Porogens addition in chitin-based chromatographic supports. Impact on chromatographic and structural properties

LAURA DANIELA PILATO; GUILLERMO JAVIER COPELLO; FEDERICO JAVIER WOLMAN

CABA

Congreso; WCCE11-11 World Congress on Chemical Engineering; 2023

World Chemical Engineering Council, Asociación Argentina de Ingenieros Químicos

Objective: The aim of this work was to develop a chitin-based material to be used as a chromatographic support for high-productivity industrial protein purification, promoting convection as the predominant mass transfer mechanism. To maximize the surface area of the materials, two possible particle porogens were studied, (CaCO3 and SiO2 < 40 µm particle diameter) in a ratio 1:10 (chitin: porogen). Materials and methods: Powder chitin was used to generate xerogels as previously described [1] with or without the aggregate of SiO2 or CaCO3 particles as porogens [2], generating three different materials: without porogen (Ch), using SiO2 (ChS) and using CaCO3 (ChC). The materials were characterized by ART-FTIR, SEM and the swelling degree was estimated. Chromatographic characterizations were carried out in batch mode employing pure chicken egg white lysozyme (Lyz) as target protein, whose interaction with chitin was already studied; The parameters maximum adsorption capacity (Qm) and dissociation constant (Kd) were estimated by performing adsorption isotherms, modeled according to Langmuir (0.078-10 mg Lyz /ml, in 20 mM carbonate pH 9), and adsorption kinetics were also performed. In addition, the percentage of elution achieved for each material used in the 0.1 M solution of acetic acid was also evaluated. Results: By ATR-FTIR the complete removal of the porogen was verified in all the materials, and the adequate structuring of the xerogel was demonstrated by SEM, where it could be seen the formation of macroporous particles with an open-pore structure. In the case of the ChC materials, major size of the pores could be observed in comparison with the ChS. All the materials showed swelling values around 80%, providing an adequate balance between the internal surfaces generated to maximize the adsorption capacity and the mechanical resistance of the material. The chromatographic studies showed highest Qm value for Ch (Ch: 59,74 ± 5,05; ChS: 37,48 ± 1,61; ChC: 48,7 ± 2,17 mg/g), and evidenced better Kd value for ChS (Ch: 3,05 ± 0,74; ChS: 0,49 ± 0,09; ChC: 1,8 ± 0,41 mg/ml). The kinetic studies showed higher adsorption capacities for the ChS materials than ChC in the experimental conditions, but interestingly the ChC matrices showed the highest adsorption velocities. Conclusion: Even though ChS material showed lower Kd, ChC showed a kinetic behavior that suggests that its open and interconnected pore structure with low tortuosity, which could promote that convective mechanism predominates the movement of the protein molecules inside the support. For the near future, it is expected to test the performance of this novel chitin-based supports under dynamic bedpacked condition.