Written by María Gerpe, PhD
July 23, 2021
As proteins are assembled, they fold into different structural orders: from primary to quaternary. The exact sequence of the primary structure (the amino acid sequence) will dictate how a protein will fold and therefore function. The importance of the primary structure has been noted in several studies, where changes in the original amino acid sequence have resulted in affinity problems, binding disruption, reduced half-life, and higher aggregation odds (Boyd et al., 2018). As commercial, therapeutic, and diagnostic antibodies are produced in animal or cellular systems, they are often prone to errors, and require validation (Lamanna et al. 2017). Problems with the original sequence stem from a variety of errors within replication, transcription, translation, and post-translation (Scott et al. 2014).
Scientists often focus on specificity-minded testing because reproducibility-oriented validation is often thought to be blind to problems such as cross-reactivity (Uhlen et al. 2016; Edfors et al. 2018). Conventional methods that test for specificity include immunohistochemistry, WB, and ELISAs, to name a few. Though these methods may highlight off-target effects, or binding issues, they do not output results that indicate whether additional chains, or another antibody is present.
Often, additional chains are thought of as non-functional, but they can actually interfere with experiments, and cause cross-reactivity problems. In a recently published study, Bradbury et al. (2018) analyzed sequencing data from 185 hybridomas chosen at random. They found that 32% of these hybridomas expressed additional functional light and heavy chains in addition to the monoclonal antibody they were thought to exclusively express. These variants have the potential of being immunogenic and therefore must be purified from the final antibody product prior to clinical development (Boyd et al. 2017).
During our REmAb® protein sequencing projects, we have also often found the presence of additional chains in purified samples whether from hybridomas or other approaches. After reporting such unexpected findings, our clients always ask us to sequence the other competing chains and antibodies so they can then verify that their ELISA, IHC and WB results corroborate to the appropriate antibody. As a result, they feel empowered with the knowledge we provide to have a more efficient optimization journey by avoiding cross-reactivity and reproducibility problems down the road.