Shahram Shahraki Zahedani"
          "autores" => array:3 [
            0 => array:2 [
              "nombre" => "Masoud"
              "apellidos" => "Keikha"
            ]
            1 => array:2 [
              "nombre" => "Mehdi"
              "apellidos" => "Zandhaghighi"
            ]
            2 => array:2 [
              "nombre" => "Shahram Shahraki"
              "apellidos" => "Zahedani"
            ]
          ]
        ]
      ]
    ]
    "idiomaDefecto" => "en"
    "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S157698872200200X?idApp=UINPBA00004N"
    "url" => "/15769887/0000002400000003/v1_202307141223/S157698872200200X/v1_202307141223/en/main.assets"
  ]
  "itemAnterior" => array:18 [
    "pii" => "S1576988723000316"
    "issn" => "15769887"
    "doi" => "10.1016/j.vacun.2023.04.003"
    "estado" => "S300"
    "fechaPublicacion" => "2023-07-01"
    "aid" => "291"
    "copyright" => "Elsevier Espa&#241;a&#44; S&#46;L&#46;U&#46;"
    "documento" => "article"
    "crossmark" => 1
    "subdocumento" => "rev"
    "cita" => "Vacunas. 2023;24:218-47"
    "abierto" => array:3 [
      "ES" => false
      "ES2" => false
      "LATM" => false
    ]
    "gratuito" => false
    "lecturas" => array:1 [
      "total" => 0
    ]
    "en" => array:13 [
      "idiomaDefecto" => true
      "cabecera" => "<span class="elsevierStyleTextfn">Review article</span>"
      "titulo" => "The status of COVID-19 vaccines in India&#58; A review"
      "tienePdf" => "en"
      "tieneTextoCompleto" => "en"
      "tieneResumen" => array:2 [
        0 => "en"
        1 => "es"
      ]
      "paginas" => array:1 [
        0 => array:2 [
          "paginaInicial" => "218"
          "paginaFinal" => "247"
        ]
      ]
      "titulosAlternativos" => array:1 [
        "es" => array:1 [
          "titulo" => "El estado de las vacunas COVID-19 en India&#58; Una revisi&#243;n"
        ]
      ]
      "contieneResumen" => array:2 [
        "en" => true
        "es" => true
      ]
      "contieneTextoCompleto" => array:1 [
        "en" => true
      ]
      "contienePdf" => array:1 [
        "en" => true
      ]
      "resumenGrafico" => array:2 [
        "original" => 0
        "multimedia" => array:8 [
          "identificador" => "f0065"
          "etiqueta" => "Fig&#46; 13"
          "tipo" => "MULTIMEDIAFIGURA"
          "mostrarFloat" => true
          "mostrarDisplay" => false
          "figura" => array:1 [
            0 => array:4 [
              "imagen" => "gr13.jpeg"
              "Alto" => 1276
              "Ancho" => 2165
              "Tamanyo" => 184800
            ]
          ]
          "detalles" => array:1 [
            0 => array:3 [
              "identificador" => "al0065"
              "detalle" => "Fig&#46; 1"
              "rol" => "short"
            ]
          ]
          "descripcion" => array:1 [
            "en" => "<p id="sp0065" class="elsevierStyleSimplePara elsevierViewall">The figure represents the publication trend of COVID-19 as well as vaccine articles in the year 2019&#44; 2020&#44; 2021 and 2022&#46; The data was obtained from NCBI and PubMed using SARS-CoV-2&#44; COVID-19&#44; Vaccine&#44; Coronavirus&#44; SARS-CoV-2 vaccine and COVID-19 vaccine as keywords&#46;</p>"
          ]
        ]
      ]
      "autores" => array:1 [
        0 => array:2 [
          "autoresLista" => "Deepak Kumar Jha, Kumar Pranay,  Samiksha, Amit Kumar, Niti Yashvardhini"
          "autores" => array:5 [
            0 => array:2 [
              "nombre" => "Deepak Kumar"
              "apellidos" => "Jha"
            ]
            1 => array:2 [
              "nombre" => "Kumar"
              "apellidos" => "Pranay"
            ]
            2 => array:1 [
              "apellidos" => "Samiksha"
            ]
            3 => array:2 [
              "nombre" => "Amit"
              "apellidos" => "Kumar"
            ]
            4 => array:2 [
              "nombre" => "Niti"
              "apellidos" => "Yashvardhini"
            ]
          ]
        ]
      ]
    ]
    "idiomaDefecto" => "en"
    "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1576988723000316?idApp=UINPBA00004N"
    "url" => "/15769887/0000002400000003/v1_202307141223/S1576988723000316/v1_202307141223/en/main.assets"
  ]
  "en" => array:19 [
    "idiomaDefecto" => true
    "cabecera" => "<span class="elsevierStyleTextfn">Desarrollo en Vacunas</span>"
    "titulo" => "The conjugation of serotype 31 pneumococcal polysaccharide and CRM197 in N&#44;N-dimethylformamide"
    "tieneTextoCompleto" => true
    "paginas" => array:1 [
      0 => array:2 [
        "paginaInicial" => "248"
        "paginaFinal" => "254"
      ]
    ]
    "autores" => array:1 [
      0 => array:4 [
        "autoresLista" => "Chengli Zong, Hongzhao Mao, Huiting Li, Shiyan Mai"
        "autores" => array:4 [
          0 => array:4 [
            "nombre" => "Chengli"
            "apellidos" => "Zong"
            "email" => array:1 [
              0 => "chengli.zong@hainanu.edu.cn"
            ]
            "referencia" => array:1 [
              0 => array:2 [
                "etiqueta" => "<span class="elsevierStyleSup">&#42;</span>"
                "identificador" => "cr0005"
              ]
            ]
          ]
          1 => array:2 [
            "nombre" => "Hongzhao"
            "apellidos" => "Mao"
          ]
          2 => array:2 [
            "nombre" => "Huiting"
            "apellidos" => "Li"
          ]
          3 => array:2 [
            "nombre" => "Shiyan"
            "apellidos" => "Mai"
          ]
        ]
        "afiliaciones" => array:1 [
          0 => array:2 [
            "entidad" => "School of Pharmaceutical Sciences&#44; Hainan Universi&#44; Hainan University School of Pharmaceutical Sciences&#44; China"
            "identificador" => "af0005"
          ]
        ]
        "correspondencia" => array:1 [
          0 => array:3 [
            "identificador" => "cr0005"
            "etiqueta" => "&#8270;"
            "correspondencia" => "Corresponding author&#46;"
          ]
        ]
      ]
    ]
    "titulosAlternativos" => array:1 [
      "es" => array:1 [
        "titulo" => "La conjugaci&#243;n del polisac&#225;rido bacteriano neumoc&#243;cico del serotipo 31 y CRM197 en N&#44;N-Dimetilformamida"
      ]
    ]
    "resumenGrafico" => array:2 [
      "original" => 0
      "multimedia" => array:8 [
        "identificador" => "f0005"
        "etiqueta" => "Fig&#46; 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
            "Alto" => 2389
            "Ancho" => 1896
            "Tamanyo" => 193343
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "al0005"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="sp0005" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleSup">1</span>H &#40;A&#41;&#44; TOCSY &#40;B&#41; NMR of activated native s-31-ps&#46; Calculation of activation level&#58; Unit D 6-methyl 1H NMR peak moved due to activation&#46; The activation level &#40;&#37;&#41; can be obtained by comparing the integration of shifted peak and the total D-6 integration&#46; Activation &#61;<span class="elsevierStyleHsp" style=""></span>100&#37;&#42;&#40;integration of 1&#46;17&#8239;ppm&#8239;&#43;&#8239;1&#46;15&#8239;ppm&#41;&#47;sum of integration&#40;1&#46;22&#8239;ppm&#8239;&#43;&#8239;1&#46;17&#8239;ppm&#8239;&#43;&#8239;1&#46;15&#8239;ppm&#41; &#61;<span class="elsevierStyleHsp" style=""></span>100&#37;&#42;&#40;0&#46;38&#8239;&#43;&#8239;0&#46;68&#41;&#47;3&#46;00&#8239;&#61;&#8239;35&#37;&#46;</p>"
        ]
      ]
    ]
    "textoCompleto" => "<span class="elsevierStyleSections"><span id="s0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">1</span><span class="elsevierStyleSectionTitle" id="st0025">Introduction</span><p id="p0005" class="elsevierStylePara elsevierViewall">Streptococcus pneumonia &#40;Spn&#41; caused invasive pneumococcal disease &#40;IPD&#41; is one of the major death causes for children under 5&#8239;years old<a class="elsevierStyleCrossRef" href="#bb0005"><span class="elsevierStyleSup">1</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#bb0010"><span class="elsevierStyleSup">2</span></a>&#46; Polysaccharide conjugate vaccine &#40;PCV&#41;&#44; including 7 serotypes &#40;PCV7&#44; Prevnar 7&#41;&#44; was approved by FDA in 2000 and recommended for all children&#46; In this vaccine&#44; purified capsular pneumococcal polysaccharide was conjugated to CRM197 to activate T-cell-dependent immune responses<a class="elsevierStyleCrossRef" href="#bb0015"><span class="elsevierStyleSup">3</span></a>&#46; Two doses are enough to provide good protection for children and people at greater risk of complications for IPD&#46; PCVs&#44; including 15 and 20 serotypes &#40;PCV15- VAXNEUVANCE and PCV20- Prevnar 20&#41; were approved by FDA in 2021&#46;</p><p id="p0010" class="elsevierStylePara elsevierViewall">Serotype 31 is a non-vaccine serotype and has increased in prevalence&#46; By using Nuclear Magnetic Resonance &#40;NMR&#41; as the primary tool&#44; we report the revised serotype 31 polysaccharide &#40;s-31-ps&#41; structure as &#91;&#8594;<span class="elsevierStyleHsp" style=""></span>3&#41;-&#946;-D-Galf-&#40;5&#47;6-OAc&#41;-&#40;1&#8239;&#8594;&#8239;3&#41;-&#946;-D-Galp-&#40;1&#8239;&#8594;&#8239;3&#41;-&#946;-L-Rhap-&#40;2-OAc&#41;-&#40;1&#8239;&#8594;&#8239;2&#41;-&#945;-L-Rhap-&#40;1&#8239;&#8594;&#8239;4&#41;-&#946;-D-GlcpA-&#40;1<span class="elsevierStyleHsp" style=""></span>&#8594;&#93;n&#46; Furthermore&#44; the reductive amination-conjugate of serotype 31 polysaccharide and cross reacting material &#40;CRM197&#41; protein was prepared in organic solvent &#40;N&#44;N-dimethylformamide&#44; DMF&#41; instead of water&#46; The reaction is faster&#44; and the DMF conjugate elicited comparable immune responses with the aqueous conjugate&#46; S-31-ps conjugate vaccine has the potential of being included in the next-generation PCV vaccines&#46;</p><p id="p0015" class="elsevierStylePara elsevierViewall">The conjugation between a large polysaccharide &#40;molecular weight up to a few million&#41; and CRM197 carrier protein &#40;58&#8239;kDa&#41; is very complex&#44; making PCV one of the most complicated drugs to manufacture&#46; Most pharmaceutical companies have been employing NaCNBH<span class="elsevierStyleInf">3</span> mediated reductive amination in water as the conjugation method&#46; It is very slow and may take days&#44; jeopardizing the protein stability<a class="elsevierStyleCrossRef" href="#bb0020"><span class="elsevierStyleSup">4</span></a>&#46; The process must be carefully monitored for a good quality control&#44; which is a significant financial and labor burden for companies&#46; Here we report the detailed procedure &#40;<a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>&#41; for the fast conjugation of s-31-ps and CRM197 by reductive amination in an organic solvent &#40;DMF&#41;&#44; using an organic solvent-soluble reducing reagent&#58; sodium triacetoxyborohydride &#40;STAB&#41;&#46;</p><elsevierMultimedia ident="sch0005"></elsevierMultimedia></span><span id="s0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">2</span><span class="elsevierStyleSectionTitle" id="st0030">Materials and methods</span><span id="s0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">2&#46;1</span><span class="elsevierStyleSectionTitle" id="st0035">Activation</span><p id="p0020" class="elsevierStylePara elsevierViewall">Vortex the solution of s-31-ps powder &#40;5&#8239;mg&#44; 1&#8239;eq &#40;based on the pentasaccharide repeating unit&#41;&#41; in water &#40;500&#8239;&#956;L&#41; in a 2&#8239;mL microcentrifuge tube for 10&#8239;min&#46; If the solution has un-dissolved material&#44; vortex the solution for 15&#8239;min&#46; If the solution is still not clear&#44; leave the solution on the bench-top overnight&#46; Usually&#44; the solution will be clear and pipettable&#46; Then 3&#46;0&#8239;M sodium acetate solution &#40;pH&#8239;5&#41; was added to the solution to achieve a final concentration of 100&#8239;mM sodium acetate&#44; followed by the addition of 100&#8239;mM NaIO4 stock solution &#40;1&#8239;eq&#44; 44&#8239;&#956;L&#41;&#46; The mixture was stirred by a magnetic bar for 5&#8239;h at room temperature&#46; Then the reaction mixture was transferred to a pre-rinsed 15&#8239;mL 30 kD mwco Amicon filter&#44; filled up with water&#44; centrifuged at 4500&#8239;rpm at 20&#8239;&#176;C until the final volume reached &#8764;<span class="elsevierStyleHsp" style=""></span>100&#8239;&#956;L&#46; The filtrate was discarded&#46;</p><p id="p0025" class="elsevierStylePara elsevierViewall">The residue may appear very viscous&#46; For the following filtrations&#44; after filling the filter up with water&#44; pipette up and down to mix&#46; Otherwise&#44; the polysaccharide may wrap the unwanted molecules and stick to the bottom of the filter&#46; The filtration step was repeated two more times&#46; Finally&#44; the residue was pipetted to a 2&#8239;mL vial&#46; Two portions of 200&#8239;&#956;L of water were used to rinse the filter&#46; The aqueous solutions were combined and freeze-dried&#46;</p></span><span id="s0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">2&#46;2</span><span class="elsevierStyleSectionTitle" id="st0040">Formulation</span><p id="p0030" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Polysaccharide formulation</span>&#58; Add 250&#8239;&#956;L water to 2&#46;5&#8239;mg of free-dried polysaccharide&#46; The solution is very viscous at the beginning&#46; Extended stirring did not alleviate the situation&#46; Then 100&#8239;&#956;L 30&#37; w&#47;v freshly prepared sucrose solution and 650&#8239;&#956;L water to reach a 2&#46;5&#8239;mg&#47;mL polysaccharide&#44; 5&#37; sucrose solution&#46; After stirring overnight&#44; the solution became clear and homogeneous&#46; The well-mixed solution was lyophilized to give a white cake&#46;</p><p id="p0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">CRM197 formulation</span>&#58; Commercially available CRM197 &#40;1&#46;25&#8239;mg in solution&#41; was spin filtered three times by a 10&#8239;k Amcon filter&#46; The 100&#8239;&#956;L residue was pipetted out and reconstituted to 1&#8239;mL 5&#8239;mM sodium phosphate pH&#8239;7&#46;2 and 5&#37; w&#47;v sucrose solution&#46; The mixture was freeze-dried to give a white cake&#46;</p></span><span id="s0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">2&#46;3</span><span class="elsevierStyleSectionTitle" id="st0045">Conjugation and purification</span><p id="p0040" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Sodium triacetoxyborohydride &#40;STAB&#41; preparation&#58;</span> acetic acid &#40;11&#46;8&#8239;&#956;L&#41; was added to the mixture of NaBH<span class="elsevierStyleInf">4</span> &#40;2&#46;22&#8239;mg&#41; in DMF &#40;1&#8239;mL&#41;&#46; Bubbles were formed upon acid addition&#46; The solution was stirred for 20&#8239;min before use&#46;</p><p id="p0045" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Polysaccharide and CRM197 dissolution in DMF</span>&#58; 1&#8239;mL DMF was added individually to the lyophilized polysaccharide and CRM197 cakes at room temperature&#46; The solutions were vortexed at low speed for 2&#8239;min&#46; Do not store the solutions for a longer time&#46;</p><p id="p0050" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Conjugation&#58;</span> The prepared solutions were combined in a 20&#8239;mL tube&#46; The freshly prepared STAB solution was added&#46; The mixture was incubated at room temperature at 500&#8239;rpm for 3&#8239;h&#46; Then 1&#8239;mg NaBH<span class="elsevierStyleInf">4</span> in 1&#8239;mL water was added to the mixture to quench any unreacted aldehyde&#46;</p><p id="p0055" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleBold">Purification&#58;</span> The solution was diluted by adding 40&#8239;mL 0&#46;1&#37; w&#47;v PS-20 in water to dilute the DMF percentage to less than 5&#37;&#44; which is the maximum DMF tolerance level for water-compatible Amicon filters&#46; Then the reaction mixture was transferred proportionally to a pre-rinsed 15&#8239;mL 100 kD mwco Amicon filter&#44; centrifuged at 4500&#8239;rpm at 20&#8239;&#176;C until the final volume reached &#8764;<span class="elsevierStyleHsp" style=""></span>100&#8239;&#956;L&#46; Then water was added to fill up the filter and centrifuged until the final volume reached 100&#8239;&#956;L&#46; The process was repeated two more times&#46; Finally&#44; the residue was pipetted to a 2&#8239;mL vial&#44; two portions of 200&#8239;&#956;L of D<span class="elsevierStyleInf">2</span>O were used to rinse the filter&#44; and the aqueous solutions were combined and analyzed by using NMR and HPLC-RI&#46;</p></span></span><span id="s0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">3</span><span class="elsevierStyleSectionTitle" id="st0050">Results and discussion</span><p id="p0060" class="elsevierStylePara elsevierViewall">The structure of s-31-ps has been reported in our previous work<a class="elsevierStyleCrossRef" href="#bb0025"><span class="elsevierStyleSup">5</span></a>&#46; The polysaccharide features a pentasaccharide repeating unit&#44; one furanose&#44; and three <span class="elsevierStyleItalic">O</span>-acetate groups with a molecular weight of 910kD and a poly-dispersity index &#40;PDI&#41; of 1&#46;42&#46; The following steps are followed to prepare s-31-ps and CRM197 conjugate&#58; the polysaccharide was activated by NaIO<span class="elsevierStyleInf">4</span>&#44; followed by formulation with sucrose in water&#44; lyophilization&#44; dissolution in DMF&#44; and finally conjugation with CRM197 by using freshly prepared STAB &#40;<a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>&#41;&#46;</p><span id="s0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">3&#46;1</span><span class="elsevierStyleSectionTitle" id="st0055">Activation</span><p id="p0065" class="elsevierStylePara elsevierViewall">S-31-ps powder was firstly dissolved in water at a concentration of 10&#8239;mg&#47;mL&#46; It should be noted that the water solution of pneumococcal bacterial polysaccharide with <span class="elsevierStyleItalic">O</span>-acetate groups is usually viscous&#44; probably due to the hydrogen bond between an acetyl-oxygen atom and hydroxyl proton&#46; Longer vortexing or storage time is required for proper dissolution&#46; Various methods are available for polysaccharide activation&#46; Here&#44; sodium periodate &#40;NaIO<span class="elsevierStyleInf">4</span>&#41; was used to break the vicinal-hydroxyls under slightly acid condition to form the aldehyde conjugation sites&#46; The aqueous solution of the activated product is even more viscous&#46;</p><p id="p0070" class="elsevierStylePara elsevierViewall">As indicated by the blue circle in <a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>&#44; there are two activation sites &#40;vicinal-hydroxyl groups&#41;&#46; To pinpoint the exact activation sites&#44; the ratio between those sites and the activation level is challenging due to the structural complexity of pneumococcal polysaccharide&#46; It is a mixture of the desired capsular polysaccharide&#44; cell wall polysaccharide&#44; residue amount of protein and other residuals from process&#46; Also&#44; the generated aldehyde group may hydrate or form other active intermediates<a class="elsevierStyleCrossRef" href="#bb0020"><span class="elsevierStyleSup">4</span></a>&#46; We attempted to estimate these information by using NMR&#46; The activation level &#40;&#37;&#41; was roughly estimated as 35&#37; by comparing the integration of shifted methyl peaks &#40;1&#46;17 and 1&#46;15&#8239;ppm&#41; and the total D-6 integration &#40;<a class="elsevierStyleCrossRef" href="#f0005">Fig&#46; 1</a>&#41; based on the assumption that the activation of ring D and E can both impact the chemical shift of D-6 proton&#46; This assumption may not be accurate&#46; Other derivation methods may be explored to more accurately quantify the activation&#46;</p><elsevierMultimedia ident="f0005"></elsevierMultimedia><p id="p0075" class="elsevierStylePara elsevierViewall">There are two vicinal-hydroxyls activation sites marked in blue circles &#40;<a class="elsevierStyleCrossRef" href="#sch0005">Scheme 1</a>&#44; unit D 3&#47;4 and unit E 2&#47;3&#41;&#46; As shown in <a class="elsevierStyleCrossRef" href="#f0005">Fig&#46; 1</a>A&#44; a few new peaks appeared in the anomeric region&#44; partially attributed to the hydrated aldehyde proton&#46; Among those peaks&#44; the highest at 5&#46;01&#8239;ppm correlates with the shifted unit D methyl peak at 1&#46;15&#8239;ppm&#44; suggesting unit D might be the major activation site &#40;<a class="elsevierStyleCrossRef" href="#f0005">Fig&#46; 1</a>B&#41;&#46;</p></span><span id="s0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">3&#46;2</span><span class="elsevierStyleSectionTitle" id="st0060">Formulation</span><p id="p0080" class="elsevierStylePara elsevierViewall">Pneumococcal bacterial polysaccharide like s-31-ps does not dissolve in an organic solvent&#44; such as DMF&#46; S-31-ps will be soluble in DMF when properly formulated with sucrose&#44; which is soluble in water and DMF&#46; First&#44; a mixture of s-31-ps &#40;2&#46;5&#8239;mg&#47;mL&#41; and 5&#37; w&#47;v sucrose was prepared and freeze-dried&#46; Similarly&#44; CRM197 was formulated with sucrose and pH&#8239;7&#46;2 5&#8239;mM sodium phosphate to stabilize the protein&#46;</p></span><span id="s0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">3&#46;3</span><span class="elsevierStyleSectionTitle" id="st0065">Conjugation</span><p id="p0085" class="elsevierStylePara elsevierViewall">With the lyophilized cakes in hand&#44; they were individually dissolved in DMF&#44; combined&#44; followed by the addition of freshly prepared STAB&#46; The mixture was stirred for 3&#8239;h&#46; A longer mixing time will lead to precipitation&#44; probably due to over-conjugation forming macromolecules&#46; The unreacted aldehyde was quenched by adding NaBH<span class="elsevierStyleInf">4</span> in water&#46; Then the solution was diluted with water and purified by spin filtration using a 100&#8239;k mwco Amicon filter to afford the s-31-ps-CRM197 conjugate&#46; Un-reacted CRM197 will be filtered off&#46;</p></span><span id="s0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">3&#46;4</span><span class="elsevierStyleSectionTitle" id="st0070">Conjugate characterization</span><p id="p0090" class="elsevierStylePara elsevierViewall">The S-31-ps and CRM197 conjugate was characterized by HPSEC-IR &#40;<a class="elsevierStyleCrossRef" href="#f0010">Fig&#46; 2</a>&#41; and NMR &#40;<a class="elsevierStyleCrossRef" href="#f0015">Fig&#46; 3</a>&#41;&#46; The HPSEC-IR data indicated the conjugate was eluted within 20&#8239;min&#44; while native s-31-ps was eluted at around 22&#8239;min<span class="elsevierStyleSup">5</span>&#44; indicating a size increase and the success of the conjugation&#46; The NMR spectrum of the conjugate &#40;<a class="elsevierStyleCrossRef" href="#f0015">Fig&#46; 3</a>C&#41; has been stacked with that of native &#40;<a class="elsevierStyleCrossRef" href="#f0015">Fig&#46; 3</a>A&#41; and activated &#40;<a class="elsevierStyleCrossRef" href="#f0015">Fig&#46; 3</a>B&#41; s-31-ps&#46; As can be seen from the spectrum&#44; the conjugate indicated protein aromatic and polysaccharide signals&#46; The O-acetate signals can still be observed at around 2&#8239;ppm&#44; indicating the conjugation condition did not touch the O-acetate groups&#44; which may impact the polysaccharide immunogenicity&#46;</p><elsevierMultimedia ident="f0010"></elsevierMultimedia><elsevierMultimedia ident="f0015"></elsevierMultimedia><p id="p0095" class="elsevierStylePara elsevierViewall">Preparation of polysaccharide samples&#58; 3&#8239;mg&#47;mL polysaccharide&#44; glucan molecular weight of 70 kD&#44; 150 kD&#44; 200 kD&#44; 500 kD&#44; and 1000 kD standards were dissolved in 1&#8239;mL water individually for further analysis&#46;</p><p id="p0100" class="elsevierStylePara elsevierViewall">The HPLC chromatographic conditions&#58; Waters e2695 coupled with RI detector &#40;2414&#41; was used for the analysis&#46; Separation was carried out using UltrahydrogelTM-120 column &#40;7&#46;8&#8239;&#215;&#8239;300&#8239;mm&#41; and UltrahydrogelTM-500 column &#40;7&#46;8&#8239;&#215;&#8239;300&#8239;mm&#41; using isocratic elution of 0&#46;05&#37; sodium azide in an aqueous solution at 0&#46;6&#8239;mL&#47;min&#46; Column and detector temperature was kept at 40&#8239;&#176;C&#46; The total run time was 15&#8239;min&#44; and the sample injection volume was 10&#8239;&#956;L&#46; Data were processed by GraphPad Prism 8&#46;0&#46;1&#46;</p><p id="p0105" class="elsevierStylePara elsevierViewall">NMR data were acquired on JOEL 600&#8239;MHz or Brucker 500&#8239;MHz instruments&#46; 600&#8239;&#956;L of the samples dissolved in deuterated solvents was usually used&#46; Lock&#44; shim&#44; tune was performed before data acquisition&#46; NMR data were processed by MestReNova version 14&#46;2&#46;0&#46; The spectrum was either referenced to DMSO internal standard or CPS choline methyl signal&#46; Water suppression was performed for serotype 31 polysaccharide-CRM197 DMF conjugate&#46;</p></span></span><span id="s0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleLabel">4</span><span class="elsevierStyleSectionTitle" id="st0075">Conclusions</span><p id="p0110" class="elsevierStylePara elsevierViewall">The conjugation of polysaccharide and carrier protein is the most challenging step for PCVs&#46; The reaction is usually in water using NaCNBH<span class="elsevierStyleInf">3</span> and may take up to several days&#46; Here we report the reductive amination of s-31-ps and CRM197 in DMF using STAB&#46; To assist the polysaccharide and CRM197 dissolution in DMF&#44; they were individually formulated with sucrose as the solubilizer&#46; The target conjugate can be successfully obtained within hours&#46; The current method provides a new polysaccharide and carrier protein conjugation strategy&#46;</p></span></span>"
    "textoCompletoSecciones" => array:1 [
      "secciones" => array:9 [
        0 => array:3 [
          "identificador" => "xres1930703"
          "titulo" => "Abstract"
          "secciones" => array:1 [
            0 => array:1 [
              "identificador" => "as0005"
            ]
          ]
        ]
        1 => array:2 [
          "identificador" => "xpalclavsec1664178"
          "titulo" => "Keywords"
        ]
        2 => array:3 [
          "identificador" => "xres1930704"
          "titulo" => "Resumen"
          "secciones" => array:1 [
            0 => array:1 [
              "identificador" => "as0010"
            ]
          ]
        ]
        3 => array:2 [
          "identificador" => "xpalclavsec1664179"
          "titulo" => "Palabras clave"
        ]
        4 => array:2 [
          "identificador" => "s0005"
          "titulo" => "Introduction"
        ]
        5 => array:3 [
          "identificador" => "s0010"
          "titulo" => "Materials and methods"
          "secciones" => array:3 [
            0 => array:2 [
              "identificador" => "s0015"
              "titulo" => "Activation"
            ]
            1 => array:2 [
              "identificador" => "s0020"
              "titulo" => "Formulation"
            ]
            2 => array:2 [
              "identificador" => "s0025"
              "titulo" => "Conjugation and purification"
            ]
          ]
        ]
        6 => array:3 [
          "identificador" => "s0030"
          "titulo" => "Results and discussion"
          "secciones" => array:4 [
            0 => array:2 [
              "identificador" => "s0035"
              "titulo" => "Activation"
            ]
            1 => array:2 [
              "identificador" => "s0040"
              "titulo" => "Formulation"
            ]
            2 => array:2 [
              "identificador" => "s0045"
              "titulo" => "Conjugation"
            ]
            3 => array:2 [
              "identificador" => "s0050"
              "titulo" => "Conjugate characterization"
            ]
          ]
        ]
        7 => array:2 [
          "identificador" => "s0055"
          "titulo" => "Conclusions"
        ]
        8 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2022-09-12"
    "fechaAceptado" => "2023-02-15"
    "PalabrasClave" => array:2 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1664178"
          "palabras" => array:3 [
            0 => "Serotype 31 pneumococcal polysaccharide"
            1 => "Polysaccharide conjugate vaccine"
            2 => "CRM197 conjugation"
          ]
        ]
      ]
      "es" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Palabras clave"
          "identificador" => "xpalclavsec1664179"
          "palabras" => array:3 [
            0 => "Polisac&#225;rido del serotipo neumoc&#243;cico 31"
            1 => "vacuna polisac&#225;rida conjugada"
            2 => "conjugaci&#243;n de CRM197"
          ]
        ]
      ]
    ]
    "tieneResumen" => true
    "resumen" => array:2 [
      "en" => array:2 [
        "titulo" => "Abstract"
        "resumen" => "<span id="as0005" class="elsevierStyleSection elsevierViewall"><p id="sp0025" class="elsevierStyleSimplePara elsevierViewall">Traditionally&#44; PCV &#40;pneumococcal conjugate vaccine&#41; was prepared by the coupling of aldehyde-activated polysaccharide&#44; with a carrier protein via NaCNBH3 &#40;sodium cyanoborohydride&#41; mediated reductive amination in water&#46; However&#44; the reaction is very slow and may take up to several days&#44; which is a significant burden for pharmaceutical companies&#46; Here we report the detailed reaction process of the reductive amination of structurally reassigned serotype 31 polysaccharide and cross reacting material &#40;CRM197&#41; in an organic solvent &#40;N&#44;N-dimethylformamide&#44; DMF&#41; by using STAB &#40;sodium triacetoxyborohydride&#41;&#46; The product has been characterized by size exclusion chromatography&#44; nuclear magnetic resonance and transmission electron microscopy&#46; Compared with the traditional method&#44; the reaction can finish within hours and elicited a comparable immune response&#46; The new strategy has the potential of being applied in the preparation of next-generation polysaccharide conjugate vaccines&#46;</p></span>"
      ]
      "es" => array:2 [
        "titulo" => "Resumen"
        "resumen" => "<span id="as0010" class="elsevierStyleSection elsevierViewall"><p id="sp0030" class="elsevierStyleSimplePara elsevierViewall">Tradicionalmente&#44; la PCV &#40;vacuna neumoc&#243;cica conjugada&#41; se ha preparado mediante acoplamiento de un polisac&#225;rido activado por aldeh&#237;do y una prote&#237;na portadora a trav&#233;s de la aminaci&#243;n reductora en agua mediada por NaCNBH3 &#40;sodium cianoborohidruro de sodio&#41;&#46; Sin embargo&#44; la reacci&#243;n es muy lenta&#44; pudiendo prolongarse varios d&#237;as&#44; lo cual supone una carga significativa para las compa&#241;&#237;as farmac&#233;uticas&#46; Reportamos aqu&#237; el proceso detallado de la reacci&#243;n de aminaci&#243;n reductora del polisac&#225;rido del serotipo 31 estructuralmente reasignado y el material de reacci&#243;n cruzada &#40;CRM197&#41; en un disolvente org&#225;nico &#40;N&#44;N-dimetilformamida&#44; DMF&#41; utilizando STAB &#40;triacetoxiborohidruro de sodio&#41;&#46; El producto ha sido caracterizado mediante cromatograf&#237;a de exclusi&#243;n de tama&#241;o&#44; resonancia magn&#233;tica nuclear y microscopio electr&#243;nico de transmisi&#243;n&#46; En comparaci&#243;n con el m&#233;todo tradicional&#44; la reacci&#243;n puede finalizar en horas&#44; suscitando una respuesta inmunol&#243;gica comparable&#46; La nueva estrategia tiene el potencial de aplicarse en la preparaci&#243;n de vacunas polisac&#225;ridas conjugadas de nueva generaci&#243;n&#46;</p></span>"
      ]
    ]
    "multimedia" => array:4 [
      0 => array:8 [
        "identificador" => "f0005"
        "etiqueta" => "Fig&#46; 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
            "Alto" => 2389
            "Ancho" => 1896
            "Tamanyo" => 193343
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "al0005"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="sp0005" class="elsevierStyleSimplePara elsevierViewall"><span class="elsevierStyleSup">1</span>H &#40;A&#41;&#44; TOCSY &#40;B&#41; NMR of activated native s-31-ps&#46; Calculation of activation level&#58; Unit D 6-methyl 1H NMR peak moved due to activation&#46; The activation level &#40;&#37;&#41; can be obtained by comparing the integration of shifted peak and the total D-6 integration&#46; Activation &#61;<span class="elsevierStyleHsp" style=""></span>100&#37;&#42;&#40;integration of 1&#46;17&#8239;ppm&#8239;&#43;&#8239;1&#46;15&#8239;ppm&#41;&#47;sum of integration&#40;1&#46;22&#8239;ppm&#8239;&#43;&#8239;1&#46;17&#8239;ppm&#8239;&#43;&#8239;1&#46;15&#8239;ppm&#41; &#61;<span class="elsevierStyleHsp" style=""></span>100&#37;&#42;&#40;0&#46;38&#8239;&#43;&#8239;0&#46;68&#41;&#47;3&#46;00&#8239;&#61;&#8239;35&#37;&#46;</p>"
        ]
      ]
      1 => array:8 [
        "identificador" => "f0010"
        "etiqueta" => "Fig&#46; 2"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr2.jpeg"
            "Alto" => 833
            "Ancho" => 1587
            "Tamanyo" => 78063
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "al0010"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="sp0010" class="elsevierStyleSimplePara elsevierViewall">HPSEC-RI of serotype 31 polysaccharide &#40;blue line&#41; and the DMSO conjugate &#40;black line&#41;&#46; &#40;For interpretation of the references to color in this fig&#46; legend&#44; the reader is referred to the web version of this article&#46;&#41;</p>"
        ]
      ]
      2 => array:8 [
        "identificador" => "f0015"
        "etiqueta" => "Fig&#46; 3"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr3.jpeg"
            "Alto" => 1944
            "Ancho" => 3348
            "Tamanyo" => 431558
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "al0015"
            "detalle" => "Fig&#46; "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="sp0015" class="elsevierStyleSimplePara elsevierViewall">1D <span class="elsevierStyleSup">1</span>H NMR of native &#40;A&#41;&#44; aldehyde activated &#40;B&#41; and CRM197 conjugate &#40;C&#41; of serotype 31 polysaccharide&#46;</p>"
        ]
      ]
      3 => array:8 [
        "identificador" => "sch0005"
        "etiqueta" => "Scheme 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "sc1.jpeg"
            "Alto" => 1771
            "Ancho" => 3548
            "Tamanyo" => 416281
          ]
        ]
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "al0020"
            "detalle" => "Scheme "
            "rol" => "short"
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="sp0020" class="elsevierStyleSimplePara elsevierViewall">The preparation of serotype 31-polysaccharide and CRM197 conjugate in DMF&#46;</p>"
        ]
      ]
    ]
    "bibliografia" => array:2 [
      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bs0005"
          "bibliografiaReferencia" => array:5 [
            0 => array:3 [
              "identificador" => "bb0005"
              "etiqueta" => "1&#46;"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Triplex direct quantitative polymerase chain reaction for the identification of <span class="elsevierStyleItalic">streptococcus pneumoniae</span> serotypes"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:1 [
                            0 => "M&#46; Ouattara"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1093/infdis/jiab056"
                      "Revista" => array:6 [
                        "tituloSerie" => "J Infect Dis"
                        "fecha" => "2021"
                        "volumen" => "224"
                        "paginaInicial" => "S204"
                        "paginaFinal" => "S208"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/34469558"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            1 => array:3 [
              "identificador" => "bb0010"
              "etiqueta" => "2&#46;"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Antibodies reactive to commensal <span class="elsevierStyleItalic">Streptococcus mitis</span> show cross-reactivity with virulent <span class="elsevierStyleItalic">Streptococcus pneumoniae</span> serotypes"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:1 [
                            0 => "S&#46; Shekhar"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:3 [
                        "tituloSerie" => "Front Immunol"
                        "fecha" => "2018"
                        "volumen" => "9"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            2 => array:3 [
              "identificador" => "bb0015"
              "etiqueta" => "3&#46;"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
                            0 => "F&#46;Y&#46; Avci"
                            1 => "X&#46; Li"
                            2 => "M&#46; Tsuji"
                            3 => "D&#46;L&#46; Kasper"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1038/nm.2535"
                      "Revista" => array:6 [
                        "tituloSerie" => "Nat Med"
                        "fecha" => "2011"
                        "volumen" => "17"
                        "paginaInicial" => "1602"
                        "paginaFinal" => "1609"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/22101769"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            3 => array:3 [
              "identificador" => "bb0020"
              "etiqueta" => "4&#46;"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "A review on the use of sodium triacetoxyborohydride in the reductive amination of ketones and aldehydes"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "A&#46;F&#46; Abdel-Magid"
                            1 => "S&#46;J&#46; Mehrman"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:3 [
                        "tituloSerie" => "ChemInform"
                        "fecha" => "2007"
                        "volumen" => "38"
                      ]
                    ]
                  ]
                ]
              ]
            ]
            4 => array:3 [
              "identificador" => "bb0025"
              "etiqueta" => "5&#46;"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Conjugate of structurally reassigned pneumococcal serotype 31 polysaccharide with CRM197 elicited potent immune response"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:1 [
                            0 => "Tiantian Sun"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.carbpol.2022.119414"
                      "Revista" => array:5 [
                        "tituloSerie" => "Carbohydrate polymers"
                        "fecha" => "2022"
                        "volumen" => "289"
                        "paginaInicial" => "119414"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/35483835"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
          ]
        ]
      ]
    ]
  ]
  "idiomaDefecto" => "en"
  "url" => "/15769887/0000002400000003/v1_202307141223/S1576988723000213/v1_202307141223/en/main.assets"
  "Apartado" => array:4 [
    "identificador" => "95988"
    "tipo" => "SECCION"
    "en" => array:2 [
      "titulo" => "Desarrollo en vacunas"
      "idiomaDefecto" => true
    ]
    "idiomaDefecto" => "en"
  ]
  "PDF" => "https://static.elsevier.es/multimedia/15769887/0000002400000003/v1_202307141223/S1576988723000213/v1_202307141223/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/"
  "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S1576988723000213?idApp=UINPBA00004N"
]

Article information

ISSN: 15769887
Original language: English

DOI:10.1016/j.vacun.2023.02.002

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 November	2	2	4
2024 October	9	6	15
2024 September	6	6	12
2024 August	8	5	13
2024 July	9	8	17
2024 June	9	9	18
2024 May	9	8	17
2024 April	7	7	14
2024 March	9	6	15
2024 February	8	3	11
2024 January	6	0	6
2023 December	9	0	9
2023 November	9	0	9
2023 October	25	0	25
2023 September	10	0	10
2023 August	12	0	12
2023 July	24	14	38
2023 June	15	0	15
2023 May	6	2	8
2023 April	1	3	4

Show all

Indexed in:

Follow us:

Subscribe:

Statistics

Indexed in:

Follow us:

Subscribe:

Statistics

Subscribe to our newsletter