(320c) Bovine Serum Albumin and Myoglobin Separation By Size Exclusion Simulated Moving Bed Technology

Proteins are found in bacteria and viruses as well as in complex organisms, vertebrates and higher mammals such as humans [1]. Protein functions depend on its structure, which is influenced by physical and chemical parameters. Additionally, proteins are already included in our daily life, being present in detergents, food, cosmetics, perfumes and others such as pharmaceutical products. They can also be used in organic chemistry, for enzymatic catalysis [2]. Therefore, proteins must be produced and purified industrially, at large scale, to be implemented into their applications. To obtain a protein, in its pure form, from a mixture of others can be a laborious and expensive task. Indeed, to develop a process and determine the optimal conditions to purify proteins is the major challenge in the area. Separation and purification of proteins by SMB started with the size-exclusion simulated moving bed (SE-SMB) [3]. This kind of separation is simple; the working pH is not relevant; neither the ionic strength nor the composition of the buffer. This is mainly because these aspects do not influence the size, which is the parameter that is responsible for the separation [4]. In this kind of separation, a mixture of proteins with different molecular masses, the path that the compounds take will be different. The smallest will be able to enter in the pores, and as a result, their concentration wave velocity in the column is slowed. The larger ones, rarely enter in the pores or if they are very large, they are not able to enter at all, being its elution in the column easier and faster [1].

Materials and Methods

The main objective of this work is to study the separation of proteins, bovine serum albumin (BSA) and myoglobin (Mb) by size-exclusion simulated moving bed.

Bovine Serum Albumin belongs to albumins family, which includes other proteins such as Human Serum Albumin and ovalbumin. Albumin is the most predominant plasma protein, and it is produced in the liver [5]. Its molecular weight is 65.4 kDa. Myoglobin is a cytoplasmic hemoprotein and belongs to the globin family, as well as hemoglobin (Hb), neuroglobin (Ngb) and cytoglobin (Cygb) [6, 7]. Its molecular weight is 16.89 kDa. Therefore, the fractionation range of the selected resin, Sephadex G-50, is from 1 to 30 kDa, which excludes the BSA totally, since its molecular weight is higher than the upper exclusion limit.

To achieve the objective of separating the two proteins, fixed bed experiments were performed to obtain the adsorption breakthrough curves in Sephadex G-50, as well as the binary breakthrough curves. A phenomenological model was developed and used to predict the obtained experimental results. SMB experiments were carried out.

Results

The results of the fixed bed experiments, in terms of the retention times of the two proteins, showed that retention time of Mb is higher than BSA, which was expected since its molecular weight is within the fractionation range of the adsorbent, so it is possible for Mb to enter in the pores. The developed mathematical model was validated with fixed bed experiments, and it was possible to conclude that these two proteins can be separated by size exclusion chromatography.

An SMB model was used to determine the separation region, for raffinate and extract purity of 99.9 %. An operating point within the mentioned region was selected to perform the separation experimentally. Thus, the appropriated flowrates and switching time were selected. Afterward, SMB experiments were performed in the FlexSMB^® unit, and the model was validated. It was concluded that the SMB model could predict the experiments and that the system can separate the BSA and Mb efficiently.

Acknowledgments

This work is a result of: Project “AIProcMat@N2020 - Advanced Industrial Processes and Materials for a Sustainable Northern Region of Portugal 2020”, with the reference NORTE-01-0145-FEDER-000006, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); Associate Laboratory LSRE-LCM - UID/EQU/50020/2019 - funded by national funds through FCT/MCTES (PIDDAC), and by FCT/PD/BD/137891/2018.

References

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