UNDERSTANDING THE RELATIONSHIPS BETWEEN LOCAL DYNAMICS AND PHYSICAL STABILITY OF IgG1 ANTIBODIES: EFFECTS OF EXCIPIENTS, SALTS AND MUTATIONS

Abstract

Immunoglobulins are large multi-domain and multi-functional proteins that are increasingly being developed as biologic drugs to bind to a wide variety of therapeutic targets. The immunoglobulin G1 class of molecules represents the largest class of therapeutic monoclonal antibodies (mAbs) currently under preclinical and clinical development. One of the major challenges in developing antibodies as drugs is their physical instability caused by various processing, storage and handling related stresses which can lead to aggregation and loss of efficacy. Immunoglobulins are dynamic molecules whose motions are important for their biological function and stability. It is currently difficult to predict the effects of minor process or formulation changes on the higher order structure and dynamics of mAbs using currently available analytical techniques. Hence new analytical techniques are needed to better understand the effects of such changes on the structure and dynamics of mAbs, especially within pharmaceutical dosage forms containing excipients. This dissertation work explored the effects of certain pharmaceutical excipients and three sodium salts from the Hofmeister series on the changes in thermal stability, storage stability and local dynamics of an IgG1 mAb. Changes in local dynamics of the mAb were explored at peptide level resolution using hydrogen-deuterium exchange mass spectrometry. Preliminary correlations were established between changes in dynamics of particular segments in the CH2 domain of the IgG1 mAb and decreased thermal stability as well as higher aggregation propensity of the IgG1 mAb in presence of destabilizing excipients and salts. This dissertation work also explored the mechanism behind the relative decrease in physical stability due to YTE (M255Y/S257T/T259E) triple mutation in the CH2 domain of a different IgG1 mAb (that was shown in previous work to result in extended in-vivo half-life due to changes in interaction with FcRn receptors). The YTE mutation induced decreases in physical stability of the IgG1 mAb which correlated with changes in local dynamics of the same particular segment in the CH2 domain described above in the presence of destabilizing excipients and salts. Hence, changes in dynamics of specific local segments within the CH2 domain of IgG1 mAbs identified in this work may serve as a general indicator for changes in physical stability for other IgG mAb drug products. By better understanding the root causes of mAb physical destabilization, more rational design of antibody formulations and/or protein engineering approaches are now possible to improve the pharmaceutical stability properties of mAbs.