Stereospecific interactions between histidine and monoclonal antibodies

Histidine is a frequently used buffer in the final formulation of many commercialized monoclonal antibodies (mAbs), with histidine helping to stabilize the antibody during storage in addition to its buffering function. The objective of this study was to examine the stereospecificity of any histidine‐antibody interactions using a combination of experimental studies and molecular dynamics simulations. Isothermal titration calorimetry provided evidence of weak stereospecific interactions, with the antibody showing approximately two to four additional interaction sites for d‐ versus l‐histidine. The greater interactions with d‐histidine were confirmed by measurements of the net protein charge using electrophoretic light scattering. The reduction in the net negative charge of the antibody in d‐histidine led to significantly different behavior during diafiltration due to Donnan exclusion effects. Molecular dynamics simulations corroborated the presence of additional d‐histidine interaction sites. These results provide the first demonstration of weak stereospecific interactions between l‐ and d‐histidine and a mAb and the implications of these interactions for antibody formulation. The authors examined the stereospecificity of histidine‐antibody interactions using a combination of experimental and theoretical approaches. Diafiltration experiments showed significantly different pH profiles in d‐ versus l‐histidine, which was directly related to a difference in net protein charge (confirmed by electrophoretic mobility data). Molecular dynamics simulations corroborated the presence of additional d‐histidine interaction sites. These results provide the first demonstration of weak stereospecific interactions between a monoclonal antibody and histidine, which is widely used as a buffer component and excipient.