Purpose: Conjugated reagents play a critical role in assay performance of Immunogenicity assay. The ECL assays for the detection of ADA mainly uses drug conjugated to Biotin and Ruthenium as the capture and reporter protein. Aggregation of these conjugated reagents leads to variation in the assay which in turn leads to detection of false negatives in the clinical samples with altered pharmacokinetic profile. The purpose of the presentation is to demonstrate the importance of storage condition of the conjugated reagents on immunogenicity assessments.
Methods: Drug Biotin was conjugated with the challenge ratio 1:12 and Drug Ruthenium was conjugated with a challenge ratio of 1:10. In the first approach, these conjugated reagents were dispensed into aliquots of 1mL and were deep frozen. Before usage each aliquot was thawed at room temperature, dispensed into single use aliquots and again deep frozen until usage contrary to the approach taken earlier by the conjugation Scientist.
Results: When the conjugated reagents were subjected to multiple freeze-thaw cycles were tested in the assay, the response of naïve drug-antibody samples varied from 90-500 RLUs. Whereas the single use aliquoted regents showed lesser variation of 90-150 RLUs in the assay consistently.
Conclusion: Repeated freeze-thaw cycles can damage protein structures, which can interfere with study protein kinetics. Upon the fast freezing small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the protein.