The range of this analytical method was 0

The range of this analytical method was 0.05C10?g/mL. ADA ImmunoassayUNISA The Common Indirect Species-Specific Assay (UNISA) was performed as previously described (8). methods that quantified bound and/or unbound TP to ADA. The ADA response in the animals was classified into bad, low, medium, and high based on the magnitude of the response. The presence of ADA in samples led to discrepant TP measurements between the methods, especially at time points where the TP concentrations were low. This could be due to ADA interference to the accurate measurement of ADA-bound TP concentrations. The TP concentration at last time point (total, PK exposure INTRODUCTION Administration of therapeutic proteins (TPs) such as humanized, fully human monoclonal antibodies or recombinant proteins can induce the formation of anti-therapeutic antibodies, commonly known as anti-drug antibodies (ADAs) in both preclinical animals and clinical Methyl Hesperidin subjects (1). The ADA can impact pharmacokinetic (PK) exposure, bioavailability of TP, and the pharmacodynamic (PD) effects depending on their distinct characteristics. These may include epitope specificity (idiotype non-idiotype), magnitude (titers or relative concentration), timing (early late onset), maturity (persistent transient), and affinity (IgM IgG) of the ADA response (1,2). During the biotherapeutic development, we often utilize bioanalytical methods designed to measure TPs that are not bound to soluble ligands or targets using either a neutralizing anti-idiotypic pair or targeted ligands (electrochemiluminescence (ECL) in Fig.?1a). Such methods are frequently referred to as free methods for unbound TP measurement. Additionally, bioanalytical methods designed to measure TPs bound to ligands or targets are used and referred to as total methods (3) (ELISA and microfluidic platform (MFP) in Fig.?1a). While the term free can also refer to TPs that are not bound to the circulating ADAs targeted against complementary determining region (CDR) of TP (Fig.?1b), the term total does not necessarily reflect the measurement of all forms of TP-ADA immune complexes due to the complex formation differential binding sites (Fig.?1). In this study, the term unbound (TPu) refers to the serum concentration of TPs not complexed to any ADAs directed against any region of the TP. The term bound (TPb) refers to the serum concentration of TP-ADA-bound immune complexes irrespective of if ADA binds CDR or Fc portions of TP. Additionally, the term bound and unbound (TPu+b) refers to both TPu and TPb complexed with ADAs (Fig.?1). Physique?1c illustrates the various forms of TPb based on the binding of the ADA to either Fc or CDR regions of TP. The inability of the ELISA and MFP platforms to detect the ADA bound TP at Fc region and ECL platform to detect the ADA bound TP at CDR region has been shown. Initially, a reference colorimetric ELISA-based method was used to measure the TPu+b using different capture and detection Methyl Hesperidin antibody Methyl Hesperidin clones (clones A and B, respectively) specific to the Fc region of human IgG. Then, a microfluidic-based platform (referred in this report as MFP) that steps the TPu+b was used. In this method, monoclonal antibody specific to the Fc region of human IgG (clone A) is used as capture and detection. Finally, an ECL-based platform that steps the TPu concentration using a pair of anti-idiotypic antibodies as capture and detection (clones 1 and 2) was employed. Open in a separate windows Fig. 1 a Schematic diagram describing the three bioanalytical methods: ELISA, MFP, and ECL platforms. ELISA and MFP have anti-human IgG Fc (clone A) as the capture reagent, but use different anti-human IgG Fc clones (clone B in ELISA and clone A in MFP) for detection. b Simplified illustrations of the TP and TP with ADA immune complexes that ELISA, MFP, or ECL methods can detect. c Simplified illustrations of ADA or TP-ADA immune complexes that each method cannot detect if the ADAs are bound to the TPs where ADA binding sites overlap ZNF143 with that of detection antibodies. The immune complexes are identified as: (TP bound to ADA targeted at different binding sites) and is TP unbound to ADA. Detection reagents: (horse radish peroxidase labeled), (Alexa Fluor 647 labeled), or (ruthenium labeled) The formation of ADA can confound the PK data interpretation by either a direct interference in the bioanalytical method or by impacting the clearance profile of the TP (immune-mediated clearance). Several factors such as soluble ligand/target, nonspecific serum components, or ADA can affect the accurate measurement of TP (4). Often, the interference of ADA in the bioanalytical method to measure TP is usually evaluated during the pre-study method validation using the monoclonal or polyclonal antibodies against TP. These antibodies are commonly cited in the literature as positive controls for immunogenicity methods. However, these antibodies may not be truly representative of an ADA response in study animals. Hence, the ADA interference testing around the PK bioanalytical method generated during pre-study method validation may not directly relate to ADA impact. Consequently, the.