Proteins on the Rack: Mechanistic Studies on Protein Particle Formation During Peristaltic Pumping.

Peristaltic pumping can cause protein particle formation. The expected causes were unfolding by heat in the pump head, oxidative stress by cavitation generated during roller movement, interfacial adsorption to the tubing wall and mechanical stress by stretching of the tubing itself. The pump head reached 28°C during experiments stayed well below the onset of the melting point of the proteins. Thus, heat may only be a relevant root cause for proteins containing domains with very low unfolding temperature. Analysis by terephthalic acid dosimetry and protein oxidation via RP-HPLC ruled out major induction of reactive hydroxyl radicals by pumping, indicating that cavitation does not play a significant role in particle generation. Addition of surfactants suppresses protein adsorption to the tubing wall and drastically reduced protein particle formation. This indicates that interfacial protein adsorption is a key element. Repeated stretching of tubing filled with protein solution led to the formation of protein particles, demonstrating that expansion and compression of the protein film on the tubing surface is the second key component for particle formation. Thus, protein particle generation during peristaltic pumping originates from the formation of a protein film on the tubing surface which gets stretched and compressed, leading to film fragments entering the bulk solution. This interplay of protein film formation and its rupture has been also observed at liquid/liquid or liquid/air interfaces.