New insights into the intrinsic tryptophan fluorescence emission spectra of IgG antibodies and the impact of protein unfolding and aggregation.

Intrinsic tryptophan (Trp) fluorescence emission (ITFE) spectroscopy is widely used in biologics development, yet interpretation remains challenging for proteins with more than one Trp, such as antibodies, and is often limited to bulk spectral shifts. For better understanding and more detailed characterization of mAbs by ITFE, we analyzed an in-house therapeutic IgG1 (mAbT) and the National Institute of Standards and Technology (NIST mAb) reference IgG1. Human serum albumin (HSA) was used as a single-Trp protein control. Steady-state, second-derivative and time-resolved fluorescence measurements combined with Molecular Dynamics simulations enabled site-resolved interpretation of ITFE signals. A limited number of dominant Trp residues, primarily near the complementary-determining regions (CDRs) in the VH domains, were identified as the main contributors to the overall emission. Long fluorescence lifetimes (>5 ns) correlated with moderate solvent accessibility (around 25%) and low quenching propensity. The direction of spectral shifts during unfolding and aggregation was governed by the environment of the individual VH domain Trp. The increase in fluorescence intensity during denaturation was primarily attributed to unfolding and was further enhanced by thermal stress induced aggregation. Protein aggregation induced by interfacial stress produced similar spectral signatures in mAbT and HSA, indicating comparable effects on ITFE. This study demonstrates that integration of ITFE with molecular dynamic simulations allows for mechanistic interpretation beyond just an empirical description. By thoroughly interpreting ITFE spectra, one can obtain information about spectral properties of variable Trps residues in IgG1 molecules and gain valuable insights into protein denaturation mechanisms.