Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development

Abstract

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed.

The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.

Introduction

Most protein therapeutics are known to be immunogenic and more often than not antibodies generated to these therapeutics have no significant impact on their safety and efficacy (Koren et al., 2008, Rosenberg and Worobec, 2004, Rosenberg and Worobec, 2005, Shankar et al., 2007, Wang et al., 2008, Worobec and Rosenberg, 2004). However on rare occasions there can be serious clinical consequences due to the development of anti-drug antibodies in patients (Arepally and Ortel, 2010, Casadevall et al., 2005, George and Aster, 2009, McKoy et al., 2008). On other occasions the anti-drug antibodies may have an impact on safety leading to minor adverse events (Rosenberg and Worobec, 2004) or have an impact on clinical efficacy/cause adverse reactions (Baert et al., 2003, Malucchi et al., 2004, Subramanyam, 2008). Therefore, it is important to develop sensitive and robust immunogenicity assays that can detect clinically meaningful anti-drug antibodies in the presence of circulating drug (EMEA, 2007, EMEA, 2009, U.S. Department of Health, 2009, Mire-Sluis et al., 2004, Shankar et al., 2008) in order to fully evaluate and understand the impact of anti-drug antibodies and consider this aspect in defining the path for clinical development of protein therapeutics.

Majority of protein therapeutics are humanized antibodies. In general, the humanized antibody drugs have long half-life (typically 10–20 days) and are often administered chronically at high dose levels without a drug wash-out period. Therefore, during clinical development and even in clinical practice, samples collected to evaluate the status of anti-drug antibodies are expected to contain high levels of circulating drug. This is especially true in preclinical toxicology studies where drug is administered at multiple-fold above the clinical dose to evaluate toxicity (Ponce et al., 2009). In such settings the presence of drug in samples interferes with the detection of anti-drug antibodies. It is recognized that drug tolerance and sensitivity parameters reported for a given immunogenicity assay may vary depending on the positive control antibody utilized in the assay. Nevertheless, evaluation of these parameters in the context of defined positive control is important in order to develop immunogenicity assays with optimal drug tolerance and sensitivity during the assay development phase before applying it in the clinical setting (EMEA, 2007, EMEA, 2009, U.S. Department of Health, 2009, Koren et al., 2008, Shankar et al., 2008). However, it is worth noting that the optimal conditions for detection of a diverse array of ADAs present in test samples may vary. Therefore, the data from immunogenicity assessment should take into consideration the limitations of positive controls.

Over the last few years, sample pre-treatment methods with acid, for dissociation of drug:anti-drug complexes along with several technology platforms have been developed and optimized to overcome drug interference in immunogenicity assays (Bourdage et al., 2007, Liang et al., 2007, Loyet et al., 2009, Patton et al., 2005, Smith et al., 2007, Spengler et al., 2009, White et al., 2008, Zhong et al., 2010). ELISA is the most commonly used assay platform even though the ECL assay platforms have become increasingly the format of choice in recent years (Liang et al., 2007, Loyet et al., 2009, White et al., 2008). However, many laboratories using the ECL platform were recently affected by the sudden withdrawal of the Bioveris ECL platform from the market place. Therefore, it became clear that development of alternative solutions that are not dependent on technology platforms from sole source providers is essential in order to achieve consistent and robust assay performance desired for immunogenicity assessment during the development life-cycle of a biologic therapeutic that may span several years to a decade (or more).

In this manuscript, we describe evaluation of several technology platforms and methods for the development of an immunogenicity assay with the goals of achieving desired drug tolerance and sensitivity. In addition, we identified an assay format for which critical reagents/equipment are commonly available and do not depend on a sole source provider. Further, we developed a method that could be universally adopted and implemented in a wide range of laboratories. In the case study presented, we also implemented DOE for optimization of sample acidification conditions leading to improved assay drug tolerance and developed the approach to overcome soluble target interference. The experimental data presented highlight important assay parameters to consider and optimize in developing a robust immunogenicity assay platform, in particular, the solution ELISA format, which can be broadly applied across many classes of protein therapeutics. It should be noted however, that the optimal conditions (including acid treatment) for a given immunogenicity assay may vary depending on the characteristics of the therapeutic, reagents and the repertoire of antibodies present in the test samples.