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1.
AAPS J ; 22(2): 56, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32166588

RESUMO

The administration of biotherapeutics has the potential to induce potent immune responses. Among these responses, the production of anti-drug antibodies (ADA), including a subset of ADA referred to as neutralizing antibodies (NAb), is of heightened concern. Aside from their capacity to alter the pharmacological profile of a given biotherapeutic, NAb can also pose significant safety risks, especially in instances where an endogenous counterpart to the drug exists. As such, the inclusion of an assay to detect NAb in clinical samples is critical to the effectiveness of a tiered approach to immunogenicity assessment. PF-06730512 is a biotherapeutic protein being developed for the treatment of primary Focal Segmental Glomerulosclerosis (FSGS). To support the immunogenicity assessment of PF-06730512, a cell-based assay was developed for the detection of NAb in FSGS serum samples. Herein, we describe the development of the assay with a focus on the challenges faced, including drug and blood collection tube interferences in NAb detection. The outcome of our efforts was a robust assay capable of detecting 1 µg/mL of a NAb positive control in the presence of clinically relevant drug concentrations up to 30 µg/mL.

2.
AAPS J ; 22(2): 42, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32020345

RESUMO

The first author's name was published incorrectly as "Gorovits Boris". The correct name is "Boris Gorovits".

3.
AAPS J ; 22(2): 19, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900604

RESUMO

After tier 1 and 2 cut points for anti-drug antibody (ADA) assays are derived during pre-study assay validation in a population, there is a need to verify the continued appropriateness of the previously derived cut points during sample analysis in the same or different populations, per FDA guidance (US HHS, FDA, CDER, CBER, 2019). Proper sample size-dependent criteria with statistical underpinning were derived and presented in this technical note to aid in assessing the appropriateness of tier 1 and tier 2 cut points, respectively.

4.
AAPS J ; 22(2): 24, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31907680

RESUMO

Viral vector-based gene therapies (GTx) have received significant attention in the recent years and the number of ongoing GTx clinical trials is increasing. A platform of choice for many of these studies is adeno-associated virus (AAV). All humans may be exposed to natural AAV infections and could mount an immune response against the virus. Consequently, there can be a high prevalence of pre-existing anti-AAV immunity. This presents a potential limitation for AAV-based GTx due to the potential for AAV-specific antibodies to reduce the efficacy of the GTx. Therefore, appropriate assessment of potential subjects enrolled in these studies should include evaluation for the presence and degree of anti-AAV immunity, including anti-AAV neutralizing antibodies (NAb). Recommendations for the development and validation of cell-based anti-AAV NAb detection methods, including considerations related to selection of appropriate cell line, surrogate vector/reporter gene, assay matrix and controls, and methodologies for calculating assay cut-point are discussed herein. General recommendations for the key assay validation parameters are provided as well as considerations for the development of NAb diagnostic tests. This manuscript is produced by a group of scientists involved in GTx therapeutic development representing various companies. It is our intent to provide recommendations and guidance to industrial and academic laboratories working on viral vector based GTx modalities with the goal of achieving a more consistent approach to anti-AAV NAb assessment.

5.
BioDrugs ; 34(1): 39-54, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31641991

RESUMO

Compounds containing two or more structural domains with a distinct mode of action relevant to functionality have been defined as multi-domain biotherapeutics (MDBs). Several modalities, including endogenous protein fusions with an antibody Fc fragment or another polypeptide, bispecific antibodies, antibody-drug conjugates, as well as polyethylene glycol conjugates have been viewed as examples of MDBs. Similar to other biotherapeutics, MDBs have the potential to induce a host immune response, commonly detected in the form of anti-drug antibodies (ADAs). The need to characterize ADA specificity to a particular domain of the MDB has been identified as a potential regulatory requirement based on the compound nature of the drug and associated immunogenicity risk factors. MDB-related immunogenicity risk factors are discussed herein. The relative risk level of each of the immunogenicity factors was analyzed based on publicly available information. It is proposed that MDB-related immunogenicity risk factors can be divided into major and minor categories. Major risk category factors include (a) presence of immunogenic structural or linear epitopes of either non-human or human sequence origin and (b) significant homology of an MDB domain to an endogenous protein with a specific and unique function. Proposed minor risk category factors include (a) epitope spread, (b) repetitive antigenic structure of MDB, and (c) hapten-like effect due to chemical conjugation or fusion with a larger protein. Detailed modality-based information on several examples of MDBs is presented to support this proposal.

6.
Pharm Res ; 36(12): 169, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654236

RESUMO

PURPOSE: The purpose of this study was to validate a ligand binding assay for the quantitation of a monoclonal antibody-based biotherapeutics (PF-57781346) in samples collected via capillary microsampling to support a regulated mouse toxicity study. METHOD: A quantitative ligand binding assay on the Gyrolab platform was developed to quantify PF-57781346 in blood samples derived from capillary mouse serial sampling. The method validation evaluated assay characteristics including accuracy and precision, influence of sample processing on drug quantitation, whole blood matrix selectivity, dilution linearity and the stability of the drug in the study sample matrix. RESULTS: The method validation demonstrated acceptable analytical characteristics. The whole blood selectivity testing demonstrated accuracy between -4.8% and 13.9% in 10 out of 10 individual whole blood samples, suggesting that drug quantitation from whole blood is not impacted by the serial sampling procedure. Short-term and long-term drug stability in study sample matrix were established to cover required stability for sample storage and analysis (accuracy between -7.3% and 6.1%). CONCLUSION: We reported a successful validation of a bioanalytical method that quantifies PF-55781346 in samples collected via capillary microsampling. The experience shared in this study could serve as a model process for bioanalytical method validation when capillary microsampling is used.


Assuntos
Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/toxicidade , Imunoensaio/métodos , Animais , Coleta de Amostras Sanguíneas/métodos , Estabilidade de Medicamentos , Ligantes , Camundongos , Modelos Animais , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Toxicocinética
7.
Bioanalysis ; 11(21): 2011-2024, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31648530

RESUMO

The number of gene therapy (GTx) modality therapies in development has grown significantly in the last few years. Adeno-associated virus (AAV)-based delivery approach has become most prevalent among other virus-based GTx vectors. Several regulatory guidelines provide the industry with general considerations related to AAV GTx development including discussion and recommendations related to highly diverse bioanalytical support of the AAV-based therapeutics. This includes assessment of pre- and post-treatment immunity, evaluation of post-treatment viral shedding and infectivity, as well as detection of transgene protein expression. An overview of the current regulatory recommendations as found in currently active and published draft US FDA and EMA guidance or guideline documents is presented herein.

8.
Bioanalysis ; 11(15): 1383-1385, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31490105

RESUMO

Biography Boris Gorovits is a Senior Director of the Bioanalytical lab at Pfizer. Boris earned his PhD in Enzymology from the Moscow State University and later completed postdoctoral research studies in protein biophysics at the Medical Center, University of Texas at San Antonio, TX, USA. In 2000, Boris joined Wyeth Research (later Pfizer Inc) to work as a bioanalytical group lead with a growing scope of responsibilities. Currently, he leads the Bioanalytical group within Biomedicine Design department which is responsible for many aspects of the Regulated and Nonregulated Bioanalytical support for the pan-Pfizer Biotherapeutic portfolio. Boris co-chairs Pfizer internal Immunogenicity Expert Working Group, which is responsible for review of the biotherapeutic immunogenicity risk assessment and mitigation strategies. Recently, Boris has been actively involved in industry discussions focusing on PK and immunogenicity assessment, bioanalytical support of various biotherapeutic modalities, including mAbs, bi-specific antibodies, antibody-drug conjugates, ADCs and gene therapy. Boris is proud to be an active member of the American Association of Pharma Scientists. This interview was conducted by Sankeetha Nadarajah, Managing Commissioning Editor of Bioanalysis, at the AAPS ICH-M10 Public Consultation Workshop (Silver Spring, MD, USA), 11 June 2019.


Assuntos
Técnicas de Química Analítica , Guias como Assunto , Consenso , Documentação , Laboratórios , Estados Unidos , United States Food and Drug Administration
9.
J Immunol Methods ; 474: 112642, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31400410

RESUMO

Proper evaluation of immunogenicity during clinical development of biotherapeutics is a major challenge to bioanalytical scientists, in part due to matrix interference in anti-drug antibody (ADA) and neutralizing antibody (NAB) assays. If not addressed, matrix interference could confound the immunogenicity assessment of a given biotherapeutic in clinical development. To support clinical development of a B cell maturation antigen (BCMA)-CD3 bispecific antibody, a cell-based NAB assay was developed as part of a tiered approach to evaluating the immunogenicity of the drug. The assay endpoint (T cell activation) was chosen based on its strong association with the mechanism of action of the drug. The BCMA-CD3 bispecific antibody activates T cells through simultaneous binding of CD3 on T cells and BCMA on target cells. In this system, T cell activation was assessed through the measurement of luciferase activity in an engineered Jurkat cell line. In the presence of NAB, the degree of T cell activation measured by the amount of luciferase activity can be reduced. During method development, soluble BCMA (sBCMA) interference in the NAB assay was apparent. The binding of sBCMA to the anti-BCMA domain of the bispecific drug led to reduced T cell activation, which caused false positive results in NAB testing. To mitigate this interference, several strategies to eliminate sBCMA were investigated. Among the procedures tested, a bead-based approach proved most effective in depleting sBCMA, while maintaining robust assay performance and achieving fit-for-purpose sensitivity. Using this sample pretreatment procedure, the NAB assay tolerated sBCMA up to 2 µg/mL, or approximately four times the estimated median sBCMA concentration in serum samples from patients with active multiple myeloma.

10.
AAPS J ; 21(5): 76, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31214862

RESUMO

Anti-drug antibody (ADA) assay selectivity is evaluated during assay validation to assess the potential for individual matrices to interfere with detection of ADA. While current EMA and FDA guideline documents suggest comparative analysis with and without matrix, they do not provide specific recommendations on the acceptance criteria such as an acceptable percent positive control (PC) recovery range or positive rate. Industry has adopted an approach where recovery of PC spiked sample is expected to fall within ± 20% (80 to 120%) vs. that for the PC material spiked in negative control matrix or assay buffer. Here, it is proposed that ADA assay selectivity evaluated using a qualitative assessment of PC recovery vs. a PK-like quantitative method may be more appropriate. The PC recovery test should focus on the reliability of the method to detect the low PC level in individual samples and avoid false-negative ADA reporting. Therefore, it is proposed that assessment of high PC level as well as the assessment of quantitative percent recovery (within ± 20%) should not be included in the test. The recovery test may be viewed as acceptable should a pre-selected number of individual samples (for example at least 8 or 9 out of 10) prepared at the low PC concentration of the assay score as ADA positive.

11.
Bioanalysis ; 11(12): 1207-1216, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31204868

RESUMO

Bioanalytical challenges were encountered during developmental and reproductive toxicity studies of tanezumab in cynomolgus monkeys. Possible changes in breast milk composition over the postpartum period potentially complicated assessment of tanezumab concentration in this matrix, requiring validation of the quantification assay across different time intervals. Immunogenicity assessment in maternal serum was complicated by apparent increases in the incidence of antidrug antibody-positive results in treatment-naive samples as pregnancy progressed that were due to changes in the concentration of nerve growth factor, tanezumab's target protein. This was overcome by employing gestational day-specific cut points throughout pregnancy. Researchers should recognize potential challenges associated with dynamic matrices/physiological conditions and anticipate that assays developed under normal conditions may require adaptation for specialized situations.


Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Testes de Química Clínica/métodos , Crescimento e Desenvolvimento/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Cinética , Limite de Detecção , Macaca fascicularis , Leite Humano/química , Reprodutibilidade dos Testes
12.
AAPS J ; 21(4): 71, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31161482

RESUMO

Assays for the detection and confirmation of anti-drug antibodies (ADA) are commonly used tools for assessing the immunogenicity of drug candidates in both clinical and nonclinical studies. During the development of such assays, it is typical to optimize the assay conditions based on factors such as sensitivity or signal/noise ratio (S/N) and is commonly done using an assay positive control (PC). However, even carefully optimized methods often suffer with problems due to low cut-point factors and failure to distinguish assay "noise" from a true biological response. In this paper, we describe an approach to assay development in which the impacts of assay conditions on the response and variability, both analytical and biological, of drug-naïve samples are tested by way of PC-independent assay condition optimization. Using two ADA methods as model systems, we examine the impact of minimum required dilution, assay reagent (labeled drug) concentrations, incubation time, assay, and wash buffer composition. We find that the choice of assay conditions, particularly the labeled drug concentration, can greatly affect the distribution of naïve sample responses and thus impact screening and confirmatory assay cut-points. In two case studies presented, screening assay cut-point (SCP) varied from 1.38 to 2.20 and 1.04 to 1.20 while the confirmatory assay cut-point (CCP) varied from 58.5 to 95.6% and 26.2 to 16.2% depending on the conditions tested. Some of the conditions produced unacceptably high CCP values. It is proposed that the degree of the observed impact of the assay conditions on SCP and CCP values depends on the compound nature and assay matrix composition and is likely connected with the diversity of interactions between drug protein and matrix components. Because it was also observed that higher assay SCP can associate with a loss of the PC-based assay sensitivity, additional assessment of the assay conditions would be required to determine an overall assay performance acceptability, including assay PC-based sensitivity, drug, and target tolerance characteristics. In conclusion, it is suggested that by assessing performance of treatment-naïve samples at various assay conditions, one can identify potential assay protocols that allow to avoid undesirably low screening (e.g., < 1.2) and confirmatory (e.g., < 25%) cut-points.

13.
BioDrugs ; 33(3): 275-284, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31069709

RESUMO

Chimeric antigen receptor (CAR) T-cell immunotherapy has gained significant attention in the past decade due to its considerable potential in the treatment of various types of malignancies, particularly hematological. While success has been achieved in a number of studies, and two CAR-T-cell products were recently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) (YESCARTA®, KYMRIAH®), this treatment modality continues to present challenges for clinical development. One major potential side effect is the ability of CAR-T products to induce host immune responses. Immunogenicity induction risk factors have been shown to be associated with the presence of non-human or partially human sequences in the CAR construct, suicide domain, or other components of the CAR-T, and also with the presence of residual viral proteins or other non-human origin proteins utilized as part of the gene editing step of CAR-T production. Both humoral (antibody-based) and cellular-type responses have been described, leading to various degrees of impact on CAR-T expansion and persistence, and therefore the overall safety and clinically meaningful response of the treatment. In this article we discuss various types of immune responses specific to CAR-T therapy, their impact on treatment outcome, and methodologies used to detect them.


Assuntos
Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Edição de Genes/métodos , Humanos , Imunoterapia/métodos , Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia
14.
AAPS J ; 21(4): 55, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30993501

RESUMO

In September 2018, the American Association of Pharmaceutical Scientists (AAPS) conducted an Annual Guidance Forum on the considerations related to immunogenicity testing for therapeutic protein products. In addition to a broad representation by the pharmaceutical industry, the event included strong representation by leading scientists from the US Food and Drug Administration (FDA). The agency and industry perspectives and updates to the guidance were presented. Specific topics that were discussed included the strategies of anti-drug antibody (ADA) assay cut-point assessments, the selection of ADA-positive controls (PCs), and the evaluation of PC performance. Assessment strategies and relevance of ADA assay attributes were also discussed, including assay drug tolerance and ADA assay sensitivity. The following is a summary of the discussion.

15.
Bioanalysis ; 11(8): 703-712, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30994012

RESUMO

Aim: Characterization of antidrug antibody (ADA)-like reactivity has emerged as critical element of bioanalytical design and assessment of compound immunogenicity risk. Materials & methods: Multiplex immunoassay was applied to detect and characterize ADA like reactivity using Photonic Ring Immunoassay platform (Genalyte). Specific binding to human IgE or human recombinant IL21-receptor-Fc fusion using exogenous reagents as surrogates for drug-specific reactivity was investigated. Results: Multiplexed assay format allowed identification of spiked antihuman IgE reactivity as murine IgG1 and endogenous antihuman recombinant IL21-receptor-Fc reactivity in rheumatoid arthritis sera as antihuman Fc-specific binding. Conclusion: The ability of a multiplex immunoassay platform to identify isotype and domain specificity of antidrug immunoglobulins was shown to be effective and should be considered when screening and characterizing pre- and post-dose ADA reactivity.


Assuntos
Anticorpos Monoclonais/imunologia , Imunoensaio/métodos , Humanos
16.
AAPS J ; 21(3): 52, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976993

RESUMO

This paper presents a systemic investigation of ADA development and ADA impact of a human coagulation factor in nonclinical species during drug development and provides insights into potential implications in human if a similar ADA occurs. FXaI16L-induced ADA response was characterized in monkey, mouse, rat, and dog in different studies, and ADA effects on pharmacokinetic and/or pharmacodynamics of FXaI16L were further examined in ADA-negative and ADA-positive animals. After repeated administrations, FXaI16L elicited a dose and exposure day-dependent ADA response which ranged from no response to a transient or persistent response. Increase in exposure day and increase in dose generally enhanced ADA incidence except for a decrease in ADA incidence was observed in monkeys after repeated high-dose administrations. The observable ADA impact on pharmacokinetics was only found in some ADA+ animals and included decrease in clearance and increase in systemic exposure but no increase in half-life. In addition, no or limited effect on pharmacodynamics by ADA was observed. The earliest ADA response was observed after three exposure days, marked elevation of drug exposure was observed in some animals at log titer > 2.0, and the highest antibody titer excited was about 4 (Log10) in all species. A correlation between ADA induction and accumulative exposure after various repeat treatments in different species was found for FXaI16L. In addition, potential immunogenicity risk and mitigation of ADA in clinics are discussed.

17.
AAPS J ; 21(3): 46, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30927117

RESUMO

Immunogenicity is a major challenge for protein therapeutics which can potentially reduce drug efficacy and safety and is often being monitored by anti-drug antibody (ADA) and neutralizing antibody (NAb) assays. Circulating targets and residual drugs in matrices can have significant impacts on accuracy of results from ADA and NAb assays, and sufficient drug and target tolerance for these assays are necessary. Here, we report the development of a competitive ligand binding (CLB) NAb assay for an anti-TFPI (tissue factor pathway inhibitor) monoclonal antibody (PF-06741086) with high drug and target tolerance to support ongoing clinical studies. A double acid affinity capture elution approach was used to mitigate drug interference, and a robust target removal strategy was employed to enhance target tolerance. The validated NAb assay has sensitivity of 313 ng/mL, drug tolerance of 50 µg/mL, and target tolerance of 1200 ng/mL. A step-by-step tutorial of assay development is described in this manuscript along with the rationale for using a high drug/target tolerant NAb assay. The NAb assay cut point factor obtained was 0.78. Other assay performance characteristics, e.g., precision and selectivity, are also discussed. This validated method demonstrated a superior drug and target tolerance to warrant specific and precise characterization of the NAb responses in support of ongoing clinical studies.

18.
Bioanalysis ; 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30488726

RESUMO

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. 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 and LBA/cell-based assays approaches. This 2018 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 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

19.
AAPS J ; 21(1): 4, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30402825

RESUMO

Insufficient drug tolerance presents a major challenge in the development of neutralizing antibody (NAb) assays for biotherapeutics. Sample pre-treatment using solid-phase extraction with acid dissociation (SPEAD) is widely reported to improve drug tolerance. In this paper, a case study is presented in which SPEAD was used in conjunction with a competitive ligand binding NAb assay format. A significant degree of biotin-drug conjugate leaching was observed resulting in the reporting of both false positive and false negative results in NAb assay. Mitigation steps have been evaluated to address drug/biotin-drug conjugate leaching. These steps included assessment of the streptavidin-coated plate in conjunction with biotin-drug conjugates at various biotin molar challenge ratios (MCR). In addition, an alternative method based on covalent capture of the drug on an aldehyde-activated plate was assessed. Both approaches were compared for the degree of drug/biotin-drug conjugate leaching during the second elution step of the SPEAD procedure. Moreover, the impact of various conditions on the assay performance was assessed, including elution pH, sample incubation time, and biotin MCR. For the covalent drug capture method, capture conditions were evaluated. Optimized conditions in both streptavidin capture and covalent capture methods enabled a significant reduction of drug/biotin-drug conjugate leaching. A streptavidin high binding capacity approach using biotin-drug conjugate with a MCR of 50:1 was chosen as the optimal method yielding a NAb assay with a fit for purpose sensitivity (153 ng/mL) and a drug tolerance of up to 50 µg/mL with 500 ng/mL PC.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Indicadores e Reagentes/química , Testes de Neutralização/métodos , Extração em Fase Sólida/métodos , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/imunologia , Produtos Biológicos/química , Produtos Biológicos/imunologia , Produtos Biológicos/farmacologia , Biotina/análogos & derivados , Biotina/química , Química Farmacêutica , Cromatografia de Afinidade/métodos , Tolerância a Medicamentos , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Estreptavidina/química , Succinimidas/química
20.
AAPS J ; 20(3): 45, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29536273

RESUMO

In ligand binding assays (LBA), the concentration to response data is a nonlinear relationship driven by the law of mass action. Four parameter logistic (4PL) and five parameter logistic (5PL) curve fitting models are two widely accepted and validated models for LBA calibration curve data. Selection of the appropriate regression model and weighting function are key components of LBA development. Assessment of selected model and weighting function should be performed during assay development and confirmed later during validation. There has been limited published work on practical approaches to determining an appropriate weighting function and selection of a regression model for ligand binding assays. Herein, a structured scheme is presented to determine both. By applying commonly available software, assay performance data were analyzed to determine weighting functions and associated choice of a curve fitting model in three presented case studies. As a result, assay ranges of quantification were improved by reducing lower limit of quantification (from 1.00 to 0.317 ng/mL in one case study and from 2.06 to 1.37 ng/mL in another) or extending both low and upper limits of quantification(e.g., 1.04 to 48.3 ng/mL improved to 0.602 to 145 ng/mL). In addition, assay calibration curve performance demonstrated improved assay accuracy (%RE) and precision (%CV). Recommendations on decision flow when determining appropriate weighting function and curve fit model are presented.


Assuntos
Modelos Teóricos , Padrões de Referência , Proteínas Sanguíneas/metabolismo , Calibragem , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Humanos , Ligantes , Limite de Detecção , Modelos Estatísticos , Farmacocinética , Ligação Proteica , Reprodutibilidade dos Testes , Software
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