Neergaard, Martin S5; Nielsen, Anders D4; Parshad, Henrik4; van de Weert, Marco6
1 Department of Pharmacy, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet2 Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet3 Drug Research Academy A, Drug Research Academy, Faculty of Pharmaceutical Sciences, Københavns Universitet4 Novo Nordisk A/S5 Drug Research Academy A, Drug Research Academy, Faculty of Pharmaceutical Sciences, Københavns Universitet6 Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet
head-to-head comparison of the IgG1 and IgG4 subclass
Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences between the IgG subclasses. Both physical and chemical stability were evaluated by applying a range of methods to measure formation of protein aggregates [size-exclusion chromatography (SEC)-HPLC and UV340 nm], structural integrity (circular dichroism and FTIR), thermodynamic stability (differential scanning calorimetry), colloidal interactions (dynamic light scattering), and fragmentation and deamidation (SEC-HPLC and capillary isoelectric focusing). The impact of pH (4-9) and ionic strength (10 and 150 mM) was investigated using highly-concentrated (150 mg/mL) mAb formulations. Lower conformational stability was identified for the IgG4 resulting in increased levels of soluble aggregates. The IgG1 was chemically less stable as compared with the IgG4 , presumably because of the higher flexibility in the IgG1 hinge region. The thermodynamic stability of individual mAb domains was also addressed in detail. The stability of our mAb molecules is clearly affected by the IgG framework, and this study suggests that subclass switching may alter aggregation propensity and aggregation pathway and thus potentially improve the overall formulation stability while retaining antigen specificity.
Journal of Pharmaceutical Sciences, 2014, Vol 103, Issue 1, p. 115-27