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Aim: Serotype-specific assays detecting pneumococcal polysaccharides in bodily fluids are needed to understand the pneumococcal serotype distribution in non-bacteremic pneumonia. Methods: We developed a urine antigen detection assay and using urine samples from adult outpatients without pneumonia developed positivity cutoffs for both a previously published 15-valent and the new 21-valent assay. Clinical sensitivity was confirmed with samples from patients with invasive pneumococcal disease. Results: Total assay precision ranged from 7.6 to 17.8% coefficient of variation while accuracy ranged between 80 and 150% recovery, except for three serotypes where recoveries ranged from 32 to 60%. Clinical sensitivity was 86.4% and specificity was 96.5% across all 30 serotypes. Conclusion: The assay could potentially assess serotype-distribution in non-infected and infected participants with pneumococcal disease.
[Box: see text].
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Selection and characterization of antibodies are critically important in establishing robust immunoassays to support the development efforts of vaccines. Plate-based ELISA can be time- and resource-intensive to select initial antibody clones or characterize downstream resupply lots while providing limited information regarding the binding characteristics of the antibodies beyond concentration-response curves. This work applied the microfluidic Gyrolab to holistically evaluate immunoassay reagents through analyses of concentration-response curves as well as antibody-antigen interactions visualized in column images and affinity estimates. We exploited the automation capability of the Gyrolab to reduce the resources (time, reagents, and scientists) required for screening and evaluating antibody reagents. Using a flexible semi-universal assay format, we compared antibody clones for selection and resupply lots of sera and monoclonal antibodies in a simple "plug-and-play" manner without antibody modifications. We found that the performance of antibodies in the Gyrolab correlated well with the trends observed in traditional ELISAs, while the Gyrolab provided additional advantages over plate-based assays such as column images of antibody binding and affinity measurements.
Assuntos
Anticorpos Monoclonais , Microfluídica , Indicadores e Reagentes , Imunoensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
Aim: Critical virus reagents in regulated bioanalytical assays require stability monitoring. Although stability at ultra-low frozen temperatures is generally assumed, published data are limited and real-time studies are time consuming. Materials & methods: The authors reviewed literature data, typical mechanisms of molecular degradation, glass transition temperatures of commonly used buffers and available real-time storage data to model frozen virus reagent stability. Results: Storage at ultra-low temperatures below the glass transition temperature was critical for virus stability. Modeling of real-time data suggested that virus potency remained within 0.5 log10 of its starting potency at a probability of >99, 90 and 73% after 10, 20 and 30 years, respectively. Conclusion: The study supports the practice of virus storage at -70°C or below for 20-30 years.
Assuntos
Congelamento , TemperaturaRESUMO
Global deployment of vaccines poses significant challenges in the distribution and use of the accompanying immunoassays, one of the standard methods for quality control of vaccines, particularly when establishing assays in countries worldwide to support testing/release upon importation. This work describes our effort toward developing an integrated, portable device to carry out affinity assays for viral particles quantification in viral vaccines by incorporating (i) aptamers, (ii) microfluidic devices, and (iii) electrochemical detection. We generated and characterized more than eight aptamers against multiple membrane proteins of cytomegalovirus (CMV), which we used as a model system and designed and fabricated electrochemical microfluidic devices to measure CMV concentrations in a candidate vaccine under development. The aptamer-based assays provided a half maximal effective concentration, EC50, of 12 U/mL, comparable to that of an ELISA using a pair of antibodies (EC50 60 U/mL). The device measured relative CMV concentrations accurately (within ±10% bias) and precisely (11%, percent relative standard deviation). This work represents the critical first steps toward developing simple, affordable, and robust affinity assays for global deployment without the need for sensitive equipment and extensive analyst training.
Assuntos
Aptâmeros de Nucleotídeos , Infecções por Citomegalovirus , Vacinas Virais , Aptâmeros de Nucleotídeos/química , Bioensaio , Humanos , Dispositivos Lab-On-A-ChipRESUMO
Analytical methods are utilized throughout the biopharmaceutical and vaccines industries to conduct research and development, and to help control manufacturing inputs and outputs. These analytical methods should continuously provide quality data to support decisions while managing the remaining of risk and uncertainty. Analytical quality by design (AQbD) can provide a systematic framework to achieve a continuously validated, robust assay as well as life cycle management. AQbD is rooted in ICH guidelines Q8 and Q9 that were translated to the analytical space through several white papers as well as upcoming USP 1220 and ICH Q14. In this white paper, we expand on the previously published concepts of AQbD by providing additional context for implementation in relation to ICH Q14. Using illustrative examples, we describe the AQbD workflow, its relation to traditional approaches, and potential pathways for ongoing, real-time verification. We will also discuss challenges with respect to implementation and regulatory strategies.
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Projetos de Pesquisa , Vacinas , Animais , Estágios do Ciclo de VidaRESUMO
Monoclonal antibodies (mAbs) are widely used as critical reagents in analytical assays. While regulatory guidelines exist for stability monitoring of biopharmaceutical antibodies, they do not apply directly to the stability of mAbs used as assay reagent. We investigated alternative approaches to real-time stability monitoring of assay reagents. We compared functional (ELISA and cell-based) and biochemical (aggregation, deamidation) assay results using temperature-stressed mAb reagents. Data from both assay groups were compared for indications of antibody degradation. Arrhenius model kinetics was used to further extrapolate stability trends. Changes detected by traditionally monitored biochemical changes were not directly predictive of assay function. Instead, monitoring of reportable results was a closer indication of changes in assay performance related to mAb degradation. Using Arrhenius kinetic modeling, we combined forced degradation of individual reagents with reportable assay results to classify reagents into risk groups with associated re-evaluation and monitoring plans. This combined approach mitigates risk by monitoring each mAb reagent individually under stressed conditions while streamlining expiry assignment through simplified Arrhenius kinetics with only limited real-time stability data.
Assuntos
Anticorpos Monoclonais/química , Desnaturação Proteica , Proteólise , Proteínas de Arabidopsis , Bioensaio/métodos , Guias como Assunto , Indicadores e Reagentes/química , Indicadores e Reagentes/normas , Laboratórios/normas , Modelos Biológicos , Proteínas Nucleares , Controle de QualidadeRESUMO
BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Memória Imunológica , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Hipermutação Somática de Imunoglobulina , Adulto , Animais , Anticorpos Antibacterianos/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Feminino , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Sorogrupo , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Streptococcus pneumoniae/imunologia , Vacinação , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêuticoRESUMO
Compared with biologics, vaccine potency assays represent a special challenge due to their unique compositions, multivalency, long life cycles and global distribution. Historically, vaccines were released using in vivo potency assays requiring immunization of dozens of animals. Modern vaccines use a variety of newer analytical tools including biochemical, cell-based and immunochemical methods to measure potency. The choice of analytics largely depends on the mechanism of action and ability to ensure lot-to-lot consistency. Live vaccines often require cell-based assays to ensure infectivity, whereas recombinant vaccine potency can be reliably monitored with immunoassays. Several case studies are presented to demonstrate the relationship between mechanism of action and potency assay. A high-level decision tree is presented to assist with assay selection.
Assuntos
Bioensaio , Avaliação Pré-Clínica de Medicamentos/métodos , Potência de Vacina , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Células Hep G2 , Humanos , Imunogenicidade da Vacina , Camundongos , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Células VeroRESUMO
Vaccine in vitro potency assays are vital regulatory tests that are used to confirm the presence and concentration of an antigen of interest in a form that directly or indirectly relates to protective activity in patients. Current assays come in many forms, but they almost exclusively use antibody reagents for selective detection of the target antigen. Antibodies provide specific recognition of vaccine antigens but also exhibit drawbacks such as stability limitations, cost, and lot-to-lot variation, which can make it challenging to maintain the reagent throughout the lifetime of the vaccine. We explored replacing antibodies with aptamers. Aptamers are macromolecules, such as nucleic acids, which can bind to their targets with high specificity and affinity, similar to that of antibodies. Some of the advantages of using aptamers over antibodies is that aptamers can be more stable, smaller, less expensive to produce, synthesized in vitro, and logistically easier to supply throughout the multi-decade lifespan of a commercial vaccine. We created modified DNA aptamers against the common vaccine carrier protein, CRM197. Several aptamers were discovered and one was chosen for further characterization. The binding kinetics of the aptamer revealed an off-rate 16-fold slower than anti-CRM197 antibodies used for comparison. The aptamers were more sensitive than available antibodies in some assay formats and comparable in others. The aptamer epitope was mapped to the receptor-binding domain of CRM197, a site adjacent to a known antibody binding site. These data address some key aspects for a path forward in replacing antibodies with aptamers for use as critical reagents in vaccine assays. We further highlight the possibility of using nucleic acid reagents to develop next generation potency assays.
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Anticorpos/imunologia , Aptâmeros de Nucleotídeos/imunologia , Antígenos/imunologia , Proteínas de Bactérias/imunologia , Bioensaio/métodos , Humanos , Ligação Proteica/imunologia , Potência de Vacina , Vacinas/imunologiaRESUMO
Measuring vaccine potency is critical for vaccine release and is often accomplished using antibody-based ELISAs. Antibodies can be associated with significant drawbacks that are often overlooked including lot-to-lot variability, problems with cell-line maintenance, limited stability, high cost, and long discovery lead times. Here, we address many of these issues through the development of an aptamer, known as a slow off-rate modified DNA aptamer (SOMAmer), which targets a vaccine antigen in the human papillomavirus (HPV) vaccine Gardasil. The aptamer, termed HPV-07, was selected to bind the Type 16 virus-like-particle (VLP) formed by the self-assembling capsid protein L1. It is capable of binding with high sensitivity (EC50 of 0.1 to 0.4 µg/mL depending on assay format) while strongly discriminating against other VLP types. The aptamer competes for binding with the neutralizing antibody H16.V5, indicating at least partial recognition of a neutralizing and clinically relevant epitope. This makes it a useful reagent for measuring both potency and stability. When used in an ELISA format, the aptamer displays both high precision (intermediate precision of 6.3%) and a large linear range spanning from 25% to 200% of a typical formulation. To further exploit the advantages of aptamers, a simplified mix and read assay was also developed. This assay format offers significant time and resource reductions compared to a traditional ELISA. These results show aptamers are suitable reagents for biological potency assays, and we expect that their implementation could improve upon current assay formats.
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Antígenos Virais/imunologia , Aptâmeros de Nucleotídeos/imunologia , Epitopos/imunologia , Papillomavirus Humano 16/imunologia , Vacinas contra Papillomavirus/imunologia , Reações Antígeno-Anticorpo , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/química , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
Dilutions are a common source of analytical error, both in terms of accuracy and precision, and a common source of analyst mistakes. When serial dilutions are used, errors compound, even when employing laboratory automation. Direct point dilutions instead of serial dilutions can reduce error but is often impractical as they require either large diluent volumes or very small sample volumes when performed with traditional liquid handling equipment. We evaluated preparation of dilution curves using a picoliter digital dispenser, the HP, Inc. / TECAN D300 which is capable of accurately delivering picoliter volumes directly into sample wells filled with assay diluent. Dilution linearity and variability of the direct dilutions were similar to or less than those generated with a traditional liquid handler as measured using a fluorophore assay and an ELISA used to measure vaccine potency. Minimum concentrations for detergent in the dispensed sample were identified but no correlation with detergent characteristics was observed. The tolerance to protein in the sample was evaluated as well with up to 5% BSA having no impact on dispense linearity and precision. We found the digital dispenser to reduce automation complexity while maintaining or improving assay performance in addition to facilitating complex plate lay-outs.
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Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Potência de Vacina , Automação Laboratorial , Calibragem , Detergentes/química , Ensaio de Imunoadsorção Enzimática/normas , Desenho de Equipamento , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/normas , Miniaturização , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de FluorescênciaRESUMO
AIM: Biologics development often requires multiple immunoassays to evaluate both assay reagents and potential drug candidates resulting in extensive analytical development. METHODOLOGY: We developed a semi-universal, 5-layer platform assay on Gyrolab using secondary antispecies or anti-isotype-specific capture and detection antibodies. We applied the assay to several multivalent vaccines. RESULTS: Method performance exhibited a median accuracy of 110%, reproducibility of 9% CV and intermediate precision of 11% CV. System suitability criteria were met for 92.5% of the samples and only one out of 31 replicate samples exhibited a %CV greater than 20%. CONCLUSION: The semi-universal Gyrolab assay allowed assay development without reagent labeling. The format could also be translated into a plate-based assay.
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Anticorpos/análise , Terapia Biológica/métodos , Ensaio de Imunoadsorção Enzimática , Vacinas/imunologia , Animais , Corantes Fluorescentes/química , Imunoglobulina G/análise , Isotipos de Imunoglobulinas/análise , Camundongos , Reprodutibilidade dos Testes , Vacinas/análiseRESUMO
BACKGROUND: Dilution bias is a major cause of immunoassay variability due to the lack of an internal standard to determine the true versus the expected dilution value. METHODOLOGY: We used an internal control to measure dilution bias in an ELISA. Acridine-orange was added at the first dilution step and monitored throughout dilutions. Assay results were corrected using the fluorescent signal ratio between samples and reference. Acridine dilution correlated with analyte-specific assay measurements (R2 = 0.987). Correction of assay results with the measured dilution factor improved both accuracy and precision resulting in a reduction of >50% %CV reduction. CONCLUSION: Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.
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Ensaio de Imunoadsorção Enzimática/métodos , Laranja de Acridina/análise , Ensaio de Imunoadsorção Enzimática/normas , Fluorescência , Corantes Fluorescentes/análise , Técnicas de Diluição do IndicadorRESUMO
Microtiter plate-based assays are a common tool in biochemical and analytical labs. Despite widespread use, results generated in microtiter plate-based assays are often impacted by positional bias, in which variability in raw signal measurements are not uniform in all regions of the plate. Since small positional effects can disproportionately affect assay results and the reliability of the data, an effective mitigation strategy is critical. Commonly used mitigation strategies include avoiding the use of outer regions of the plate, replicating treatments within and between plates, and randomizing placement of treatments within and between plates. These strategies often introduce complexity while only partially mitigating positional effects and significantly reducing assay throughput. To reduce positional bias more effectively, we developed a novel block-randomized plate layout. Unlike a completely randomized layout, the block randomization scheme coordinates placement of specific curve regions into pre-defined blocks on the plate based on key experimental findings and assumptions about the distribution of assay bias and variability. Using the block-randomized plate layout, we demonstrated a mean bias reduction of relative potency estimates from 6.3 to 1.1 % in a sandwich enzyme-linked immunosorbent assay (ELISA) used for vaccine release. In addition, imprecision in relative potency estimates decreased from 10.2 to 4.5 % CV. Using simulations, we also demonstrated the impact of assay bias on measurement confidence and its relation to replication strategies. We outlined the underlying concepts of the block randomization scheme to potentially apply to other microtiter-based assays.
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Ensaio de Imunoadsorção Enzimática/instrumentação , Vacinas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Distribuição AleatóriaRESUMO
Quality by design (QbD) expanded in recent years from pharmaceutical processing into analytical chemistry. Beyond online process analytical technology, off-line assays including immunoassays are also starting to benefit from QbD. Although analytical QbD is still a relatively new development, underlying concepts, such as target-oriented development and statistical quality control have been applied to diagnostic assays under the term 'design control' for several years. We reviewed how QbD and statistical quality control concepts have been applied both to diagnostic and bioanalytical immunoassays ranging from the use of individual tools to end-to-end QbD-based workflows. We will discuss some of the results of the different approaches to immunoassays, how they fit into the QbD framework and how individual tools may complement a QbD workflow.
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Imunoensaio/métodos , Estatística como Assunto/métodos , Controle de QualidadeRESUMO
BACKGROUND: As quality by design (QbD) for pharmaceutical product development is being expanded to include analytical methods, we applied QbD to the development of antigenicity assays measuring in vitro relative potency of a quadrivalent vaccine candidate. RESULTS: After establishing development targets together with customers, immunoassays were developed to meet objectives. Statistical design of experiments was used to optimize method parameters and establish a design space. Systematic risk analysis enabled identification of potential risks to method performance that were mitigated by investigating the available design space around risk factors and by establishing appropriate control strategies. CONCLUSION: We found QbD-based method development was a more efficient and systematic approach that could also potentially facilitate assay transfers and life cycle management.
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Antígenos/análise , Análise Fatorial , Técnica Direta de Fluorescência para Anticorpo/normas , Técnica Indireta de Fluorescência para Anticorpo/normas , Vacinas/análise , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Coelhos , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e Especificidade , Vacinas/imunologia , Estudos de Validação como AssuntoRESUMO
After more than 40 years, immunoassays are still the backbone of protein biomarker analysis in clinical diagnostics and drug development. They have come a long way since their inception, incorporating technical developments including monoclonal antibodies, novel labels and lately microfluidics. A number of microfluidic platforms have been tested, such as centrifugational compact disc assays, lab-on-a-chip, arrays and digital electrochemical assays. This review focuses on commercial applications of microfluidic immunoassays with reference to some applied academic examples of interest. Advantages and disadvantages of the platform technologies are discussed in general.
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Imunoensaio/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Miniaturização/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Miniaturização/métodosRESUMO
BACKGROUND: Dalotuzumab (MK 0646), an anti-IGF1R antibody intended for cancer therapy, has progressed to Phase III clinical trials. To evaluate pharmacokinetic properties, we developed and compared two ELISAs to measure dalotuzumab in human serum and validated the second method following regulatory guidelines for ligand-binding assays. RESULTS: After an IGF1R-mediated capture step, dalotuzumab was detected by either an antihuman IgGFc- or by an antihuman IgG1-specific antibody. The assay range was 20 to 2000 ng/ml with mean inter-day accuracy of controls ranging from 97 to 108% (method A) and 83 to 97% (method B), respectively. Mean assay precision was ≤20% CV both intra- and inter-day. Other parameters that were validated included dilution linearity, stability, interferences and incurred sample reanalysis. In addition, application of both assay formats to clinical sample analysis was demonstrated establishing time-concentration curves. CONCLUSION: As the methods rely on commercial reagents, they may be applicable to other anti-IGF1R antibodies and facilitate the development of new therapeutics.
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Anticorpos Monoclonais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Receptor IGF Tipo 1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Biotina/química , Biotina/metabolismo , Estabilidade de Medicamentos , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Estreptavidina/química , Estreptavidina/metabolismoRESUMO
Pharmacokinetic data derived from assays that accurately and precisely quantitate a therapeutic drug in circulation are critical to appropriately designing suitable dosing schedules. This manuscript describes the validation and implementation of methods to quantitate a therapeutic anti-human PCSK9 monoclonal antibody in rat and monkey sera as well as immunogenicity methods to screen the possible presence of rat and monkey antibodies directed against the antibody. As soluble, endogenous PCSK9 can interfere with a PCSK9-mediated capture step in ELISA, an indirect target-capture assay was used that potentially could capture free and target-engaged therapeutic mAb. Immunogenicity assays were based on a standard bridge ELISA using the therapeutic antibody for capture and detection. Both pharmacokinetic and immunogenicity assays were implemented in preclinical studies of the therapeutic antibody. The methods presented here may enable further pharmacokinetic studies.
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Anticorpos Monoclonais/análise , Serina Endopeptidases/análise , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Ensaio de Imunoadsorção Enzimática/métodos , Haplorrinos , Humanos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Serina Endopeptidases/imunologia , Serina Endopeptidases/farmacocinética , SolubilidadeRESUMO
INTRODUCTION: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays. METHODS: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support. RESULTS: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10µL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less. DISCUSSION: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.