In this study, we report the isomerization of an aspartic acid residue in the complementarity-determining region (CDR) of crizanlizumab as a major degradation pathway. The succinimide intermediate and iso-aspartic acid degradation products were successfully isolated by ion exchange chromatography for characterization. The isomerization site was identified at a DG motif in the CDR by peptide mapping. The biological characterization of the isolated variants showed that the succinimide variant exhibited a loss in target binding and biological activity compared to the aspartic acid and iso-aspartic acid variants of the molecule. The influence of pH on this isomerization reaction was investigated using capillary zone electrophoresis. Below pH 6.3, the succinimide formation was predominant, whereas at pH values above 6.3, iso-aspartic acid was formed and the initial amounts of succinimide dropped to levels even lower than those observed in the starting material. Importantly, while the succinimide accumulated at long-term storage conditions of 2 to 8°C at pH values below 6.3, a complete hydrolysis of succinimide was observed at physiological conditions (pH 7.4, 37°C), resulting in full recovery of the biological activity. In this study, we demonstrate that the critical quality attribute succinimide with reduced potency has little or no impact on the efficacy of crizanlizumab due to the full recovery of the biological activity within a few hours under physiological conditions.
Aspartic Acid , Succinimides , Aspartic Acid/chemistry , Isomerism , Succinimides/analysis , Succinimides/chemistry , Complementarity Determining Regions/chemistry , Hydrogen-Ion Concentration
Pre-filled syringes have simplified parenteral administration of protein drugs. To ensure an easy and consistent movement of the plunger, the inner glass container surface is typically siliconized. For bake-on siliconization, emulsions are sprayed on and heat treated. Due to the European Union regulation REACh (Regulation concerning theRegistration,Evaluation,Authorisation and Restriction ofChemicals) the use of certain emulsion components, partially constituting the gold standard LiveoTM 365 35% Dimethicone NF Emulsion (LiveoTM 365), becomes restricted and LiveoTM 366 35% Dimethicone NF Emulsion (LiveoTM 366) has been introduced as an alternative. This change may affect the handling properties as well as the silicone layer formed. The purpose of these studies was to identify any differences that may influence the stability and safety of the final drug/device combination product to enable the use of the new emulsion. We compared silicone emulsions LiveoTM 365 and LiveoTM 366 and dilutions focusing on 1) their general physical stability, 2) the thermal degradation process of the emulsions and their components, and 3) the resulting silicone layer concerning chemistry, morphology, and functionality. The results were linked to the assessment of the final product regarding particle formation and short-term stability. A comparison of the emulsions LiveoTM 365 and LiveoTM 366 for bake-on siliconization is presented to support the transition of the latter as it becomes mandatory with REACh. Our studies show that the two emulsions do not significantly differ with respect to handling and stability, the resultant silicone layer characteristics as well as its functionality. We conclude that the transition to the new emulsion will not significantly impact the final product or the layer performance upon storage and with respect to particle formation.
Silicones , Syringes , Emulsions , Hot Temperature , Proteins , Silicones/chemistry
Pre-filled syringes have simplified parenteral administration of protein drugs. To ensure an easy and consistent movement of the plunger, the inner glass container surface is typically siliconized. For bake-on siliconization emulsions are sprayed on and heat treated. Due to the European Union regulation REACh (Regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals) the use of certain emulsion components, partially constituting the gold standard Liveo™ 365 35% Dimethicone NF Emulsion (Liveo™ 365), becomes restricted and Liveo™ 366 35% Dimethicone NF Emulsion (Liveo™ 366) has been introduced as an alternative. This change may affect the handling properties as well as the silicone layer formed. The purpose of these studies was to identify any differences that may influence the stability and safety of the final drug/device combination product to enable the use of the new emulsion. We compared silicone emulsions Liveo™ 365 and Liveo™ 366 and dilutions focusing on I) their general physical stability, II) the thermal degradation process of the emulsions and their components, and III) the resulting silicone layer concerning chemistry, morphology, and functionality. The results were linked to the assessment of the final product regarding particle formation and long-term stability. A comparison of the emulsions Liveo™ 365 and Liveo™ 366 for bake-on siliconization is presented to support the transition of the latter as it becomes mandatory with REACh. Our studies show that the two emulsions do not significantly differ with respect to handling and stability, the resultant silicone layer characteristics as well as its functionality. We conclude that the transition to the new emulsion will not significantly impact the final product or the layer performance upon storage and with respect to particle formation.
