Peptides represent a large and growing sector of pharmaceutical products that are used to treat a variety of diseases. The immunogenicity risk of peptides is generally lower than protein therapeutic products, but immunogenicity must be evaluated in compliance with regulatory requirements and to mitigate product risk. Peptide anti-drug antibody (ADA) assays present several unique challenges to method developers. Specifically, labeled reagents can be difficult to generate and positive control antibodies may not react strongly to the peptide.

One particularly difficult challenge during peptide ADA assay development is the generation of conjugated peptide reagents. ADA assays need at least one, preferably two, labeled drug conjugate reagents to ensure assay specificity. The most common label used to capture drug: ADA complexes is biotin. A reporter molecule (e.g., ruthenium) is then conjugated to the drug to detect drug:ADA complexes in a conventional bridging assay format. Due to the low molecular weight of peptides and the relative paucity of primary amines compared with proteins, using standard N-Hydroxysuccinimide (NHS) conjugation chemistry may not be feasible and alternative labeling approaches must be utilized.

One approach that has facilitated labeling and positive control binding is the use of an extended spacer arm on the conjugate label. In direct comparisons performed in the laboratory, low molecular weight drugs conjugated with long spacer arms had better dynamic signal range and relative sensitivity compared with drugs conjugated with standard length spacer arms. Reporter labels also may not be efficiently conjugated on peptides. When labeling difficulties arise, it may be beneficial to investigate alternative labeling strategies, such as label incorporation during peptide synthesis.

The conjugated label on the drug also may interfere with positive control antibody binding. This can impact the apparent sensitivity of the assay and can be mitigated in several ways. Alternative assay formats may be investigated. For example, the assay format can be changed from a bridging format to a passive coat capture step, that allows the capture reagent to remain unconjugated. Using a longer spacer arm or conjugation to a specific site during synthesis also can mitigate potential steric hindrance to antibody binding.

It is also important to screen multiple positive control antibodies, preferably a polyclonal antibody, to ensure robust binding to the labeled drug. Peptides are not strongly immunogenic; therefore, it may be difficult to generate an antibody that reacts with the required sensitivity in the ADA assay. Often, antibodies that have screened positive in a direct ELISA after they are produced do not react in the bridging ADA assay format. Poor positive control performance sometimes can be improved by affinity purification, but this is not always effective.

Peptide ADA assays present a unique set of challenges to the bioanalytical method development scientist. These difficulties can be overcome via non-standard conjugation techniques, assay format changes and thorough careful reagent evaluation. Collaboration between bioanalytical, clinical development and manufacturing scientists during peptide ADA method development facilitates the development of strategies that are appropriate for each peptide’s chemistry and immunogenic profiling.