Impact of stirring material on formation of submicron and subvisible aggregates in mAbs by quantitative laser diffraction, dynamic light scattering and background membrane imaging

Aggregation of monoclonal antibodies (mAbs) is the driving force for their undesirable immunogenic effects. There are multiple factors responsible for aggregation in therapeutic proteins. One significant cause is the process-related shear and interfacial stress generated due to impellers and stirrers. This investigation focuses on understanding the possible aggregation arising upon stirring mAb formulations using stirrers made of different materials. We used quantitative laser diffraction (qLD) to monitor and quantify the stirring induced formation of submicron and subvisible aggregates in the size range from 100 nm to 10 µm. We analysed various aspects of aggregate generation, such as onset of aggregation, particle size distribution, and concentration of aggregates generated using stirrers of different materials. We observed that mixing with stainless steel stirrers resulted in a quicker onset of aggregation and led to significantly higher concentrations of aggregates. Analysis of the stirred samples using dynamic light scattering (DLS) and background imaging technique (BMI) were conducted to complement the qLD analysis. All the three techniques resulted in a similar trend, showing presence of larger and higher quantities of aggregates in steel stirred samples, as compared to those stirred using PEEK and glass. Additionally, we performed SEC-HPLC to quantify the soluble fraction of monomer and recorded that the least amount was present in the steel stirred samples. This work highlights the need for optimizing the materials used for fabricating the stirrers/impellers.