Johanna Mora

Application of the Gyrolab™ platform to ligand-binding assays: a user’s perspective

In many areas of drug discovery and development, scientists are in a constant search for methods and platforms to reduce assay time and cost. The Gyrolab™ microfluidics platform that we describe here promises to deliver faster ligand-binding assays with lower reagent and sample consumption, while maintaining good accuracy and precision. Due to its limited track record, we evaluated its performance on assays currently used to support pharmacokinetic and immunogenicity studies, and detection of host cell protein impurities in samples from biotechnology processes. This article summarizes our preliminary conclusions about the utility of the Gyrolab microfluidics platform from Gyros AB.

Next generation ligand binding assays-review of emerging technologies' capabilities to enhance throughput and multiplexing.

The purpose of this manuscript is to provide a summary of the evaluation done by the Throughput and Multiplexing subteam on five emerging technologies: Single molecule array (Simoa™), Optimiser™, CyTOF® (Mass cytometry), SQIDLite™, and iLite™. Most of the information is presented with a minimum amount of published data and much is based on discussions with users and the vendor, to help provide the reader with an unbiased assessment of where the subteam sees each technology fitting best in the bioanalysis of large molecules. The evaluation focuses on technologies with advantages in throughput and multiplexing, but is wide enough to capture their strengths in other areas. While all platforms may be suited to support bioanalysis in the discovery space, because of their emergent nature, only Optimiser and SQIDLite are currently ready to be used in the regulated space. With the exception of Optimiser, each instrument/technology requires an up-front investment from the bioanalytical lab that will need justification during capital budget discussions. Ultimately, the platform choice should be driven by the quality of data, project needs, and the intended use of the data generated. In a time- and resource-constrained environment, it is not possible to evaluate all emergent technologies available in the market; we hope that this review gives the reader some of the information needed to decide which technology he/she may want to consider evaluating to support their drug development program in comparison to the options they already have in their hands.

Biotherapeutic bioanalysis: a multi-indication case study review

A thorough understanding of the structure and biology of a biotherapeutic is crucial to defining a suitable strategy for pharmacokinetic characterization in proof-of-concept disease models, toxicology species as well as the healthy and disease indication patient populations. This manuscript summarizes parameters that impact bioanalytical strategy for over 50 biotherapeutics indicated for the treatment of oncology, rheumatoid arthritis, allergy, multiple sclerosis, hematology, metabolism and infectious disease. We have addressed numerous therapeutic modalities including chimeric, humanized and fully human monoclonal antibodies, replacement proteins, peptides and fusion proteins, including polyethylene glycol and Fc fusions, as well as antibody-drug conjugates. With the rapid evolution of biotherapeutics over the last 20 years and the contraction of the pharmaceutical and biotechnology labor force, efficient workflow management becomes a crucial bioanalytical component. Thus, we have also addressed new technologies that have demonstrated either increased throughput or enhanced characterization, including Meso Scale Discovery, Gyrolab and affinity MS.

Development and validation of an LC–MS/MS assay for the quantitation of a PEGylated anti-CD28 domain antibody in human serum: overcoming interference from antidrug antibodies and soluble target

AIM To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study. MATERIALS & METHODS The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28. RESULTS & CONCLUSION The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.

Addressing matrix effects in ligand-binding assays through the use of new reagents and technology.

BACKGROUND Ligand-binding assays (LBAs) used in the quantification of biotherapeutics for pharmacokinetic determinations rely on interactions between reagents (antibodies or target molecule) and the biotherapeutic. Most LBAs do not employ an analyte extraction procedure and are susceptible to matrix interference. Here, we present a case study on the development of a LBA for the quantification of a PEGylated domain antibody where matrix interference was observed. The assay used to support the single ascending dose study was a plate-based electrochemiluminescent assay with a lower limit of quantification of 80 ng/mL. To meet sensitivity requirements of future studies, new reagents and the Gyrolab™ Workstation were evaluated. RESULTS Assay sensitivity improved nearly threefold in the final method utilizing new antibody reagents, a buffer containing blockers to human anti-animal antibodies, and the Gyrolab Workstation. CONCLUSION Experimental data indicate that all factors changed played a role in overcoming matrix effects.

High-sensitivity detection methods for low-abundance RNA species: applications for functional genomics research

Gene expression analysis has facilitated a more complete understanding of the molecular biology of cellular processes and how variations of RNA expression are useful for the classification of various diseases. Furthermore, recent analysis of a variety of noncoding RNAs, such as microRNAs, has demonstrated that these RNAs play an important role in many cellular events, including cell differentiation and death, and may also serve as biological markers for disease. Besides helping in the understanding of diseases, RNA analysis is used in drug discovery, patient prognosis and treatment evaluation. One obstacle left to overcome is the amount of material required for the analysis, particularly when trying to extract information from precious, limited, clinical samples. Here we review the many approaches scientists take to either amplify the amount of RNA or amplify the signal generated from small amounts of RNA.

Evaluation of Acoustic Membrane Microparticle (AMMP) Technology for a Sensitive Ligand Binding Assay to Support Pharmacokinetic Determinations of a Biotherapeutic

Achieving the required sensitivity can be a challenge in the development of ligand binding assays for pharmacokinetic (PK) determinations of biotherapeutics. To address this need, BioScale’s Acoustic Membrane Microparticle (AMMP) technology was evaluated for the quantification of a PEGylated domain antibody (dAb) biotherapeutic. Previous uses of this technology had shown utility in biomarker and process development applications and this is the first application, to our knowledge, for PK determinations. In this evaluation, AMMP was capable of delivering a sensitivity of 0.750 ng/mL, which surpasses the sensitivity requirements for the majority of assays to support PK determinations. This evaluation demonstrates that this emerging technology has the ability to produce the required sensitivity, reproducibility, and selectivity needed to meet the industry’s standards for PK analysis.

Pharmacokinetic, Pharmacodynamic, and Safety Profile of a Novel Anti‐CD28 Domain Antibody Antagonist in Healthy Subjects

We report pharmacokinetics, pharmacodynamics, and safety of a novel anti‐CD28 domain antibody antagonist (lulizumab pegol) in healthy subjects following single‐ or multiple‐dose administration. A minimal anticipated biological effect level approach was used to select a 0.01 mg starting dose for a single‐ascending‐dose (SAD), double‐blind, first‐in‐human study. Part 1 included 9 intravenous (IV; 0.01‐100 mg) and 3 subcutaneous (SC; 9‐50 mg) doses or placebo. In part 2, a keyhole limpet hemocyanin (KLH) immunization was performed in 16 subjects/panel, who received 1 of 3 IV doses (9‐100 mg) or placebo. In a double‐blind, multiple‐ascending‐dose (MAD) study, subjects received SC lulizumab 6.25 mg every 2 weeks, 12.5 mg weekly, 37.5 mg weekly, or placebo. Among 180 treated subjects, 169 completed the studies. Peak concentrations and areas under the curve from time 0 to infinity increased dose proportionally. Estimated SC bioavailability was 68.2%. Receptor occupancy of approximately ≥80% was maintained for ≥2 weeks at ≥9‐mg doses (SAD) and throughout the dosing interval (MAD). IV doses ≥9 mg inhibited antibody production against KLH for 2 weeks. No significant cytokine or immune cell changes were observed. No immunogenicity responses persisted, and there was no correlation to adverse events. Headache occurred in 21 SAD and 4 MAD subjects receiving lulizumab; in the MAD study 5 lulizumab subjects experienced infections. Lulizumab IV or SC was safe at all doses studied, without evidence of cytokine release.

Emerging technologies for biotherapeutic bioanalysis from a high-throughput and multiplexing perspective: insights from an AAPS emerging technology action program committee

This manuscript aims to provide insights and updates on emerging technologies from a throughput and multiplexing perspective and to update readers on changes in previously reported technologies. The technologies discussed range from nascent (ultrasensitive Cira, Intellicyt®, Dynaxi and Captsure™) to the more established (Ella and SQIDlite™). For the nascent technologies, there was an emphasis on user interviews and reviews, where available, to help provide an unbiased view to our readers. For the Ella, a review of published user data as well as author and other user experiences are summarized. Due to their emergent nature, all the technologies described are applicable in the early drug development stage, may require an upfront investment of capital and may not perform as expected.

Ligand Binding Assays in the Regulated Bioanalytical Laboratory

This chapter covers the use of ligand binding assays (LBAs) in a regulated bioanalysis environment. The various platforms employed in the analysis of protein therapeutics are varied and selection of the appropriate platform for a particular application is highlighted. The suitability, procurement and implementation of critical reagents are of major importance for LBAs and considerations for their careful selection and use are provided. In addition, various strategies, including Design of Experiments (DoE) are discussed as a means of optimizing assay conditions to produce robust and rugged assays during method development. The key parameters for the validation of LBAs are discussed in depth as well as many considerations, including automation, that can be employed when analyzing study samples in a production environment.