Comparative Evaluation of Surfactants and Stabilizers in Preventing Protein Aggregation in Insulin Formulations

INTRODUCTION: Protein aggregation is an issue that affects the stability and effectiveness of biopharmaceutical products. Proteins easily undergoes denaturation, due to mechanical agitation and prolonged storage times. Although polysorbates have been extensively used to stabilize proteins, there is still room to identify other highly effective additives. This research aimed to compare the efficacy of hydroxypropyl-β-cyclodextrin (HP-β-CD) in preventing protein aggregation in recombinant human insulin with polysorbate 20 (PS-20), polysorbate 80 (PS-80), and Poloxamer 188 (P188). METHODS: Recombinant human insulin formulations with the addition of PS-20, PS-80, P188, or HP-β-CD, or the absence of any additive (control group), were prepared. The prepared samples were subjected to mechanical stress through shaking at 100 rpm for 24 hours. Thereafter, a sixmonth accelerated stability test was performed at 25±2°C/65±5% RH. Protein aggregation and particle formation were measured using size-exclusion high-performance liquid chromatography (SEHPLC) to detect HMWP, dynamic light scattering (DLS) for submicron aggregate size, and light obscuration (LO) of subvisible particles. RESULTS: Mechanical agitation caused protein aggregation in all formulations; however, the degree of aggregation depended on the stabilizer used. HP-β-CD showed the highest stabilization activity, with only a 1.2% increase in HMWP level, whereas the non-stabilized control increased by 12.3%. LO analysis showed that formulations containing HP-β-CD had the least subvisible particles. In addition, DLS analysis showed no changes in aggregate size. Accelerated stability study results showed even better performance of HP-β-CD, with HMWP levels ranging from 2.60% to 2.64% over six months of storage, exceeding the performance of PS-20, PS-80, and P188. DISCUSSION: The stabilization effect exerted by HP-β-CD could be assumed due to its capacity to decrease protein unfolding and prevent aggregation by the interfacial stress. Compared with the other surfactants, HP-β-CD provided better protection against mechanical and storage-related degradation of the protein while limiting particle formation. CONCLUSION: Under the conditions of agitation and storage stress, the use of HP-β-CD provides more efficient stabilization of recombinant human insulin as compared with polysorbate surfactants and Poloxamer 188.