A Pre-formulation study of recombinant human Interleukin-1 receptor antagonist for emergency use

Abstract

Abstract A methylated recombinant form of the natural anti-inflammatory mediator, interleukin-1 receptor antagonist (rhIL-1Ra) is known to competitively bind with type I receptors (IL-1RI) for IL-1. It is currently formulated in liquid dosage form as Kineret® to treat rheumatoid arthritis. Preclinical evidence exists indicating rhIL-1Ra can be used to treat brain stroke. However, the formulation is unstable at ambient temperature and is hence stored at 2°C to 8°C. In addition, the manufacturer (Amgen PLC) recommends avoiding shaking, freezing and exposure to sunlight. These recommendations for storage are incompatible, if the formulation is to be used to treat stroke outside of hospital. Therefore, the overall aims were to study thermal stability of the rhIL-1Ra and identify the root causes of instability, with an anticipation to design a more suitable pre-formulation which addresses the concerns of storage at ambient temperature. Thermal stability of Kineret (rhIL-1Ra) and new pre-formulations were studied using different techniques such as SLS, DLS, SDS-PAGE, SEC-HPLC, CD spectroscopy, fluorescence spectroscopy and “UNcle Machine”. SLS results at elevated temperature of 37°C showed a sigmoidal kinetic profile starting with a short nucleation phase of approximately 20 min, followed by more rapid aggregation to reach a saturation phase. DLS measurements indicated nano-sized particle growth within the nucleation phase. Combined DLS, SDS-PAGE, and SEC-HPLC data also suggests the presence of nanosized particle in the original formulation. CD-spectroscopy and Dye (THT) tracer assay at high temperature of 37°C reported structural changes involving formation of new non-native secondary structures. Reducing and non-reducing SDS-PAGE confirmed existence of intermolecular disulfide bonds as well as non-covalent interactions resulting in oligomer formation. UNcle and fluorescence spectroscopic data highlighted that the thermally induced aggregation of rhIL-1Ra, in CSEP buffer, is related to protein folding, which is facilitated by self-assembly of monomers, possibly via cation-π interactions among others. A series of pre-formulations were designed and tested along with three chemically modified versions of rhIL-1Ra to counteract formation of covalent and non-covalent aggregates by inhibiting either conformational changes in protein structure using structural stabilisers or by supressing disulfide bonding using reducing agents. A variety of excipients including sugar, polymers, sorbitol and amino acids were used for these purposes. Three pre-formulations and one chemically modified form of rhIL-1Ra; F3, F11, F21 and citraconylated rhIL-1Ra, gave superior performance to Kineret in terms of shelf-life at room temperature. The biological activity of the rhIL-1Ra in F3, F11 and F21 pre-formulations remained comparable to Kineret while the citraconylated form caused loss in activity which could be recovered by reversing the modification. Finally, F11 and F21 formulations could be transformed to solid dosage form which could be reconstituted in liquid dosage form by a quick dissolution step.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Harmesh Aojula (Supervisor) & Stephen Hopkins (Supervisor)