Dissertation
Dissertation > Industrial Technology > Chemical Industry > Reagents and the production of pure chemicals > Adsorbent

Preparation of Mixed-mode Adsorbent for Expanded Bed Adsorption and Its Application to Proteins Separation

Author GaoDong
Tutor YaoShanZuo
School Zhejiang University
Course Biochemical Engineering
Keywords Expanded bed adsorption Mixed-mode Salt-independent adsorption Patch-controlled adsorption Oriented adsorption Immunoglobulin of Egg Yolk
CLC TQ424
Type PhD thesis
Year 2007
Downloads 239
Quotes 0
Download Dissertation

Mixed-mode adsorption is a novel bioseparation technique, which is especially suitable for expanded bed adsorption (EBA) to directly capture of target protein from moderate ionic strength feedstock without the need of dilution or other additives. Based on our previous work, the high-density cellulose-stainless steel powder (SSP) composite matrix was chosen as the adsorbent base, and benzylamine as the ligand to prepare the anion-exchange mixed mode adsorbent, which contains both hydrophobic and ionic groups. The adsorption isotherm, chromatographic retention, adsorption kinetics, and breakthrough behaviors were investigated systematically. In addition, the prepared adsorbent was used to isolate immunoglobulin (IgY) from dilute egg yolk.First, cellulose-SSP composite matrix was developed with the method of water-in-oil suspension thermal regeneration. After activation with allyl bromide, bromohydrin with N-bromosuccinimide and coupling with benzylamine, the matrix was derived to function as an anion-exchange mixed-mode adsorbent (named Cell-SSP-BA). Under the optimized conditions, the prepared matrix had regular sphericity, and the coupling efficiency was above 75% under the given conditions. The maximum ligand density could reach 200 μmol/ml adsorbent. A series of adsorbents with different ligand densities have been prepared with the control of preparation procedure.Second, with bovine serum albumin (BSA) as the model protein, the isotherm adsorption behavior and the chromatographic retention of Cell-SSP-BA were investigated under different conditions. The results indicate that the electrostatic interactions might be the most important contributor to the protein adsorption when the electrostatic attractive interactions exist between protein and adsorbent. When there are some amounts of electrostatic repulsion protein-adsorbent interactions, the adsorption process is patch controlled due to the uneven distribution of charge on the surface of protein molecule. The adsorption capacity decreases firstly with increasing salt concentration (corresponding electrostatic shielding) until a minimum is reached, then further increases in salt concentration results in the increase of adsorptioncapacity due to the hydrophobic contribution. The relation of retention factor vs salt concentration is similar to that of adsorption capacity under different salt concentrations, and the curve of retention factor as the function of salt concentration shows a typical "U" shape.Third, the effects of liquid phase conditions, salt concentration and pH value, on the BSA adsorption kinetics to Cell-SSP-BA were studied. The experimental data were fitted with PDM model. It was found that there were specific salt concentration and pH to result in the maximum effective pore diffusivity. Little dependence of BSA diffusivity on ligand density was found at low salt concentration. When there is general electrostatic repulsion interaction between protein and adsorbent, the increase of De value was considered due to the decrease of electrostatic repulsion and increase of hydrophobic interaction between BSA and adsorbent surface with the increase of salt concentration.Fourth, the expansion characteristics, liquid mixing performance and BSA breakthrough curves of the prepared adsorbents were studied in an expanded bed under different conditions. A simplified mathematical model for protein adsorption was used for describing the breakthrough curves of protein. Under the conditions tested, the flow in expanded bed was proved to be plug flow for the composite matrices prepared. The dynamic binding capacity was determined by the equilibrium adsorption capacity and the adsorption rate, and an optimal value of dynamic binding capacity could be obtained by altering the ionic strength, which affected the adsorption rate. A stronger desorption condition would be required for the adsorbent with higher ligand density in the elution process, and an optimal yield could be found under an appropriate ionic strength.Fifth, with the commercial adsorbent of Streamline Direct HST as the typical cation-exchange mixed-mode adsorbent, the effects of liquid phase conditions, pH and salt concentration, on the adsorption behaviors of BSA were studied and the corresponding adsorption mechanisms were also discussed. The results indicate that the maximum binding capacity of BSA on Streamline Direct HST occurs near the isoelectric point of BSA. The conformational change of protein molecule couldinfluence the adsorption process. When the electrostatic attractive interactions exist between protein and adsorbent, the adsorption isotherm obeys the Langmuir adsorption equation. When there are some amounts of electrostatic repulsion protein-adsorbent interactions, the multilayer adsorption process could be found, which might be due to the oriented adsorption through the protein/protein interactions. High salt concentration does not favor the protein adsorption under the electrostatic repulsion conditions.Finally, the prepared mixed-mode adsorbent, Cell-SSP-BA, was introduced to isolate immunoglobulin from diluted egg yolk. After optimizied the separation conditions, it was found that the total recovery was up to 82%-86% with a purification factor of 2.7, which demonstrated that the mixed-mode expanded bed adsorption has a potential application for the primary isolation of target biomolecules directly from complicate feedstock.The results in the present work would be helpful for understanding the adsorption mechanism of mixed-mode chromatography, and useful for the optimization of separation conditions and process design.

Related Dissertations
More Dissertations