(T1230-02-07) Highly Specific Bridging ECL Assay for Small Peptide Drug Modalities and Their Use as an Alternative to a Direct ELISA Approach

Purpose: Herein we present the complete development of an ECL bridging assay for drug A, a small peptide drug. We present the critical development steps, including labeling of an under 15 amino acid polypeptide drug. This method is an alternative to the classic direct ELISA approach used for small drug modalities due to typical standard hinderance observed.

Methods: The method for drug A, a small ˂ 15 amino acids peptide drug, was designed to avoid the original assay’s non-specific binding issues. Method development was initiated with the investigation of the labeling potency of drug A, as the ECL assay design required at least one labeled reagent. Due to the small size, labeling proved challenging as it required unconventional considerations for the purification steps of the critical reagent. Following the reagent conjugations, both homogenous and semi-homogenous bridging assays were attempted. As purification steps were challenging, ruthenylation, required for a homogenous ECL assay, was abandoned, moving the assay to a semi-homogenous method. Excess free biotin in the labeled reagent was resolved by blocking steps instead of purification based on drug size while steric hinderance challenges were overcome by carrier conjugation, allowing the final assay bridging format. The resulting method was a robust, highly specific ADA assay.

Results: Biotinylation of drug A was performed at different ratios, with close consideration to avoid high excess of free biotin in the final critical reagent. Overall, the final ratio selected allowed good specific to non-specific responses. In the absence of purification to remove excess free labels, blocking of the critical reagent was performed by co-incubation of carrier protein to capture excess labels, resulting in low background noise. Steric hinderance, due to the small size of drug A, was overcome with the use of carrier-conjugation, allowing the bridge formation between the two drug molecules. In the absence of carrier, the assay exhibited nearly no specific response, while the optimized carrier step boosted the signal to noise ratios of the high positive control above 20. The resulting assay sensitivity was under 20 ng/mL. The final assay exhibited no non-specific binding in human serum samples, robust precision and high sensitivity. Furthermore, based on the bridging nature of the assay, it required a low total MRD in accordance with regulator agencies guidance.

Conclusion: Small drug modalities are typically performed via direct ELISA methods due to their size limitations. Although widely used, these direct methods present multiple challenges and are typically less robust than bridging assays. Herein we demonstrate the use of a highly specific and sensitive semi-homogenous ECL bridging assay for small drug immunogenicity analysis. We provide data, insight and critical considerations for the development and validation of these methods.


Impact of Drug-BSA conjugation on assay feasibility. The semi-homogenous bridging assay was tested using both a native Drug capture format and a Drug-BSA conjugate capture format. Under native conditions, steric hinderance and drug availability for capture render the assay non-viable. BSA conjugation proved to be critical for assay feasibility.


Pre-incubation of Biotinylated peptide drug to reduce non-specific background caused by free biotin. Based on small drug size ( < 1.5 kDA), standard purification procedures to remove excess biotin reagent cannot be used. To bypass complex HPLC purification steps, a 10 to 20-minutes preincubation, in assay buffer, step was added to biotinylated drug preparation to capture excess free biotin. This simple step reduced background by over 50%, further increasing assay sensitivity.


Robustness of Drug-Biotin conjugation in the absence of purification step to remove excess free biotin. All conjugation steps were carried out in parallel, except for preparation A.