You are invited to view and download the poster, originally presented in Orlando, FL during the 15th annual WRIB in 2021
Salt precipitation: the method used for the drug tolerance assays creates a salt precipitation not long after the addition of Acid at pH 2.5 to the MgCl2 sample dilution. In order to test what was precipitating and if the precipitate went fully back into solution, the following method was used. In a non-binding plate samples containing either BSA, serum, plasma, sucrose, ovalbumin, 1x PBS, glucose, fibrogen, fibrogen extracted matrix, NaCl, histidine, or IgG (human IgG1-4) were added at equal volumes to 2 M MgCl2. To this solution 400 mM Glycine was added at 1:2.5 dilution for a total of 1:5 dilution. The plate(s) were read at 490, 540, 656 and 280 nm as a kinetic read every 5 minutes for 30 minutes. After the precipitate was formed 250 mM Tris +3% BSA, pH 8.0 was added to return the solution to a neutral pH range. Kinetic reads at 490, 540, 656 and 280 nm were performed every 5 minutes for 30 minutes.
Drug Tolerance: Multiple experiments with a non-drug tolerant monoclonal anti-drug antibody were performed using 3 M MgCl2 in comparison to the acetic acid dissociation in the original method. The assay was performed using a 1:5 dilution of sample in either 300 mM Acetic Acid or 3 M MgCl2 for a 30 minute incubation prior to neutralization (250 mM Tris, pH 8.0 for the acetic acid and 1X PBS+Blocking buffer for the MgCl2). The MgCl2 dissociation did have a higher RLU signal overall for the antibody, as expected based on the HISDA paper by Jordan et. al 1; however, we did not have a significant increase in drug tolerance using MgCl2 alone. It is of note that the HISDA method uses 4 M MgCl2. In our laboratory we have found that high concentrations of MgCl2 caused significant loss in antibody signal overtime in this assay. Thus selection of the appropriate MgCl2 concentration is critical for assay development.
The low drug tolerance led to the development of a stepwise procedure treating first with MgCl2 then with Acid (developed in multiple assays with differing acids, drugs, and ADAs). Drug tolerance was improved from none to minimal drug tolerance up to concentrations of 1000 μg/mL for the drug evaluated depending on antibody concentration and method of removing the unbound drug.
This novel two-step acid and ionic dissociation method uses salt precipitation to aid in removing the newly dissociated drug from the system. The temporary shift to the tertiary and quaternary structures improves the drug tolerance, allowing the remaining free ADAs to be added to a variety of assay types for antibody detection. This method has been successful in achieving drug tolerance without compromising assay sensitivity for the monoclonal assay and increased sensitivity for the polyclonal assay as non-specific binding was decreased.
1.Gregor Jordan 1, Alexander Pohler 1, Florence Guilhot 2 et al. – High ionic strength dissociation assay (HISDA) for high drug tolerant immunogenicity testing – Bioanalysis (2020) 12(12), 857866″.
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