The role of mixing and surface hydrophobicity on the operation of a continuous tubular slug flow crystalliser for lysozyme

Continuous crystallisation is currently seen as a more economic and efficient alternative compared to standard techniques for the purification of biopharmaceutical proteins. Despite being promising as continuous crystalliser platforms, tubular slug flow crystallisers pose their challenges due to the tendency of proteins to accumulate at solid surfaces, to form amorphous precipitates at high protein concentrations and to undergo slow diffusive mass transport. This work investigates the effect of equipment surface chemistry and physical mixing on the design and operability of a tubular slug flow crystalliser for lysozyme under laminar flow conditions (Re similar to 1). Firstly, glass tubes with different surface functional groups, -OH or -CH3, with water contact angles between 9 degrees and 99 degrees, were investigated. CH3 surfaces resulted in a up to 33% delayed onset of nucleation, demonstrating lower heterogeneous nucleation rates, and therefore are better suited to prevent fouling. However, a surface chemistry-independent deposition of lysozyme was found, altering the water contact angle by up to 56 degrees, resulting in an unstable slug flow and a reduction of the onset of nucleation by up to 11.5-fold. To achieve a stable slug flow and controlled nucleation, the surface functional groups were recovered by implementing surface-specific cleaning protocols comprising NaOH or liquid detergent. To overcome poor mixing and amorphous precipitation, a two-step mixing approach, consisting of an intermediate mixing step was developed. This novel mixing approach reduced the mixing time from > 10 min to < 10 s, allowing the achievement of an instantaneous homogeneous solution under laminar flow conditions. Overall, the findings of this study are therefore of crucial relevance to the future design and operation of tubular slug flow crystallisers as purification platforms for biopharmaceutical proteins.