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1.
Bioanalysis ; 14(13): 923-933, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36066084

RESUMO

Aim: Assessment of pre-existing anti-drug antibody (preADA) reactivity at early drug development stages can be beneficial for candidate selection. We investigated the applicability of a generic immune-complex anti-drug antibody (ADA) assay for early preADA assessment as an easily available alternative to the commonly used ADA bridging assay. Results: The results confirmed the expected assay difference regarding isotype detectability. Tested drug candidates were identified as preADA-reactive using the immune-complex ADA assay despite its limitation of not being able to detect IgM-type preADAs. Conclusion: We recommend a purpose-driven use of the two assay formats. For the purpose of ranking different Pro329Gly mutation-bearing drug candidates, the immune-complex ADA assay is preferred in the phase before selecting a drug for clinical development.


Assuntos
Anticorpos Monoclonais , Complexo Antígeno-Anticorpo
2.
Bioanalysis ; 14(16): 1085-1093, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36125042

RESUMO

In this manuscript, the European Bioanalysis Forum reports back on their discussions on practical and scientific considerations related to bioanalytical applications of quantitative polymerase chain reaction. This publication follows an earlier publication in which the European Bioanalysis Forum recommends to consider principles of context of use when defining assay acceptance criteria for method validation criteria and sample analysis.


Assuntos
Bioensaio , Laboratórios , Bioensaio/métodos , Reação em Cadeia da Polimerase , Projetos de Pesquisa , Relatório de Pesquisa
3.
iScience ; 25(2): 103746, 2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35118359

RESUMO

Monoclonal IgG antibodies are the fastest growing class of biologics, but large differences exist in their plasma half-life in humans. Thus, to design IgG antibodies with favorable pharmacokinetics, it is crucial to identify the determinants of such differences. Here, we demonstrate that the variable region sequences of IgG antibodies greatly affect cellular uptake and subsequent recycling and rescue from intracellular degradation by endothelial cells. When the variable sequences are masked by the cognate antigen, it influences both their transport behavior and binding to the neonatal Fc receptor (FcRn), a key regulator of IgG plasma half-life. Furthermore, we show how charge patch differences in the variable domains modulate both binding and transport properties and that a short plasma half-life, due to unfavorable charge patches, may partly be overcome by Fc-engineering for improved FcRn binding.

4.
Bioanalysis ; 13(23): 1723-1729, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34708666

RESUMO

Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to a wide array of basic research purposes. COVID-19 infection testing has brought the three-letter PCR abbreviation into the vocabulary of billions of people, making it likely the most well-known laboratory test worldwide. With new modalities and translational medicine gaining importance in pharmaceutical research and development, PCR or more specifically, quantitative PCR (qPCR) is now becoming a standard tool in the (regulated) bioanalytical laboratory, driving the bioanalytical community to define best practices for method development, characterization and validation. In absence of specific guidance from health authorities, qPCR may be vulnerable to scope creep from pharmacokinetics (PK) assay validation as defined in bioanalytical method validation guidance/guidelines. In this manuscript, the European Bioanalysis Forum builds a rationale for applying context of use principles when defining requirements for qPCR assay performance and validation criteria.


Assuntos
Bioensaio/métodos , Reação em Cadeia da Polimerase/métodos , Europa (Continente) , Humanos , Projetos de Pesquisa
5.
Haemophilia ; 27(6): 984-992, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34480814

RESUMO

INTRODUCTION: Emicizumab is a humanised, bispecific monoclonal antibody mimicking the cofactor function of activated factor (F)VIII. It is indicated for routine prophylaxis of bleeding episodes in persons with haemophilia A (PwHA) with/without FVIII inhibitors. AIM: To evaluate the development of anti-emicizumab antibodies and their impact on pharmacokinetics (PK), pharmacodynamics (PD), efficacy and safety in PwHA. METHODS: Data from seven completed or ongoing phase 3 studies were pooled. The assessment of the immunogenicity profile of emicizumab included anti-drug antibody (ADA) measurement and the association of ADAs with PK, PD, bleeding events, and adverse events. RESULTS: Of 668 PwHA evaluable for immunogenicity analysis, 34 (5.1%) developed ADAs after exposure to emicizumab. ADAs were transient in 14/34 PwHA (41.2%). ADAs were neutralising in vitro in 18/34 PwHA (52.9%) and associated with decreased emicizumab concentration in 4/668 evaluable PwHA (.6%); of those, one (.1%) discontinued emicizumab due to loss of efficacy. ADAs without decreased exposure did not impact emicizumab efficacy. The proportion of PwHA who had injection-site reactions (ISRs) was higher in ADA-positive PwHA (29.4% vs. 20.8%); however, the safety profile was similar between ADA-positive and ADA-negative PwHA, overall. No cases of anaphylaxis or hypersensitivity were reported in ADA-positive participants. CONCLUSION: The immunogenicity risk of emicizumab in phase 3 studies was low. ADAs, including in vitro neutralising ADAs, were not associated with a change in safety profile. Routine surveillance is, therefore, not warranted; however, in cases where a loss and/or waning of efficacy are observed, prompt evaluation by a healthcare provider should be sought.


Assuntos
Anticorpos Biespecíficos , Hemofilia A , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator VIII , Hemofilia A/tratamento farmacológico , Humanos
6.
Bioanalysis ; 13(10): 829-840, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33890493

RESUMO

Aim: Stabilization of critical reagents by freeze-drying would facilitate storage and transportation at ambient temperatures, and simultaneously enable constant reagent performance for long-term bioanalytical support throughout drug development. Freeze-drying as a generic process for stable performance and storage of critical reagents was investigated by establishing an universal formulation buffer and lyophilization process. Results: Using a storage-labile model protein, formulation buffers were evaluated to preserve reagent integrity during the freeze-drying process, and to retain functional performance after temperature stress. Application to critical reagents used in pharmacokinetics and anti-drug antibodies assays demonstrated stable functional performance of the reagents after 11 month at +40°C. Conclusion: Stabilization and storage of critical assay reagents by freeze-drying is an attractive alternative to traditional deep freezing.


Assuntos
Estabilidade de Medicamentos , Liofilização/métodos , Indicadores e Reagentes/química , Humanos
7.
Clin Cancer Res ; 27(14): 4036-4053, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33771854

RESUMO

PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Antígenos CD40/agonistas , Endopeptidases/efeitos dos fármacos , Imunoterapia/métodos , Proteínas de Membrana/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Camundongos , Células Tumorais Cultivadas
8.
Free Neuropathol ; 22021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37284635

RESUMO

Background: In some people with Parkinson's disease (PD), α-synuclein (αSyn) accumulation may begin in the enteric nervous system (ENS) decades before development of brain pathology and disease diagnosis. Objective: To determine how different types and severity of intestinal inflammation could trigger αSyn accumulation in the ENS and the subsequent development of αSyn brain pathology. Methods: We assessed the effects of modulating short- and long-term experimental colitis on αSyn accumulation in the gut of αSyn transgenic and wild type mice by immunostaining and gene expression analysis. To determine the long-term effect on the brain, we induced dextran sulfate sodium (DSS) colitis in young αSyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses. Results: A single strong or sustained mild DSS colitis triggered αSyn accumulation in the submucosal plexus of wild type and αSyn transgenic mice, while short-term mild DSS colitis or inflammation induced by lipopolysaccharide did not have such an effect. Genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αSyn. Remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-Serine 129 αSyn in the midbrain (including the substantia nigra), in 21- but not 9-month-old αSyn transgenic mice. This increase in midbrain αSyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons. Conclusions: Our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of PD.

9.
MAbs ; 12(1): 1683432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31769731

RESUMO

The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell-surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell-surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.


Assuntos
Cromatografia/métodos , Células Endoteliais/metabolismo , Imunoglobulinas Intravenosas/química , Animais , Células CHO , Cricetulus , Heparina/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulinas Intravenosas/metabolismo , Taxa de Depuração Metabólica , Pinocitose , Ligação Proteica , Proteólise , Receptores Fc/metabolismo
10.
Bioanalysis ; 11(24): 2207-2244, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31820675

RESUMO

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.


Assuntos
Bioensaio/métodos , Biomarcadores/metabolismo , Citometria de Fluxo/métodos , Terapia Genética/métodos , United States Food and Drug Administration/normas , História do Século XXI , Humanos , Estados Unidos
11.
Bioanalysis ; 10(4): 197-204, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29345496

RESUMO

European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.


Assuntos
Anticorpos Neutralizantes/análise , Produtos Biológicos/imunologia , Tolerância a Medicamentos/imunologia , Imunoensaio/normas , Produtos Biológicos/uso terapêutico , Descoberta de Drogas , Guias como Assunto , Humanos
12.
Regul Toxicol Pharmacol ; 88: 125-137, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28624430

RESUMO

With the emergence of novel biotherapeutic formats and immunostimulatory biotherapeutics in cancer immunotherapy, an understanding of immune-complex (IC) mediated hypersensitivity reactions in toxicology studies - and their differentiation from pharmacology - remains key to the preclinical evaluation of these drugs. In this review we provide an in-depth evaluation and comparison of case examples where IC-mediated hypersensitivity reactions were observed in cynomolgus monkeys. We provide details of the parameters evaluated in each study to substantiate and guide the interpretation of these findings. Five study cases (1 therapeutic protein, 4 monoclonal antibodies) are discussed for which effects ranged from minor to fatal. Common characteristics are the high incidence of clinical signs, detectable antidrug antibodies, and accelerated drug clearance up to virtual loss of exposure. In our experience, measurement of cytokine levels in vivo and detection of complement (split products) were supportive markers in situations where coagulopathy was suspected to play a role in the observed effects. Recommendations are outlined to prepare for root-cause analysis of suspected hypersensitivity reactions. Overall, a thorough analysis of the findings has helped to start clinical trials despite major findings. The hypersensitivity reactions with our human(ized) immunoglobulins have not proven to be predictive for humans.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Terapia Biológica/efeitos adversos , Hipersensibilidade/imunologia , Imunoglobulina G/imunologia , Imunoterapia/efeitos adversos , Animais , Anticorpos Monoclonais/imunologia , Citocinas/sangue , Humanos , Imunoglobulina G/administração & dosagem , Macaca fascicularis
13.
Mol Cell Proteomics ; 16(3): 451-456, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28062799

RESUMO

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct FcRn interactions with both the Fc region and the Fab regions of briakinumab, and correlate the occurrence of excessive FcRn binding to an unusually strong Fab-FcRn interaction.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Medição da Troca de Deutério/métodos , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica
14.
Biologicals ; 44(5): 291-305, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27461239

RESUMO

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Controle de Qualidade , Animais , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
15.
Proc Natl Acad Sci U S A ; 112(19): 5997-6002, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918417

RESUMO

Here, we investigated the influence of the variable fragment (Fv) of IgG antibodies on the binding to the neonatal Fc receptor (FcRn) as well as on FcRn-dependent pharmacokinetics (PK). FcRn plays a key role in IgG homeostasis, and specific manipulation in the crystallizable fragment (Fc) is known to affect FcRn-dependent PK. Although the influence of the antigen-binding fragment (Fab) on FcRn interactions has been reported, the underlying mechanism is hitherto only poorly understood. Therefore, we analyzed the two IgG1 antibodies, briakinumab and ustekinumab, that have similar Fc parts but different terminal half-lives in human and systematically engineered variants of them with cross-over exchanges and varied charge distribution. Using FcRn affinity chromatography, molecular dynamics simulation, and in vivo PK studies in human FcRn transgenic mice, we provide evidence that the charge distribution on the Fv domain is involved in excessive FcRn binding. This excessive binding prevents efficient FcRn-IgG dissociation at physiological pH, thereby reducing FcRn-dependent terminal half-lives. Furthermore, we observed a linear correlation between FcRn column retention times of the antibody variants and the terminal half-lives in vivo. Taken together, our study contributes to a better understanding of the FcRn-IgG interaction, and it could also provide profound potential in FcRn-dependent antibody engineering of the variable Fab region.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Receptores Fc/química , Animais , Anticorpos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Reações Antígeno-Anticorpo , Cromatografia de Afinidade , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Eletricidade Estática , Ressonância de Plasmônio de Superfície , Ustekinumab , Microglobulina beta-2/química
16.
MAbs ; 6(5): 1229-42, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25517308

RESUMO

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Fragmentos Fc das Imunoglobulinas/metabolismo , Metionina/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Cromatografia de Afinidade , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/sangue , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Taxa de Depuração Metabólica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxirredução/efeitos dos fármacos , Ligação Proteica , Receptores Fc/genética , Receptores Fc/metabolismo , Espectrometria de Massas por Ionização por Electrospray
17.
Lab Anim (NY) ; 43(3): 95-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24552915

RESUMO

In pharmacokinetic studies, intravenous (i.v.) administration of antibodies to mice is usually done via the lateral tail vein. This approach can cause stress to the mice and has a high rate of failure because it is challenging to perform correctly. Administration via the retro-orbital venous sinus has been suggested as a good alternative to tail vein i.v. administration of antibodies. Evidence is still needed, however, to determine whether the route of administration has an effect on the absorption or the pharmacokinetic activity of the injected antibody. The authors compared serum concentrations and pharmacokinetic parameters of a therapeutic antibody administered via tail vein injection or via retro-orbital injection. The findings suggest that there is no difference in the absorption or pharmacokinetic activity of therapeutic antibodies when administered via the lateral tail vein versus the retro-orbital venous sinus.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Cavidades Cranianas , Imunoglobulina G/administração & dosagem , Imunoglobulina G/sangue , Cauda/irrigação sanguínea , Animais , Anticorpos Monoclonais/sangue , Área Sob a Curva , Feminino , Meia-Vida , Injeções Intravenosas , Masculino , Camundongos
18.
J Med Microbiol ; 63(Pt 4): 508-517, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24430251

RESUMO

Pseudomonas aeruginosa is a common cause of nosocomial infections and is associated with high rates of mortality. In order to facilitate rapid and sensitive identification of the most prevalent serotypes of P. aeruginosa, we have developed a 4-valent real-time PCR-based assay using oligonucleotides specific for open-reading frames constituting the O-antigen-specific lipopolysaccharide loci of P. aeruginosa. The assay simultaneously detects and differentiates between each of the four serotypes IATS-O1, -O6, -O11 and serogroup 2 (IATS-O2, -O5, and -O16) with high sensitivity and specificity in a single-tube reaction. No cross-reactivity was observed with other serotypes of P. aeruginosa or other microbial species, and reproducibility was demonstrated regardless of template, i.e. purified DNA, bacterial culture and clinical specimens (broncho-alveolar lavage). The limit of detection of the assay was approximately 100 copies per reaction for IATS-O1, -O2 and -O11, and 50 copies per reaction for IATS-O6. Comparison of the assay specificity with a commercially available slide agglutination kit showed consistent results; however, the number of non-typable isolates was reduced by 15 % using the genotyping assay. Use of the 4-valent genotyping assay in the context of a clinical trial resulted in identification of pneumonia patients positive for the IATS-O11 serotype, and hence eligible for therapy with panobacumab (an investigational monoclonal antibody against the O-polysaccharide of serotype IATS-O11).


Assuntos
Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Bacteriana/diagnóstico , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Genótipo , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
MAbs ; 5(4): 576-86, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23765230

RESUMO

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.


Assuntos
Anticorpos Monoclonais Murinos/análise , Cromatografia de Afinidade/métodos , Antígenos de Histocompatibilidade Classe I/química , Imunoglobulina G/química , Receptores Fc/química , Animais , Anticorpos Monoclonais Murinos/genética , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Camundongos Transgênicos , Mutação , Receptores Fc/genética , Receptores Fc/imunologia , Ressonância de Plasmônio de Superfície
20.
J Med Microbiol ; 57(Pt 5): 601-604, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18436593

RESUMO

The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMérieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.


Assuntos
Neoplasias Hematológicas/complicações , Neutropenia/complicações , Sepse/complicações , Sepse/diagnóstico , Adulto , Idoso , Técnicas Bacteriológicas , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sepse/sangue
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