As one might imagine, selecting the right assay to assess bioactivity during the entire life cycle of these follow on biologics —from early development until market-released product—is difficult and requires a lot of expertise, in part because there are so many different ways and stages of assessing these compounds in vitro (see image below). Consider Rituximab, a mAb approved for the treatment of lymphoma and rheumatoid arthritis. Rituximab works by recognizing a specific protein, CD20, that sits on the surface of B cells. When the mAb targets the protein, it signals the human immune system to kill it.
Four different in vitro tests designed to analyze drug effectiveness—three that reflect potential MoAs and one measuring binding to target—can provide different methods of characterizing how effective biobetters of Rituximab with an altered glycosylation pattern are compared to the innovator.
In a typical example of testing a biobetter of Rituximab, the binding assay showed that the modified mAb was able to bind to CD20—showing that it recognized and docked its target like the innovator mAb. Three other assays that measure how well the mAb reflects the in vivo function on the immune system, mainly how well they target tumor cells, found increasing levels of antibody-dependent cellular cytoxicity (ADCC)—usually an indication of drug effectiveness and potency—and lower levels of complement dependent cytoxicity (CDC) —suggesting lower toxic side effects. The behavior of the follow-on biologic in a direct apoptosis test is moderate but with tendency to enhanced efficacy compared to the innovator mAb.
These different assays all capture important pieces of information about the in vitro evidence for in vivo efficacy in inducing unwanted immune reactions, which goes a long way in determining early on whether a mAb is suitable for further development.
Choosing the most appropriate in vitro test for routine testing throughout the life cycle of the mAb requires an intimate knowledge of how well the drug works in vivo. Or to return to that car analogy, it means lifting the hood and fully vetting the chain reaction of events that make a particular drug run smoothly.