Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays.

Robert J Kubiak,Sophia V Levitskaya,Wendy I White,Nancy Lee,Varghese Abraham,William R Franch,Peter F Akufongwe,Surekha R Krishnan,Christopher J Larkin,Yuan Zhu

doi:10.1155/2016/1485615

Abstract

Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.

Highlights

Protein-based therapeutic drugs have an inherent potential to elicit undesired immune response in human subjects
Based on this report and our own accumulated experience with the ECL immunogenicity assays, we evaluated reagent aggregation and its impact on anti-drug antibody (ADA) assays focusing on the ruthenylated conjugate as being more critical for assay performance than its biotinylated counterpart
Chromatograms and results of the High Performance Size-Exclusion Chromatography (HPSEC) analysis of the ruthenylated antibody stored in histidine-sucrose buffer (HSB) or in phosphate-buffered saline (PBS) and subjected to 0, 1, 3 and 5 freeze/thaw (F/T) cycles are shown in Figure 1 and Table 1

Summary

Introduction

Protein-based therapeutic drugs have an inherent potential to elicit undesired immune response in human subjects. Regulatory agencies mandate testing for the presence of ADA in all phases of clinical development and require assessments of potential impact on safety, drug exposure, and efficacy [1,2,3,4,5]. Assay, which typically provides high levels of sensitivity and drug tolerance combined with ability to detect most ADA isotypes. In this format, the ECL signal is generated by ADA simultaneously binding two different conjugated forms of the drug: one biotinylated and one conjugated with a ruthenium complex. Conjugation chemistry requires the protein being labeled to be in a buffer free of primary and secondary amines. The use of PBS for long-term storage of proteins is fairly common as evidenced by the many commercially available antibodies formulated in PBS and stored at −20∘C or below

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Discussion

Conclusion