Bioanalytical Assay Development and Validation: Reproducible and Reliable Quantitation of Analytes

Bioanalytical assay development helps determine the design implications, limitations, favorable conditions, and suitability of a drug for the intended treatment. Before assay development and validation can be initiated, it is recommended to understand the physiochemical properties of the analyte. Bioanalytical assay development is considerably informed by the details on protein binding and in vivo/in vitro metabolism along with essential aspects of prior work on the method. Bioanalytical assays play a crucial role in the discovery and development of pharmaceutical products given their ability to discern purity, identity, performance, and potency of these compounds. Furthermore, the involved techniques allow reproducible and reliable quantitation of analytes in various biological matrices, such as plasma, urine, blood, or serum.

Assay validation is performed for the analysis of drugs, metabolites, and biomarkers to ensure that the test results are precise and accurate, and the generated data is reasonably robust in supporting drug development and other intended purposes. Several method characteristics play a significant role during each stage of assay development and validation, including specific chemistry of analyte, sample clean-up to analysis methodology, and desired method sensitivity. The selectivity and sensitivity of bioanalytical assays developed for drug and metabolite quantitation thoroughly impact the success of preclinical and clinical studies. Thus, careful sample preparation, HPLC column, and mobile phase selection, and choice of detector and quantitation method are critical during LC-MS assay development.

The purpose of assay development and validation is to find a suitable method applicable to a chosen analyte in a specific biological matrix. The analysis outcome might be inaccurate if the assay is not validated correctly and deemed unreliable. This assay inadequacy could be due to low sensitivity, selectivity, precision, accuracy or any other reason. Given the significance of this topic, the FDA has published guidance to provide guardrails around assay development and validation discussions. Generally, the assay can be used with full confidence to produce reliable data if the FDA method development guidance is followed and all its outlined criteria are met. Thus, assay development and validation are carried out with appropriate technology so that compliance with regulatory and scientific requirements can be achieved. Additionally, analytical information obtained from already existing bioanalytical assays may sometimes be effectively utilized.

Assay Development and Validation: From Drug Discovery and Preclinical to Clinical Phases

During preclinical development, bioanalytical assays assess the impact of New chemical entity (NCE) as a potential treatment on various animal species. Further assay development and validation is required to evaluate pharmacokinetic parameters, toxicity, and safety profile in humans of the drug compound that reaches the clinical stage. Sample collection, processing, and preparation should be thoroughly assessed before starting assay development to mitigate future risk factors at a later stage and validation phase.

Simply put, bioanalytical assays are imperative in improving a drug candidate’s success rate in preclinical animal studies. Robust assay development and validation during early preclinical studies is time well spent as satisfactory assays can be carried out further into development stages with minimal effort. Bioanalytical assays during clinical studies in human volunteers involve quantitation of analytes and corresponding metabolites as well as an assessment of various matrices. This analysis provides significant guidance on toxicokinetic and pharmacokinetic parameters evaluated through assay development and validation.

Bioanalytical Assay Services by NorthEast BioLab

Bioanalytical Assay Services by NorthEast BioLab

NorthEast BioLab offers comprehensive bioanalytical assay services at every stage of drug development during drug discovery, preclinical and clinical studies. Our scientists assist in assay development and validation for quantitation of analyte and metabolites for blood, plasma, serum, urine, and cerebrospinal fluid (CSF) samples among other standard matrices. Our bioanalytical method development services can be utilized for small and large molecules, such as NCE, biosimilars, peptides, antibody drug conjugates (ADC), monoclonal antibodies (mAb), and oligonucleotides.

Custom Biomarker Assay Development, Validation, and Analysis

Our custom biomarker development services support analysis using proprietary or commercially available materials, such as anti-ID antibodies or antibody pairs from the sponsor. Depending on study needs, our scientists can set-up your biomarker assays individually (single-plex assay) or combined to simultaneously analyze multiple analytes (multi-plex assay) on MSD ECL or Luminex Multiplex platform. Each project begins by consulting with our biomarker lab experts to determine the analytes(s) of interest, the species/sample matrix, sample quantity, QC requirements, dynamic range, and assay platform. Afterward, a preliminary run consisting of a standard curve and QC samples is performed to assess project feasibility with desired multiplexing or dilution.

Once method development and protocol optimization are complete, we perform biomarker validation services to measure bioanalytical parameters such as accuracy, precision, and recovery. Afterward, NorthEast BioLab issues a hyperlinked biomarker method validation pdf report documenting your eCTD submission-ready assay. Henceforth, your fully validated and robust biomarker assay is reliable for all phases of the drug development, from GLP preclinical studies to clinical biomarker testing.

Similarly, we understand that you may have validated ELISA assays to be transferred on a multiplexing biomarker assay platform. Both the Luminex and MSD platforms benefit from the traditional ELISA assay but offer significant advantages of using smaller sample volumes to analyze multiple analytes simultaneously. Consequently, these platforms lead to increased throughput, reduced costs, and more flexibility. Our team of scientists can also seamlessly transfer your existing ELISA assays to either the Luminex or MSD multiplex platforms. The transferred assay can then be used alone or combined with other biomarkers to create a panel to meet your unique drug development and research needs.

ELISA Assay: PK, BABE, TK, Immunogenicity, Biomarkers

The Enzyme-Linked Immunosorbent Assay (ELISA) helps determine and quantify macromolecules, such as proteins and peptides. Here, a capture and detection antibody pair are connected to the enzyme that accomplishes catalysis of a chemiluminescent or colorimetric substrate. Our veteran scientists at NorthEast BioLab utilize ELISA assays for determining the concentration and presence of the analyte in biological samples for a variety of drug research and development studies.

Biomarker Testing Assay: LC-MS/MS, ELISA, MSD, Luminex, qPCR

We provide comprehensive biomarker assay development and validation services, given our deep expertise in analyzing various analytes in multiplex and singleplex formats such as LC-MS/MS, ELISA, MSD ECL Assay, Luminex Assay, or qPCR for multiple therapeutic areas and species. Our biomarker testing services include method development and assay validation for novel biomarkers, development of tissue and fluid-based biomarkers, as well as GLP and GCP biomarker assessments.

Immunogenicity Testing Assay: ELISA, MSD

At NorthEast BioLab, we offer a comprehensive solution for immunogenicity testing including ADA screening, confirmation, titration, and isotyping. Our scientists diligently assess the immunogenicity of your drug product, calculate the analysis cutpoint, evaluate the formation of anti-drug antibodies, and measure the persistence of the neutralizing fractions of anti-drug antibodies.

ADME Assay

At NorthEast BioLab, we utilize in vitro and in vivo ADME studies to evaluate the dosage regimen, administration routes, and safety assessment of the drug on different types of species. A dedicated study is developed by our lab analysts to cater to the requirements of your program.

Cell-Based Proliferation Viability Assay

NorthEast BioLab performs various cell-based proliferation viability assay for metabolic activity analysis, DNA replication, antigen recognition, proliferation markers, membrane integrity, and ATP measurements. Our services in this domain include signaling and secretion, apoptosis and cell death, and cell proliferation assay.

Biomarker Testing Services using Commercial Assays and Kits

NorthEast BioLab provides biomarker testing services with an exhaustive menu of 600+ analytes in the usual biological matrices, given the attractive risk/reward profile of commercial assays. We offer biomarker analysis services using a singleplex or multiplex panel for a wide range of applications such as immunology/inflammation, cardiology, toxicology, and metabolic disorders. Furthermore, our preclinical and clinical biomarker services include cytokine testing in humans, mice, rats, dogs, pigs, non human primates (NHP), and other species.

NorthEast BioLab’s committed to delivering the best possible outcome for your biomarker services on MSD and Luminex platforms, keeping pace with the latest technological advances that allow multiplexing from a single sample with a small volume. Our scientists closely partner with industry-leading kit providers (Meso Scale Discovery, Millipore Sigma, Thermo Fisher Scientific, R&D Systems, Biorad, etc.) to accurately analyze your biomarker study samples. Thus, our vendors’ technical support empowers us to provide reproducible and reliable results using pre-validated biomarker panels or custom assay panels even for your most challenging biomarker services.

We can perform these biomarker lab services according to manufacture specifications or customize protocol to meet your bespoke research and development goals. Before biomarker analysis on your precious study samples, our team conducts a pre-validation run to optimize assay, such as determine minimum required dilution (MRD) and assess quality controls against standard curve to gauge % recovery. Per your regulatory requirement, we happily perform biomarker validation services on these commercial assays to thoroughly analyze bioanalytical parameters such as accuracy, precision, sensitivity, dilution effects, etc.

One of our most popular biomarker services is Cytokine testing, which is a crucial part of many research studies capturing the expression of immune and inflammatory responses. We offer multiple cytokine assay profiling services on both Meso Scale Discovery (MSD) and the Luminex platforms. These biomarker development and validation services are fully customizable and range from small panels containing a few cytokines of interest to larger pre-validated cytokine profile panels. Below, please find some of our standard cytokine testing panels.

Panel Assay Platform Analytes
V-plex Mouse Cytokine 19-plex MSD IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-15, IL-17A/F, IL-27p28/IL-30, IL-33, IP-10, KC/GRO, MCP-1, MIP-1α, MIP-2, TNF-α
V-plex Human Biomarkers 54-plex MSD CRP, Eotaxin, Eotaxin-3, FGF (basic), GM-CSF, ICAM-1, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-21, IL-22, IL-23, IL-27, IL-31, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α, PlGF, SAA, TARC, Tie-2, TNF-α, TNF-β, TSLP, VCAM-1, VEGF-A, VEGF-C, VEGF-D, VEGFR-1/Flt-1
Multiplex Mouse Cytokine/Chemokine Panel Luminex xMAP GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12, (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG,MIP-1α, MIP-1β,MIP-2,
Milliplex Human Cytokine/Chemoskine/Growth Factor Panel Luminex xMAP sCD40L, EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1, MCP-3, M-CSF, MDC (CCL22), MIG, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNFα, TNFβ, VEGF-A

In summary, NorthEast BioLab offers diverse biomarker testing on multiple biomarker assay platforms. We can analyze client samples for one or two analytes individually using traditional ELISA methods or multiplex biomarkers to create panels to maximize study ROI with high integrity data using small sample volumes. All biomarker studies are validated with a fit for purpose approach to fulfill your regulatory needs. Contact us today to discuss your biomarker research needs, whether you are at the discovery phase or getting ready for clinical analysis.

Basis of Bioanalysis

The Basics of Bioanalysis

Bioanalysis is an essential tool in drug discovery and development for determining the concentration of drugs and their metabolites as well as various pharmacodynamics biomarkers in biological fluids. In these analyses, scientists use developed an….

Suitable Bioanalytical Assay Development and Validation to prove your Drug's Safety and Efficacy

At NorthEast Biolab, we recognize the pivotal significance of bioanalytical assays in drug discovery and development for measuring the efficacy and safety profile of your drug compounds in biological samples. Accordingly, our scientists develop reliable analytical methods suitable for the various stages of your drug development. For example, we offer expedited assay development and sample analysis for your drug discovery and molecule screening needs. Similarly, we perform full assay validation with strict adherence to bioanalytical method validation (BMV) regulatory guidelines during preclinical safety, toxicokinetic assessments, and clinical trials.

Additionally, we initiate our bioanalytical assay services only after developing a thorough understanding of your study requirements to yield high-quality results promptly. For small molecules, we widely utilize the LC-MS/MS method that doesn’t need extensive sample preparation. In this case, a suitable mass spectrometry setup is chosen based on the required assay sensitivity and specificity. For large molecules, we have in-depth expertise in assay validation of both customized ELISA and commercially available ELISA kits as needed. Here, the calibration range of the assay is chosen based on the expected concentration to minimize the reanalysis of study samples. Our team offers you outstanding support and maintains constant communication to smoothly meet your project-critical deadlines utilizing our robust techniques and scalable sample analysis platforms.

Related FAQs

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