A common point of precipitate formation during a platform monoclonal antibody purification process is the neutralization step following the low pH viral inactivation hold. While precipitates can be removed by absolute or depth filtration, there is a lack of understanding of the specific physicochemical pathways that lead to their formation and of the species that are involved in the process, whether they be host cell proteins, DNA or other impurities. Such information, especially the growth pathways, is important in rationally designing processes as they affect precipitate growth rates and particle sizes. Here we present characterization of the precipitation that occurs during the purification of an Fc-fusion protein on neutralization after the viral inactivation hold. Under quiescent conditions, the precipitates grow to 5 – 20 µm in size in approximately 3 hours. They comprise small, roughly spherical subunits that have an average radius of ~ 1 µm. Their internal structures are consistent with mass-fractals of fractal dimension 2 as measured by transmission electron microscopy and kinetic growth experiments. While the mass-fractal structural motif is expected from classical colloidal aggregation theory, the precipitation behavior is distinctly atypical of colloid aggregation as the introduction of salt can inhibit the process and can partially dissolve the precipitate once it is formed. There appears to be a specific ion effect that alters the pH at which precipitation occurs and initial mass spectrometry results reveal that the precipitate comprises of over 100 unique host cell protein species.

Mechanisms of precipitation on pH adjustment

253rd National Meeting (2017)

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