Judy Shih

Application of multi-factorial design of experiments to successfully optimize immunoassays for robust measurements of therapeutic proteins

Developing a process that generates robust immunoassays that can be used to support studies with tight timelines is a common challenge for bioanalytical laboratories. Design of experiments (DOEs) is a tool that has been used by many industries for the purpose of optimizing processes. The approach is capable of identifying critical factors and their interactions with a minimal number of experiments. The challenge for implementing this tool in the bioanalytical laboratory is to develop a user-friendly approach that scientists can understand and apply. We have successfully addressed these challenges by eliminating the screening design, introducing automation, and applying a simple mathematical approach for the output parameter. A modified central composite design (CCD) was applied to three ligand binding assays. The intra-plate factors selected were coating, detection antibody concentration, and streptavidin-HRP concentrations. The inter-plate factors included incubation times for each step. The objective was to maximize the logS/B (S/B) of the low standard to the blank. The maximum desirable conditions were determined using JMP 7.0. To verify the validity of the predictions, the logS/B prediction was compared against the observed logS/B during pre-study validation experiments. The three assays were optimized using the multi-factorial DOE. The total error for all three methods was less than 20% which indicated method robustness. DOE identified interactions in one of the methods. The model predictions for logS/B were within 25% of the observed pre-study validation values for all methods tested. The comparison between the CCD and hybrid screening design yielded comparable parameter estimates. The user-friendly design enables effective application of multi-factorial DOE to optimize ligand binding assays for therapeutic proteins. The approach allows for identification of interactions between factors, consistency in optimal parameter determination, and reduced method development time.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3 – LBA, biomarkers and immunogenicity)

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 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 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

Automating bioanalytical sample analysis through enhanced system integration

BACKGROUND In the bioanalytical laboratory, challenges associated with manual, repetitive, labor-intensive processes can be addressed by powerful automated liquid handlers; however, their implementation has been difficult due to lack of efficient integration into laboratory workflows. Faster throughput is afforded to ligand binding assay (LBA) technologies via enhanced automation, but the upstream sample processing still remains a bottleneck. To be truly efficient, these technologies must be incorporated into a laboratory information management system (LIMS) to streamline data analysis and reporting. RESULTS Three off-the-shelf technologies that aid in improving bioanalytical laboratory efficiencies were utilized with minimal customization to streamline the sample analysis process. Information extracted via a sequence file from the LIMS run was utilized to perform the sample processing on the automated liquid handler. A file conversion tool converted the sequence files that allowed for sample processing and preparing the assay ready plate. The plate was then transferred to the LBA microfluidics platform to run the experiments. The integration was tested using a LBA PK assay that demonstrated good sample dilution and assay performance. CONCLUSION We successfully integrated LIMS with an automated liquid handler and a microfluidics platform to automate the sample analysis process in the bioanalytical laboratory. The utilization of off-the-shelf technologies with minimal customization requires minimal resources from laboratory scientists. It may be possible to implement this approach for other analytical platforms.

Implementation of a universal analytical method in early-stage development of human antibody therapeutics: application to pharmacokinetic assessment for candidate selection

BACKGROUND Effective bioanalytical support for pharmacokinetic assessment of therapeutics in early development remains challenging. Concurrent evaluation of multiple candidates per program is typical, requiring efficient characterization in various preclinical species; an ambitious effort often complicated by assay reagent unavailability and limited sample volume. Accordingly, a universal anti-human Fc assay for human monoclonal antibody and derived therapeutics was developed using a microfluidics platform to address these bioanalytical challenges. RESULTS The universal assay with standardized format was qualified for quantitation of human IgG Fc-containing biotherapeutics in matrices from commonly used preclinical species. Results from this assay compared well with those from traditional colorimetric immunoassays. Furthermore, result comparison between the universal and target-specific assays provided additional information on the effect of antidrug antibodies and in vivo drug catabolism. CONCLUSION This assay has wide applicability as a default bioanalytical approach in therapeutic candidate selection and preliminary pharmacokinetics evaluation during early-stage therapeutic development.

A practical approach to automate randomized design of experiments for ligand-binding assays

BACKGROUND Design of experiments (DOE) is utilized in optimizing ligand-binding assay by modeling factor effects. To reduce the analysts workload and error inherent with DOE, we propose the integration of automated liquid handlers to perform the randomized designs. RESULTS A randomized design created from statistical software was imported into custom macro converting the design into a liquid-handler worklist to automate reagent delivery. An optimized assay was transferred to a contract research organization resulting in a successful validation. CONCLUSION We developed a practical solution for assay optimization by integrating DOE and automation to increase assay robustness and enable successful method transfer. The flexibility of this process allows it to be applied to a variety of assay designs.

Strategic approaches for assessment and minimization of matrix effect in ligand-binding assays.

BACKGROUND Although substantial advances have been made in ligand-binding assays (LBA) for biotherapeutics in the past decade, there are still gaps that need to be addressed, especially in the context of understanding matrix effect and its root causes. Critical and in-depth characterization of matrix effect can provide valuable knowledge of the LBA limitations for proper results interpretation. RESULTS This article illustrates several strategic approaches with regard to identifying the root cause of matrix effect and practical solutions, including recognizing the confounding factors associated with matrix effect, selection of proper reagents to avoid matrix effect, and a systematic approach in dealing with matrix effect in method development and validation. CONCLUSION These strategic approaches have enhanced the management of matrix effect in LBA.

Current Trends in Ligand Binding Real-Time Measurement Technologies

Numerous advances in ligand binding assay (LBA) real-time measurement technologies have been made within the last several years, ranging from the development of novel platforms to drive technology expansion to the adaptation of existing platforms to optimize performance and throughput. In this review, we have chosen to focus on technologies that provide increased value to two distinct segments of the LBA community. First, experimentally, by measuring real-time binding events, these technologies provide data that can be used to interrogate receptor/ligand binding interactions. While overall the platforms are not new, they have made significant advances in throughput, multiplexing, and/or sensitivity. Second, clinically, these point-of-care (POC) technologies provide instantaneous information which facilitates rapid treatment decisions.

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics.

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.

Specific method validation and sample analysis approaches for biocomparability studies of denosumab addressing method and manufacture site changes.

Manufacturing changes during a biological drug product life cycle occur often; one common change is that of the manufacturing site. Comparability studies may be required to ensure that the changes will not affect the pharmacokinetic properties of the drug. In addition, the bioanalytical method for sample analysis may evolve during the course of drug development. This paper illustrates the scenario of both manufacturing and bioanalytical method changes encountered during the development of denosumab, a fully human monoclonal antibody which inhibits bone resorption by targeting RANK Ligand. Here, we present a rational approach to address the bioanalytical method changes and provide considerations for method validation and sample analysis in support of biocomparability studies. An updated and improved ELISA method was validated, and its performance was compared to the existing method. The analytical performances, i.e., the accuracy and precision of standards and validation samples prepared from both manufacturing formulation lots, were evaluated and found to be equivalent. One of the lots was used as the reference standard for sample analysis of the biocomparability study. This study was sufficiently powered using a parallel design. The bioequivalence acceptance criteria for small molecule drugs were adopted. The pharmacokinetic parameters of the subjects dosed with both formulation lots were found to be comparable.