ALBUMIN IMPRINTED AGAROSE MATRIX WITH COPPER (II) CHELATED 3-(2-IMIDAZOLIN-1- YL)PROPYL(TRIETHOXYSILANE) MODIFIED SILICA-BASED CORE FOR HUMAN BLOOD PLASMA PURIFICATION

• Main Author: Ratna Wulan, Dyah
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/69667
• Institution: Institut Teknologi Bandung

Human serum albumin (HSA), a plasma protein, is widely used for clinical therapy, stem cell media and diagnostic tools. In 2020, 1,018 tons of HSA are needed worldwide, growing of 6% from 2020 to 2027. Most of the HSA comes from the purification of human blood plasma. HSA purification by Cohn method is requires plasma from thousand of donors. For archipelago countries such as Indonesia, the process of blood plasma pooling especially during the pandemic, remains a problem. Hence, the development of a simple and rapid method to purify HSA in a small volume of human blood plasma is essential. The chromatography method that has been developed requires several steps to produce HSA with a relatively higher purity than the Cohn method. One stage chromatography method has also been developed. However, this single-stage chromatography was limited for the purpose of HSA depletion. A single-stage HSA purification method from blood plasma using molecular imprinted polymer (MIP), a selective material, is being developed. However, MIP has not been able to purify HSA optimally, and the process using a flow system has not been studied. Therefore, this study aims was developed MIP called HSA-imprinted microsphere (HSAIM) matrix that specifically binds HSA which were generated by encapsulating SiO2@3- (2-imidazolin-1-yl)propyl(triethoxysilane)-Cu(II)-HSA into agarose gel, analyzed the physicochemical properties of HSAIM, and applied HSAIM column for HSA purification from human blood plasma. There were four steps used in this study. Firstly, preliminary research was studied the interaction of affinity ligands that will be used in HSAIM, namely 3-(2- imidazolin-1-il)propyl(triethoxystilan) (IMEO) with HSA protein, analog proteins (BSA), and competitor proteins in blood plasma (IgG) through a multispectroscopic method approach (UV, FT-IR, and flouresen) and molecular interactions modeling using autodock software. Secondly, HSAIM and n-HSAIM as negative controls were synthesized. HSAIM was optimized by varying the agarose concentration, stirring speed during encapsulation, and SiO2@IMEO-Cu(II)-HSA core mass to agarose mass ratio. In addition, the optimization of the HSA purification process using HSAIM was carried out through the selection of leaching buffer, adsorption bufferand desorption buffer. Thirdly, HSAIM performance was measured and evaluated to determine the physico-chemical properties of the batch system by examining the adsorption capacity, adsorption isotherm, and specific binding activity. The performance of HSAIM on the flow system was evaluated through examining dynamic binding capacity and re-usability. Finally, the HSAIM column was used practically for the purification of HSA from human blood plasma. Preliminary research is used to determine the interaction of IMEO and HSA to understand the reason IMEO purify HSA by 88.3% in prefiouse study. Flourocene, UV and FT-IR spectra of the IMEO-HSA and IMEO-BSA mixtures showed decrease in flourocene intensity, UV wavelength shifts of 208 and 280 nm towards larger wavelengths and wave number shifts on the HSA and BSA spectra successively at 1659 and 1535 cm–1 which shifted towards a larger wave number indicating that IMEO interacted with HSA or BSA. Stern Volmer plot on the IMEO-HSA and IMEO-BSA interactions indicated a stronger IMEO-HSA interaction than the IMEO-BSA interaction. However, from multispectroscopic analysis, no interaction was found between IMEO and IgG. The interaction of IMEO and HSA (PDB 1h9z) using autodock software shows that no hydrogen bond is formed, but there is a hydrophilic and electrostatic interaction of IMEO with residues D108, Y148, R197, C200, C24 on the IIA subdomain of the HSA with a binding energy calculation value of –1.89 kcal/mol. This shows that IMEO is not selective only binding to HSA, so in addition to electrostatic interactions, other interactions are needed to increase the purity of HSA, through the interaction of the chelate and stereospecific cavity through the HSAIM approach to be able to purify the HSA from a complex matrix. In this study, sphere-shaped HSAIMs with an average diameter of 51.2±6.1 ?m have been successfully synthesized. In batch systems, HSAIMs had a maximum adsorption of 5.83 mg HSA g–1 with the printing factor (IF) for HSA was 2.83 and the selectivity factor BSA, thrombin and IgG are 1.05, 1.71 and 3.88, respectively. The HSA adsorption kinetics on the HSAIM was fitted the pseudo-second-order mechanism, and the binding characteristics of the HSA was fitted the Sips isotherm model. In the flow system, HSAIMs had a dynamic binding capacity (DBC10%) of 3.95 mg HSA g–1, with kinetic characteristics was fitted the Thomas model, and HSAIMs can be reused for up to 10 cycles with a final HSA gain of 55.92%. In practice, the initial blood plasma containing 24.9 ± 2.5 mg of protein was precipitated using ethanol, yielding 14.5 ± 4.6 mg of protein, and further purification with the HSAIM column yielded 3.6 ± 1.1 mg of protein. After purification with the HSAIM column, one protein was obtained, HSA, from 149 proteins present in human blood plasma detected by LC-MS/MS. As a comparison product, commercial infusion HSA for therapy contains 27 impurities. This suggests the HSA can be stereospecically bound to the HSAIM. Therefore, HSAIM has the potential to be applied in the process of purifying HSA from blood plasma.