Abstract Accurately modeling antibody-antigen interactions requires distinguishing intrinsic binding affinity (“ protein-interaction ”) from protein biophysical properties (“ protein-quality ”), including folding, stability, and expression. However, high-throughput mutational measurements commonly used to train and benchmark computational models often conflate these effects, obscuring the true determinants of molecular recognition. Here, we present an experimental and analytical framework to disentangle protein-interaction effects from protein-quality effects in single-domain antibody (VHH)-antigen binding. Using a large-scale deep mutational scanning (DMS) dataset spanning four VHH-antigen complexes, with single and double mutations in both partners, we introduce control binders to quantify protein-quality changes independently of protein-interaction . This enables decomposition of experimentally measured affinity into protein-interaction and protein-quality components at scale. Leveraging the disentangled dataset, we evaluated state-of-the-art structure- and sequence-based models for protein-quality and protein-interaction prediction and show that their performance largely reflects protein-quality rather than protein-interaction effects. Our results highlight a major confounder in current datasets and suggest that accounting for protein-quality will be essential for training next-generation affinity-prediction models. Nomenclature Antibody related terms Primary VHH : The VHH of a VHH-antigen complex for which the paratope and the epitope weremutated. Control VHH : A second VHH that binds to the same antigen as the primary VHH but has non-overlapping epitope positions and therefore does not bind to any of the mutated antigen positions. Affinity-related terms Real Affinity : “The strength of the interaction between two […] molecules that bind reversibly (interact)” 1 . In the context of antibody-antigen binding, it quantifies interactions between active proteins (which are expressed and correctly folded 2 and are therefore functionally and biologically active (see below). It is commonly quantified by the equilibrium dissociation constant, K D . Observed affinity (°K D ): The interaction strength experimentally measured between two molecules. Unlike real affinity, this value is confounded by the biophysical properties of the individual binding partners, specifically their folding, stability, and expression levels. Consequently, the observed affinity often differs from the real/intrinsic affinity if a significant fraction of the protein population is inactive 3 . NOTE: Unless otherwise specified, °K D is reported in - log10 space. For example, a °K D of -9 corresponds to 10 -9 M or 1nM. Change in observed affinity (Δ°K D ) : The shift in the observed affinity between two proteins upon mutation, reported as the log 10 -transformed fold change. A value of 1 reflects a 10-fold difference, a value of 2 a 100-fold difference, etc. This aggregate change resolves into two distinct biophysical components 2, 4 : Protein-interaction change: The change in the intrinsic thermodynamic affinity between the two binding partners, each in its active state (i.e., the specific change in interface Gibbs free energy because both enthalpy and entropy are considered). Protein-quality change: The change in the fraction of the mutated protein population that is biologically active - meaning it is expressed, correctly folded, and stable 2, 5 . Folding: The process that guides the polypeptide chain toward its native conformation, which is a prerequisite for forming a functional binding site. Stability: The thermodynamic capacity to maintain the folded structure over time and under physiological conditions. Stability (decrease in Gibbs free energy from the unfolded to the folded state) ensures the binding interface remains intact and prevents competing processes such as aggregation 6 . Expression: The steady-state abundance of the protein. This is largely dependent on proper folding and stability, as cellular quality control mechanisms degrade proteins that fail to fold or remain stable at functional concentrations. Change in relative affinity (ΔΔ°K D ) : the differen
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