Abstract
The crystallization of Anti-CD20, a full-length monoclonal antibody, has been studied in the PEG400/Na2SO4/Water system near Liquid-Liquid Phase Separation (LLPS) conditions by both sitting-drop vapour diffusion and batch methods. In order to understand the Anti-CD20 crystallization propensity in the solvent system of different compositions, we investigated some measurable parameters, normally used to assess protein conformational and colloidal stability in solution, with the aim to understand the aggregation mechanism of this complex biomacromolecule. We propose that under crystallization conditions a minor population of specifically aggregated protein molecules are present. While this minor species hardly contributes to the measured average solution behaviour, it induces and promotes crystal formation. The existence of this minor species is the result of the LLPS occurring concomitantly under crystallization conditions.
Highlights
Colloidal and conformational stabilities are the two main factors that govern the existence of a protein aggregate in a solution[9,10]
Anti-CD20 is a full-length monoclonal antibody and it is a therapeutic protein extensively used for treatment of chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma, which is commercialized with the brand name of Rituxan or Mabthera
Since the batch composition does not vary with time, due to the absence of solvent evaporation, it is evident that the solution is supersaturated immediately upon mixing the protein and precipitant solutions, which results in a shorter crystallization time (12–18 h) than that observed with the sitting drop set up (36–48 h)
Summary
Colloidal and conformational stabilities are the two main factors that govern the existence of a protein aggregate in a solution[9,10]. We focus on the relationships between the crystallization propensity of Anti-CD20 in the Na2SO4/PEG400 solvent system of different compositions and its relation with some measurable parameters normally used to assess protein conformational and colloidal stability in solution, with the aim to understand the aggregation mechanism of this complex biomacromolecule. The pathway for the formation of crystal nuclei drastically changes near the metastable low density–high density liquid region coexistence, with the free-energy barrier for crystal nucleation being strongly reduced In this scenario, the crystal nucleation rate increases by many orders of magnitude over that predicted from classical nucleation theory[28], generating a fast and diffuse crystallization that often results in poor-quality crystals. The results of this investigation allowed us to gain further insights into the mechanism governing the nucleation-crystallization of a complex system such as the Anti-CD20 mAb
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