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Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Dependovirus , Terapia Genética , Hemofilia A , Humanos , Dependovirus/inmunología , Dependovirus/genética , Masculino , Hemofilia A/inmunología , Hemofilia A/terapia , Adulto , Estudios Longitudinales , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Terapia Genética/métodos , Inmunidad Adaptativa , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Persona de Mediana Edad , Prevalencia , Adulto JovenRESUMEN
Mucopolysaccharidosis III type A (MPS IIIA; Sanfilippo syndrome), a genetic lysosomal disorder causing a deficiency of heparan N-sulfatase (HNS), leads to progressive cognitive decline from an early age. An effective enzyme replacement therapy (ERT) for MPS IIIA requires central nervous system (CNS) biodistribution. Recombinant human heparan N-sulfatase (rhHNS), an investigatory ERT for MPS IIIA, has been formulated for intrathecal (IT) administration since intravenous (IV) administration cannot cross the blood brain barrier (BBB) in sufficient amounts to have a therapeutic effect. In this study, systemic and CNS distribution of rhHNS in cynomolgus monkeys following IV and IT administration was evaluated by quantitation of rhHNS in serum, cerebral spinal fluid (CSF) and various tissues, and positron emission tomography (PET) imaging of live animals. Following IV administration, rhHNS levels were low to non-detectable in the CSF, and systemic clearance was rapid (≤2 h). With IT administration, rhHNS was observable in CNS tissues in ≤1 h, with varying Tmax (1-24 h). Appreciable systemic distribution was observed up to 7 days. This provides evidence that in this animal model, intrathecal administration of rhHNS delivers the replacement enzyme to therapeutically relevant tissues for the treatment of Sanfilippo Syndrome type A. Penetration into grey matter and cortex was 3-4 times greater than concentrations in white matter and deeper parenchymal regions, suggesting some limitations of this ERT strategy.
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Sistema Nervioso Central/química , Sulfatasas/administración & dosificación , Sulfatasas/farmacocinética , Administración Intravenosa , Animales , Sistema Nervioso Central/diagnóstico por imagen , Modelos Animales de Enfermedad , Humanos , Inyecciones Espinales , Macaca fascicularis , Masculino , Mucopolisacaridosis III/tratamiento farmacológico , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Distribución TisularRESUMEN
PURPOSE: Approximately two-thirds of patients with the lysosomal storage disease mucopolysaccharidosis II have progressive cognitive impairment. Intravenous (i.v.) enzyme replacement therapy does not affect cognitive impairment because recombinant iduronate-2-sulfatase (idursulfase) does not penetrate the blood-brain barrier at therapeutic concentrations. We examined the safety of idursulfase formulated for intrathecal administration (idursulfase-IT) via intrathecal drug delivery device (IDDD). A secondary endpoint was change in concentration of glycosaminoglycans in cerebrospinal fluid. METHODS: Sixteen cognitively impaired males with mucopolysaccharidosis II who were previously treated with weekly i.v. idursulfase 0.5 mg/kg for ≥6 months were enrolled. Patients were randomized to no treatment or 10-mg, 30-mg, or 1-mg idursulfase-IT monthly for 6 months (four patients per group) while continuing i.v. idursulfase weekly. RESULTS: No serious adverse events related to idursulfase-IT were observed. Surgical revision/removal of the IDDD was required in 6 of 12 patients. Twelve total doses were administrated by lumbar puncture. Mean cerebrospinal fluid glycosaminoglycan concentration was reduced by approximately 90% in the 10-mg and 30-mg groups and approximately 80% in the 1-mg group after 6 months. CONCLUSIONS: These preliminary data support further development of investigational idursulfase-IT in MPS II patients with the severe phenotype who have progressed only to a mild-to-moderate level of cognitive impairment.Genet Med 18 1, 73-81.
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Iduronato Sulfatasa/administración & dosificación , Mucopolisacaridosis II/tratamiento farmacológico , Niño , Preescolar , Terapia de Reemplazo Enzimático , Femenino , Glicosaminoglicanos/líquido cefalorraquídeo , Humanos , Iduronato Sulfatasa/efectos adversos , Iduronato Sulfatasa/líquido cefalorraquídeo , Iduronato Sulfatasa/farmacocinética , Inyecciones Espinales , Masculino , Mucopolisacaridosis II/sangre , Mucopolisacaridosis II/líquido cefalorraquídeo , Mucopolisacaridosis II/metabolismoRESUMEN
OBJECTIVE: This was an open-label, phase 1/2 dose-escalation, safety trial of intrathecal recombinant human heparan-N-sulfatase (rhHNS) administered via intrathecal drug delivery device (IDDD) for treating mucopolysaccharidosis IIIA (NCT01155778). STUDY DESIGN: Twelve patients received 10, 45, or 90mg of rhHNS via IDDD once monthly for a total of 6 doses. Primary endpoints included adverse events (AEs) and anti-rhHNS antibodies. Secondary endpoints included standardized neurocognitive assessments, cortical gray matter volume, and pharmacokinetic/pharmacodynamic analyses. RESULTS: All patients experienced treatment-emergent AEs; most of mild-to-moderate severity. Seven patients reported a total of 10 serious AEs (SAEs), all but one due to hospitalization to revise a nonfunctioning IDDD. No SAEs were considered related to rhHNS. Anti-rhHNS antibodies were detected in the serum of 6 patients and in the cerebrospinal fluid (CSF) of 2 of these. CSF heparan sulfate levels were elevated at baseline and there were sustained declines in all tested patients following the first rhHNS dose. No impact of anti-rhHNS antibodies on any pharmacodynamic or safety parameters was evident. 4 of 12 patients showed a decline in developmental quotient, 6 were stable, and 2 patients had only a single data point. No dose group showed a clearly different response pattern. CONCLUSIONS: rhHNS administration via IDDD appeared generally safe and well tolerated. Treatment resulted in consistent declines in CSF heparan sulfate, suggesting in vivo activity in the relevant anatomical compartment. Results of this small study should be interpreted with caution. Future studies are required to assess the potential clinical benefits of rhHNS and to test improved IDDD models.
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Heparitina Sulfato/líquido cefalorraquídeo , Mucopolisacaridosis III/tratamiento farmacológico , Sulfatasas/administración & dosificación , Adolescente , Anticuerpos/sangre , Anticuerpos/líquido cefalorraquídeo , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inyecciones Espinales/instrumentación , Masculino , Mucopolisacaridosis III/líquido cefalorraquídeo , Sulfatasas/efectos adversos , Sulfatasas/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
The formation of antidrug antibodies (ADA) can interfere with the accurate quantitation of therapeutic proteins, leading to significantly underestimated drug concentrations and confounded pharmacokinetic (PK) data interpretation. Although highly desirable, development of ADA-tolerant bioanalytical methods enabling unbiased measurement of both free and ADA-bound drug presents a considerable challenge. We report herein the development and validation of a robust LC-MS assay capable of quantifying therapeutic protein immunoglobulin A1 protease (IgAP) in human serum in the presence of pre-existing anti-IgAP antibodies. The procedure included sodium dodecyl sulfate (SDS) denaturation and chemical reduction of serum proteins to dissociate ADA-drug bindings, followed by tryptic digestion of protein pellets and subsequent LC-MS analysis of the surrogate IgAP peptide using stable isotope labeled peptide internal standard. Substantial enhancements in the sensitivity and selectivity were achieved by the combination of online two-dimensional reversed-phase LC (2D-LC) operated in high and low pH buffers, respectively, for efficient enrichment and quantitation of the surrogate peptide by multiple-reaction monitoring (MRM) mass spectrometry. Unlike ligand-binding assay, our method is not prone to interferences from ADA, allowing accurate and precise measurement of the IgAP in the range of 0.05 to 10 µg/mL in 25 µL of human serum with a wide range of anti-IgAP antibody levels. The intra- and inter-run precision (coefficient of variation (CV%)) was within 11.5% and 10.5%, respectively, and the bias was within ±7.1% for all quality control (QC) concentrations. With little modification, the described method can readily be applicable to the quantitation of other biotherapeutic proteins in the ADA-positive clinical matrices.
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Anticuerpos/sangre , Serina Endopeptidasas/sangre , Espectrometría de Masas en Tándem , Anticuerpos/inmunología , Isótopos de Carbono/química , Cromatografía Líquida de Alta Presión , Humanos , Marcaje Isotópico , Péptidos/química , Estándares de Referencia , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/normas , Dodecil Sulfato de Sodio/química , Espectrometría de Masas en Tándem/normasRESUMEN
The East Asian common octopus (Octopus sinensis) is an economically important species among cephalopods. This species exhibits a strict dioecious and allogamous reproductive strategy, along with a phenotypic sexual dimorphism, where the third right arm differentiates into hectocotylus in males. However, our understanding of the molecular mechanisms that underlie sex determination and differentiation in this species remains limited. In the present study, we surveyed gene-expression profiles in the immature male and female gonads of O. sinensis based on the RNA-seq, and a total of 47.83 Gb of high-quality data were generated. Compared with the testis, we identified 8302 differentially expressed genes (DEGs) in the ovary, of which 4459 genes were up-regulated and 3843 genes were down-regulated. Based on the GO enrichment, many GO terms related to sex differentiation were identified, such as sex differentiation (GO: 0007548), sexual reproduction (GO: 0019953) and male sex differentiation (GO: 0046661). A KEGG classification analysis identified three conserved signaling pathways that related to sex differentiation, including the Wnt signaling pathway, TGF-ß signaling pathway and Notch signaling pathway. Additionally, 21 sex-related DEGs were selected, of which 13 DEGs were male-biased, including Dmrt1, Foxn5, Foxj1, Sox30, etc., and 8 DEGs were female-biased, including Sox14, Nanos3, ß-tubulin, Suh, etc. Ten DEGs were used to verify the expression patterns in the testis and ovary using the RT-qPCR method, and the results showed that the expression level shown by RT-qPCR was consistent with that from the RNA-seq, which confirmed the reliability of the transcriptome data. The results presented in this study will not only contribute to our understanding of sex-formation mechanisms in O. sinensis but also provide the foundational information for further investigating the molecular mechanisms that underline its gonadal development and facilitate the sustainable development of octopus artificial breeding.
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Octopodiformes , Diferenciación Sexual , Transcriptoma , Animales , Femenino , Masculino , Octopodiformes/genética , Diferenciación Sexual/genética , Transcriptoma/genética , Ovario/metabolismo , Ovario/crecimiento & desarrollo , Testículo/metabolismo , Testículo/crecimiento & desarrollo , Transducción de Señal/genética , Perfilación de la Expresión Génica/métodos , Procesos de Determinación del Sexo/genética , Pueblos del Este de AsiaRESUMEN
ABSTRACT: Rurioctocog alfa pegol is an extended-half-life full-length recombinant factor VIII (FVIII) bound to 20-kDa polyethylene glycol (PEG) that has been shown to be well tolerated and efficacious in the treatment and prevention of bleeding events in previously treated patients with severe hemophilia A. Here, we present a comprehensive analysis of immunogenicity data collected during 6 clinical studies of rurioctocog alfa pegol, including a total of 360 unique previously treated patients with severe hemophilia A. The analysis included treatment-emerging FVIII-neutralizing antibodies (FVIII inhibitors); preexisting and treatment-emerging antibodies binding to FVIII, PEG-FVIII, or PEG; and treatment-emerging antibodies binding to Chinese hamster ovary host cell proteins. Moreover, the potential association between the presence of these binding antibodies and adverse events (AEs) observed in patients was investigated, and the potential impact of these antibodies on the incremental recovery of rurioctocog alfa pegol in patients was analyzed. Overall, the data indicate that rurioctocog alfa pegol is not associated with any unexpected immunogenicity characteristics. Of 360 patients, 1 patient developed a transient FVIII inhibitor with a titer of 0.6 Bethesda units per mL, which was not associated with any serious AEs. Antibodies binding to FVIII, PEG-FVIII, or PEG were not detected at the time when the inhibitor was present. Moreover, 54 of 360 patients either entered the clinical studies with preexisting binding antibodies or developed these antibodies after exposure to rurioctocog alfa pegol. These antibodies were transient in most patients and did not show any causal relationship to either AEs or spontaneous bleeding episodes.
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Factor VIII , Hemofilia A , Polietilenglicoles , Humanos , Factor VIII/inmunología , Factor VIII/uso terapéutico , Factor VIII/efectos adversos , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Hemofilia A/sangre , Polietilenglicoles/uso terapéutico , Masculino , Anticuerpos Neutralizantes/inmunología , Adulto , Adolescente , Animales , Proteínas Recombinantes/uso terapéutico , Proteínas Recombinantes/inmunología , Niño , Adulto JovenRESUMEN
Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication.
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Anticuerpos Neutralizantes , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , HumanosRESUMEN
The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.
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Biomarcadores , Tratamiento Basado en Trasplante de Células y Tejidos , Vacunas , Humanos , Biomarcadores/análisis , Vacunas/inmunología , Citometría de Flujo , Bioensayo/métodos , Unión Europea , BlancoRESUMEN
Fe and Zn ions are essential enzymatic cofactors across all domains of life. Fe is an electron donor/acceptor in redox enzymes, while Zn is typically a structural element or catalytic component in hydrolases. Interestingly, the presence of Zn in oxidoreductases and Fe in hydrolases challenge this apparent functional dichotomy. In hydrolases, Fe either substitutes for Zn or specifically catalyzes certain reactions. On the other hand, Zn can replace divalent Fe and substitute for more complex Fe assemblies, known as Fe-S clusters. Although many zinc-binding proteins interchangeably harbor Zn and Fe-S clusters, these cofactors are only sometimes functional proxies.
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Coenzimas , Oxidorreductasas , Oxidorreductasas/metabolismo , Coenzimas/metabolismo , Oxidación-Reducción , Hidrolasas , Zinc/químicaRESUMEN
The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.
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Medicamentos bajo Prescripción , Tecnología , Bioensayo/métodos , Biomarcadores/análisis , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.
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Anticuerpos , Anticuerpos NeutralizantesRESUMEN
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.
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Receptores Quiméricos de Antígenos , Vacunas , Biomarcadores/análisis , Sistemas CRISPR-Cas , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Activa , Reacción en Cadena de la PolimerasaRESUMEN
Clinical studies involving enzyme replacement therapies (ERTs) have increasingly utilized enzymatic activity assays to monitor efficacy and biofunction of the drug; as a result, these assays have become an important part of pharmacokinetic (PK) and pharmacodynamic assessments in ERT trials. This paper presents a two-step enzymatic activity assay for iduronate-2-sulfatase (I2S) (EC 3.1.6.13) which we have optimized to fit in 1 day and to complete in less than 6 h. The rapid assay presented here is a significant improvement over the original two-step method with run time of 24 h which spanned 2 days. The resulting 1 day assay is efficient, robust, reproducible, and better suited for use in pharmacokinetic studies. The method was fully validated in accordance with regulatory agency guidelines so that it could be implemented in PK studies. Validation of the method required additional modifications to circumvent limitations surrounding the calculation of accuracy. This challenge was overcome by developing strategies to determine both the expected and the measured values of validation samples in activity units. Subsequently, the method was validated in accordance with the FDA guidance for the validation of quantitative ligand binding assays (LBAs). Results of method development and optimization with focus on evaluations aimed at reducing the total assay run time as well as a summary of method validation performance are presented in this publication.
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AIM: The study aimed to develop an LC-MS/MS assay to measure dermatan sulfate (DS) in human cerebrospinal fluid (CSF). METHODS & RESULTS: DS was quantified by ion pairing LC-MS/MS analysis of the major disaccharides derived from chondroitinase B digestion. Artificial CSF was utilized as a surrogate for calibration curve preparation. The assay was fully validated, with a linear range of 20.0-4000 ng/ml, accuracy within ±20%, and precision of ≤20%. CSF samples from mucopolysaccharidoses (MPS) II patients showed an average of 11-fold increase in DS levels compared with controls. CONCLUSION: The described assay is capable of differentiating DS levels in the CSF of MPS II patients from controls and can be used to monitor disease progression and therapeutic responses.
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Cromatografía Liquida/métodos , Pruebas de Química Clínica/métodos , Dermatán Sulfato/líquido cefalorraquídeo , Mucopolisacaridosis II/líquido cefalorraquídeo , Espectrometría de Masas en Tándem/métodos , Animales , Biomarcadores/líquido cefalorraquídeo , Calibración , Reproducibilidad de los Resultados , PorcinosRESUMEN
AIM: Development of drug-related surrogate positive controls for isotype anti-drug antibodies assay remains challenging. Efforts on antibody engineering or chemical crosslinking have been made. However, multiple epitope recognition, purity and stability are often of concern. To tackle these challenges, we used LC-SPDP/SMPB crosslinking to conjugate polyclonal anti-drug IgG and human immunoglobulin isotype (hIgE, hIgA, hIgM or hIgG4) with optimized conditions. RESULTS: The final product was a hybrid of anti-drug IgG cross-linked to human isotype immunoglobulin through stable thioether bond. The characteristics of the hybrids and their performance as assay positive controls were further evaluated. CONCLUSION: The results demonstrate that LC-SPDP/SMPB chemical crosslinking method is suitable for generating stable hybrids to be used as assay positive controls in isotype anti-drug antibodies assay.
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Anticuerpos/análisis , Cromatografía en Gel , Isotipos de Inmunoglobulinas/análisis , Anticuerpos/química , Anticuerpos/metabolismo , Complejo Antígeno-Anticuerpo/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Isotipos de Inmunoglobulinas/química , Maleimidas/química , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Sulfuros/químicaRESUMEN
AIM: Legacy methods with complex testing scheme for characterization of anti-idursulfase antibodies (ADA) were simplified and optimized in order to meet current regulatory guidance and provide more timely and cost-effective support for routine patient care. RESULTS: To compare the performance of the original and updated methods, patient samples receiving commercially prescribed Elaprase treatment were analyzed by both test methods. The ADA and neutralizing antibody results obtained by both methods were highly correlated and the updated method had an overall higher ADA and neutralizing antibody positive rates and higher ADA titers. CONCLUSION: The updated methods and test schemes are much simpler, more sensitive, but are also highly comparable with the original methods for the measurement of total and neutralizing ADA.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Análisis Químico de la Sangre/métodos , Iduronato Sulfatasa/inmunología , Análisis Químico de la Sangre/economía , Análisis Costo-Beneficio , HumanosRESUMEN
Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors.
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Anticuerpos/sangre , Biotina , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/inmunología , Factor I del Crecimiento Similar a la Insulina/inmunología , Ácidos , Anticuerpos Neutralizantes/sangre , Humanos , Inmunoglobulina G/sangre , Proteínas Recombinantes/inmunología , Sensibilidad y EspecificidadRESUMEN
In mucopolysaccharidoses (MPS), glycosaminoglycans (GAG) accumulate in tissues. In MPS II, approximately two-thirds of patients are cognitively impaired. We investigated levels of GAG in cerebrospinal fluid (CSF) in different populations from four clinical studies (including NCT00920647 and NCT01449240). Data indicate that MPS II patients with cognitive impairment have elevated levels of CSF GAG, whereas those with the attenuated phenotype typically have levels falling between those of the cognitively affected patients and healthy controls.
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OBJECTIVE: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder due to deficient activity of arylsulfatase A (ASA) that causes accumulation of sulfatide and lysosulfatide. The disorder is associated with demyelination and axonal loss in the central and peripheral nervous systems. The late infantile form has an early-onset, rapidly progressive course with severe sensorimotor dysfunction. The relationship between the degree of nerve damage and (lyso)sulfatide accumulation is, however, not established. METHODS: In 13 children aged 2-5 years with severe motor impairment, markedly elevated cerebrospinal fluid (CSF) and sural nerve sulfatide and lysosulfatide levels, genotype, ASA mRNA levels, residual ASA, and protein cross-reactive immunological material (CRIM) confirmed the diagnosis. We studied the relationship between (lyso)sulfatide levels and (1) the clinical deficit in gross motor function (GMFM-88), (2) median and peroneal nerve motor and median and sural nerve sensory conduction studies (NCS), (3) median and tibial nerve somatosensory evoked potentials (SSEPs), (4) sural nerve histopathology, and (5) brain MR spectroscopy. RESULTS: Eleven patients had a sensory-motor demyelinating neuropathy on electrophysiological testing, whereas two patients had normal studies. Sural nerve and CSF (lyso)sulfatide levels strongly correlated with abnormalities in electrophysiological parameters and large myelinated fiber loss in the sural nerve, but there were no associations between (lyso)sulfatide levels and measures of central nervous system (CNS) involvement (GMFM-88 score, SSEP, and MR spectroscopy). INTERPRETATION: Nerve and CSF sulfatide and lysosulfatide accumulation provides a marker of disease severity in the PNS only; it does not reflect the extent of CNS involvement by the disease process. The magnitude of the biochemical disturbance produces a continuously graded spectrum of impairments in neurophysiological function and sural nerve histopathology.