Any treatment with biotherapeutic drugs can lead to an immunogenic response in the form of Anti-Drug Antibodies (ADA). ADA may inhibit the effect of the drugs and can generate unfavourable immune response in patients. In this blog we describe the development of three variants of generic rodent ADA assays for the Gyrolab® platform and assess the impact of varying drug concentrations on the detection of ADA in preclinical species.
Treatment with biotherapeutic drugs can lead to an immunogenic response in the form of anti-drug antibodies (ADA). The effect of the drug may be hampered or even eliminated by ADA, and patients’ symptoms can range from mild (skin reaction) to adverse (fatal allergic reaction).
ADA measurements should be evaluated in pre-clinical studies when
Since samples may not be evaluated for immunogenicity until called for by one of these, developing a drug specific assay for each biotherapeutic candidate can be time consuming. Having a generic method for ADA detection will save time and may also allow for testing for pre-existing antibodies in preclinical animals.
In this blog we describe the development of three variants of generic rodent ADA assays for the Gyrolab platform and assess the impact of varying drug concentrations on the detection of ADA in preclinical species.
Drug-specific bridging assays for ADA detection using antibody drug labeled with biotin (capture) and Alexa Fluor™ 647 (detection) require that the ADA dissociate from unlabeled drug molecules. This is typically achieved by an acid treatment step.
The generic ADA assay in rodents instead was designed to detect the ADA-drug complex by using an anti-human IgG capture reagent and an anti-rodent IgG detection antibody, targeting different parts of the ADA-drug complex. The assay can therefore be used for any human antibody drug (containing a κ light chain) in rodents.
Microfluidic immunoassay technology utilizes automated delivery of samples and reagents by centrifugal force and capillary action. Analytes are captured by biotinylated reagents on streptavidin beads in flow-through 15 nL-columns on discs and detected by Alexa Fluor™ 647 labeled detection reagents.
The generic Gyrolab ADA assay was assessed in mouse serum using the biotherapeutic drug golimumab and mouse anti-human polyclonal antibodies (pAb) as ADA surrogates. The results showed that the generic ADA assay selectively detected the immune complex and no signal above background was detected in absence of biotherapeutic drug.
Signal to background (S/B) results for select concentration of pAb (ADA-surrogate) and positive control (PC) in presence or absence of drug.
Since the generic ADA assay selectively detects the immune complex, different strategies need to be employed to detect ADA in the absence of drug. To address this, two additional assays were developed; the 4-step method detecting free ADA, and the Capture Mix method detecting free and complexed ADA. Adding biotherapeutic to the column prior to the sample as shown, enables detection of ADA in absence of drug while retaining high drug tolerance. This demonstrates the versatility of the generic ADA as a tool for all timepoints of preclinical immunogenicity assessment, including washout experiments.
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