Abstract: Objective To evaluate the stability and binding efficiency of CH3-CH3 heterodimer assembly generated by knobsinto-holes (KIH) method, and reveal the contribution of interface amino acids to the binding of CH3-CH3 heterodimer. Methods Molecular dynamics simulations coupled with molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) analysis were performed to study the interaction of CH3-CH3 heterodimer produced by KIH method. The binding efficiency of heavy-heavy chains was examined by SDS-PAGE analysis. Results Compared with wild-type (WT) at 355 K temperature, the thermal stability of CH3-CH3 heterodimer generated by KIH decreased. Root-mean-square deviation (RMSD) of backbone atoms in WT and KIH antibody CH3 domain were about 1.0 Å and 2.0 Å. Significant root-mean-square fluctuation (RMSF) increases could be observed at 3-27, 45-52 and 70-80 residues of the knob part. Moreover, CH3-CH3 heterodimer through KIH exhibited higher relative free binding energy than WT (107.008 2 kJ/mol vs 79.864 9 kJ/mol). Ten residues located at the CH3 binding interface produced by KIH had little positive contribution to the binding of CH3-CH3. In line with these findings, the binding efficiency of CH3-CH3 heterodimer was just about 80%, which was significantly lower than WT. Conclusion The thermal stability and binding efficiency of CH3-CH3 heterodimer produced by KIH method are not as good as WT. Residues lack of stability and had little contribution to the binding have been found. Our findings will be beneficial to the further optimization of CH3-CH3 heterodimer and improvement of the stability of bispecific antibodies designed by KIH method.