RESUMEN
Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination. Polysaccharide activation is a crucial step that affects vaccine product critical attributes including conjugate size and structure. Therefore, it is highly desired to have robust analytical methods to well characterize this activation process. In this study, using pneumococcal serotype 6A as the model, we present two complimentary analytical methods for characterization of activated polysaccharide. First, a size exclusion chromatography (SEC) method was developed for quantitative measurement of polysaccharide activation levels. This SEC method demonstrated good assay characteristics on accuracy, precision and linearity. Second, a gold nanoparticle labeled cryo-electron microscopy (Cryo-EM) technique was developed to visualize activation site distribution along polysaccharide chain and provide information on activation heterogeneity. These two complimentary methods can be utilized to control polysaccharide activation process and ensure consistent delivery of conjugate vaccine products.
RESUMEN
Multivalent pneumococcal vaccines have been developed successfully to combat invasive pneumococcal diseases (IPD) and reduce the associated healthcare burden. These vaccines employ pneumococcal capsular polysaccharides (PnPs), either conjugated or unconjugated, as antigens to provide serotype-specific protection. Pneumococcal capsular polysaccharides used for vaccine often contain residual levels of cell wall polysaccharides (C-Ps), which can generate a non-serotype specific immune response and complicate the desired serotype-specific immunity. Therefore, the C-P level in a pneumococcal vaccine needs to be controlled in the vaccine process and the anti C-P responses need to be dialed out in clinical assays. Currently, two types of cell-wall polysaccharide structures have been identified: a mono-phosphocholine substituted cell-wall polysaccharide C-Ps1 and a di-phosphocholine substituted C-Ps2 structure. In our effort to develop a next-generation novel pneumococcal conjugate vaccine (PCV), we have generated a monoclonal antibody (mAb) specific to cell-wall polysaccharide C-Ps2 structure. An antibody-enhanced HPLC assay (AE-HPLC) has been established for serotype-specific quantification of pneumococcal polysaccharides in our lab. With the new anti C-Ps2 mAb, we herein extend the AE-HPLC assay to the quantification and identification of C-Ps2 species in pneumococcal polysaccharides used for vaccines.
RESUMEN
Streptococcus pneumoniae is a highly invasive bacterial pathogen that can cause a range of illnesses. Pneumococcal capsular polysaccharides (CPS) are the main virulence factors that causes invasive pneumococcal disease (IPD). Pneumococcal CPS serotype 7F along with a few other serotypes is more invasive and likely to cause IPD. Therefore, 7F is a target for pneumococcal vaccine development, and is included in the two recently approved multi-valent pneumococcal conjugated vaccines, i.e. VAXNEUVANCE and PREVNAR 20.To support process and development of our 15-valent pneumococcal conjugated vaccine (PCV15), chromatographic methods have been developed for 7F polysaccharide and conjugate characterization. A size-exclusion chromatography (SEC) method with UV, light scattering and refractive index detections was employed for concentration, size and conformation analysis. A reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was used for analysis of conjugate monosaccharide composition and degree of conjugation. The collective information obtained by these chromatographic analysis provided insights into the pneumococcal conjugate and conjugation process.
Asunto(s)
Infecciones Neumocócicas , Humanos , Serogrupo , Serotipificación , Infecciones Neumocócicas/prevención & control , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae , Vacunas Neumococicas , Vacunas Conjugadas , Antígenos BacterianosRESUMEN
Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.
RESUMEN
A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.
Asunto(s)
Cápside , Virus Oncolíticos , Cápside/metabolismo , Virus Oncolíticos/metabolismo , Proteínas Virales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismoRESUMEN
Infections by Streptococcus pneumoniae can cause serious pneumococcal diseases and other medical complications among patients. Polysaccharide-based vaccines have been successfully developed as prophylactic agents against such deadly bacterial infections. In the 1980s, PNEUMOVAX® 23 were introduced as the first pneumococcal polysaccharide vaccines (PPSV). Later, pneumococcal polysaccharides were conjugated to a carrier protein to improve immune responses. Pneumococcal conjugate vaccines (PCV) such as PREVNAR® and VAXNEUVANCE™ have been developed. Of the more than 90 pneumococcal bacteria serotypes, serotype 1 (ST-1) and serotype 4 (ST-4) are the two main types that cause invasive pneumococcal diseases (IPD) that could lead to morbidity and mortality. Development of a novel multi-valent PCV against these serotypes requires extensive biophysical and biochemical characterizations of each monovalent conjugate (MVC) in the vaccine. To understand and characterize these high molecular weight (Mw) polysaccharide protein conjugates, we employed the multi-angle light scattering (MALS) technique coupled with size-exclusion chromatography (SEC) separation and asymmetrical flow field flow fractionation (AF4). MALS analysis of MVCs from the two orthogonal separation mechanisms helps shed light on the heterogeneity in conformation and aggregation states of each conjugate.
RESUMEN
Oncolytic virus immunotherapy is emerging as a novel therapeutic approach for cancer treatment. Immunotherapy clinical drug candidate V937 is currently in phase I/II clinical trials and consists of a proprietary formulation of Coxsackievirus A21 (CVA21), which specifically infects and lyses cells with overexpressed ICAM-1 receptors in a range of tumors. Mature Coxsackievirus virions, consisting of four structural virion proteins, (VPs) VP1, VP2, VP3, and VP4, and the RNA genome, are the only viral particles capable of being infectious. In addition to mature virions, empty procapsids with VPs, VP0, VP1, and VP3, and other virus particles are produced in V937 production cell culture. Viral protein VP0 is cleaved into VP2 and VP4 after RNA genome encapsidation to form mature virions. Clearance of viral particles containing VP0, and quantification of viral protein distribution are important in V937 downstream processing. Existing analytical methods for the characterization of viral proteins and particles may lack sensitivity or are low throughput. We developed a sensitive and robust reverse-phase ultra-performance chromatography method to separate, identify, and quantify all five CVA21 VPs. Quantification of virus capsid concentration and empty/full capsid ratio was achieved with good linearity, accuracy, and precision. ClinicalTrials.gov ID: NCT04521621 and NCT04152863.
Asunto(s)
Cápside , Virus Oncolíticos , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cromatografía , Virus Oncolíticos/genética , Virus Oncolíticos/metabolismo , ARN Viral/análisis , ARN Viral/metabolismo , Proteínas ViralesRESUMEN
Streptococcus pneumoniae bacterial infection can cause serious diseases. Among more than 90 known streptococcus pneumoniae serotypes, more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Initially, a 23-valent polysaccharide vaccines (PPSV) PNEUMOVAX®23, was developed to generate an antigen-specific immune response and prevent diseases caused by these pneumoniae serotypes. Later, pneumococcal conjugate vaccines (PCV), such as PREVNAR® and VAXNEUVANCE™ have been developed to offer a more robust immune response in the pediatric population. In our effort to develop novel pneumococcal conjugate vaccines, each serotype of pneumococcal polysaccharide (Ps) is conjugated to a detoxified diphtheria toxin carrier protein CRM197 to form a monovalent conjugate (MVC). MVCs from multiple serotypes are formulated with vaccine adjuvant to form a multi-valent vaccine drug product. During the product development, critical attributes including conjugate molecular weight (Mw), protein and polysaccharide concentration, have been used to monitor process and product quality. To measure these attributes, a size-exclusion chromatography (SEC) method was developed with a series of in-line detectors including UV, multi-angle light scattering (MALS) and refractive index (RI). This SEC-UV-MALS-RI method is employed to characterize and monitor process intermediates and product during process development and for product release and stability testing. With this, we have expanded the multi-attribute SEC method to a 15-valent pneumococcal conjugate vaccine.
Asunto(s)
Infecciones Neumocócicas , Refractometría , Niño , Cromatografía en Gel , Vacuna Neumocócica Conjugada Heptavalente , Humanos , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas , Vacunas ConjugadasRESUMEN
V937 is an oncolytic virus immunotherapy clinical drug candidate consisting of a proprietary formulation of Coxsackievirus A21 (CVA21). V937 specifically binds to and lyses cells with over-expressed ICAM-1 receptors in a range of tumor cell types and is currently in phase I and II clinical trials. Infectious V937 particles consist of a â¼30 nm icosahedral capsid assembled from four structural viral proteins that encapsidate a viral RNA genome. Rapid and robust analytical methods to quantify and characterize CVA21 virus particles are important to support the process development, regulatory requirements, and validation of new manufacturing platforms. Herein, we describe a size-exclusion chromatography (SEC) method that was developed to characterize the V937 drug substance and process intermediates. Using a 4-in-1 combination of multi-detectors (UV, refractive index, dynamic and static light scattering), we demonstrate the use of SEC for the quantification of the virus particle count, the determination of virus size (molecular weight and hydrodynamic diameter), and the characterization of virus purity by assessing empty-to-full capsid ratios. Through a SEC analysis of stressed V937 samples, we propose CVA21 thermal degradation pathways that result in genome release and particle aggregation.
RESUMEN
A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1 selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both centrally and peripherally. Administration of a highly selective CB2 agonist in a rat model of analgesia was ineffective despite substantial CNS exposure, while administration of a moderately selective CB2/CB1 agonist exhibited significant analgesic effects.
Asunto(s)
Analgésicos/química , Piridinas/química , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas , Analgésicos/síntesis química , Analgésicos/uso terapéutico , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/farmacología , Humanos , Hiperalgesia/tratamiento farmacológico , Piridinas/síntesis química , Piridinas/uso terapéutico , Ratas , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
In our ongoing efforts to develop CGRP receptor antagonists for the treatment of migraine, we aimed to improve upon telecagepant by targeting a compound with a lower projected clinical dose. Imidazoazepanes were identified as potent caprolactam replacements and SAR of the imidazole yielded the tertiary methyl ether as an optimal substituent for potency and hERG selectivity. Combination with the azabenzoxazinone spiropiperidine ultimately led to preclinical candidate 30 (MK-2918).
Asunto(s)
Azepinas/síntesis química , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Imidazoles/síntesis química , Imidazoles/farmacología , Analgésicos no Narcóticos/síntesis química , Analgésicos no Narcóticos/química , Analgésicos no Narcóticos/farmacología , Animales , Azepinas/química , Azepinas/farmacología , Disponibilidad Biológica , Caprolactama/química , Células Cultivadas , Perros , Humanos , Imidazoles/química , Concentración 50 Inhibidora , Macaca mulatta , Trastornos Migrañosos/tratamiento farmacológico , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
Several novel spiropiperidine-based CGRP receptor antagonists have been developed that maintain good potency and have reduced potential for metabolism.
Asunto(s)
Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Péptido Relacionado con Gen de Calcitonina/farmacología , Fragmentos de Péptidos/farmacología , Piperidinas/farmacología , Compuestos de Espiro/farmacología , Secuencia de Aminoácidos , Animales , Péptido Relacionado con Gen de Calcitonina/química , Dicroismo Circular , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Piperidinas/química , Unión Proteica , Transducción de Señal , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
The palladium-catalyzed Suzuki-Miyaura reaction has been utilized as one of the most powerful methods for C-C bond formation. However, Suzuki reactions of electron-deficient 2-heterocyclic boronates generally give low conversions and remain challenging. The successful copper(I) facilitated Suzuki coupling of 2-heterocyclic boronates that is broad in scope is reported. Use of this methodology affords greatly enhanced yields of these notoriously difficult couplings. Furthermore, mechanistic investigations suggest a possible role of copper in the catalytic cycle.
Asunto(s)
Ácidos Borónicos/química , Cobre/química , Compuestos Heterocíclicos/química , CatálisisRESUMEN
Two novel routes have been developed to the (3 R,6 S)-3-amino-6-(2,3-difluorophenyl)-1-(2,2,2-trifluoroethyl)azepan-2-one 2 of the CGRP receptor antagonist clinical candidate telcagepant (MK-0974, 1). The first employs a ring-closing metathesis of the styrene 7 as the key reaction, while the second makes use of a highly diastereoselective Hayashi-Miyaura Rh-catalyzed arylboronic acid addition to nitroalkene 16. The latter route has been implemented to produce multigram quantities of telcagepant for extensive preclinical evaluation.
Asunto(s)
Azepinas/síntesis química , Imidazoles/síntesis química , Antagonistas del Receptor Peptídico Relacionado con el Gen de la CalcitoninaRESUMEN
Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).
Asunto(s)
Azepinas/síntesis química , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Imidazoles/síntesis química , Trastornos Migrañosos/tratamiento farmacológico , Administración Oral , Animales , Azepinas/farmacocinética , Azepinas/farmacología , Disponibilidad Biológica , Células CACO-2 , Permeabilidad de la Membrana Celular , Perros , Humanos , Imidazoles/farmacocinética , Imidazoles/farmacología , Macaca mulatta , Ratas , Flujo Sanguíneo Regional/efectos de los fármacos , Piel/irrigación sanguínea , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.
Asunto(s)
Aflatoxina B1/química , Aductos de ADN/química , Daño del ADN , Replicación del ADN , Pirimidinas/química , Aflatoxina B1/metabolismo , Aductos de ADN/metabolismo , Isomerismo , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Pirimidinas/metabolismoRESUMEN
In our continuing effort to identify CGRP receptor antagonists for the acute treatment of migraine, we have undertaken a study to evaluate alternative 4-substituted piperidines to the lead dihydroquinazolinone 1. In this regard, we have identified the piperidinyl-azabenzimidazolone and phenylimidazolinone structures which, when incorporated into the benzodiazepine core, afford potent CGRP receptor antagonists (e.g., 18 and 29). These studies produced a potent analog (18) which overcomes the instability issues associated with the lead structure 1. A general pharmacophore for the 4-substituted piperidine component of these CGRP receptor antagonists is also presented.
Asunto(s)
Benzodiazepinas/farmacología , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Trastornos Migrañosos/tratamiento farmacológico , Piperidinas/farmacología , Benzodiazepinas/síntesis química , AMP Cíclico/antagonistas & inhibidores , Estabilidad de Medicamentos , Humanos , Piperidinas/síntesis química , Unión Proteica , Relación Estructura-ActividadRESUMEN
Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.
Asunto(s)
Antitrombinas/química , Antitrombinas/farmacología , Inhibidores del Factor Xa , Piridinas/química , Piridinas/farmacología , Cristalografía por Rayos X , Modelos Moleculares , Estructura MolecularRESUMEN
In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).