Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques.

A critical step in monoclonal antibody (mAb) screening and formulation selection is the ability of the mAb to resist aggregation following exposure to environmental stresses. Regulatory authorities welcome not only information on the presence of micron-sized particles, but often any information on sub-visible particles in the size range obtained by orthogonal sizing techniques. The present study demonstrates the power of combining established techniques such as dynamic light scattering (DLS) and micro-flow imaging (MFI), with novel analyses such as raster image correlation spectroscopy (RICS) that offer to bridge existent particle sizing gaps in this area. The influence of thermal and freeze-thaw stress treatments on particle size and morphology was assessed for a bi-specific antibody (mAb2). Aggregation of mAb2 was confirmed to be concentration- and treatment-dependent following thermal stress and freeze-thaw cycling. Particle size and count data show concentration- and treatment-dependent behaviour of aggregate counts, morphological descriptors and particle size distributions. Complementarity in particle size output was observed between all approaches utilised, where RICS bridged the analytical size gap (∼0.5-5 μm) between DLS and MFI. Overall, this study highlights the potential of orthogonal image analyses such as RICS (analytical size gap) and MFI (particle morphology) for formulation screening.