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Separation of Immunoglobulin and Monoclonal Antibody by Aqueous Two-phase Extraction

Author: WuQiang
Tutor: LinDongQiang
School: Zhejiang University
Course: Biochemical Engineering
Keywords: Aqueous two-phase system Extraction Immunoglobulin G Human serumalbumin Monoclonal antibody Mammalian cell culture broth Mixed-mode ligand
CLC: R392
Type: PhD thesis
Year: 2014
Downloads: 107
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Antibodies have been widely used for the therapeutic drug, diagnostic reagent and immunoaffinity separation, which has a broad market demand and prospects. Antibodies are normally separated from animal blood, ascites and mammalian cell culture. The manufacturing of monoclonal antibody from mammalian cell culture has been applied in large-scale production. As we know, the high-cost downstream process for antibody production has become one bottleneck of antibody industry. It is of great importance to develop new techniques with low cost and high efficiency for antibody separation. As new bioseparation technology, aqueous two-phase extraction (ATPE) has some advantages such as good biocompatibility, mild condition, relative low cost, easy to scale-up and so on. In this thesis, the feasibility of antibody separation with ATPE was investigated, which aimed at two typical processes of the separation of immunoglobulin G (IgG) from human serum albumin (HSA) containing feedstock and the purification of monoclonal antibody from mammalian cell culture broth.Main contents are summerized as follows:(1) Aqueous two-phase system (ATPS) was studied for the extraction of IgG from HSA containing feedstock. The partition behaviors of IgG in different types of ATPSs were compared firstly, and polyethylene glycol (PEG)/hydroxypropyl starch (HPS) ATPS was chosen as the suitable system. The effects of the concentrations of PEG, HPS and NaCl and pH on the partition of IgG and HSA in PEG/HPS ATPS were investigated. With the yield of IgG in top phase (Ytop) and the purification factor (PF) as the objective parameters, the response surface methodology (RSM) was used to evaluate the effects of some key factors and the separation conditions were optimized. The optimal conditions were obtained as12%(w/w) PEG4000,18%(w/w) HPS and10%(w/w) NaCl at pH8.0, and Ytop was99.2%with PF of5.28. The back extraction of IgG with PEG/phosphate ATPS were studied, the optimal conditions were10%phosphate (pH7) and the mass ratio of top phase of the first extraction to phosphate stock solution (40%w/w) of1.6:1. After two steps of ATPE, the total yield of IgG was84.3%with PF of5.73. The results demonstrated that IgG could be efficiently separated from HSA containing feedstock with suitable ATPE process.(2) The mechanisms of NaCl effects on the partition and solubility of IgG in ATPS were investigated. It was found that the addition of NaCl could lead to a shift on the binodal curve of the phase diagram of PEG/HPS ATPS, which caused the increase on the differences of hydrophobicity of two phases. The effect of NaCl addition on the surface hydrophobicity of IgG molecule was determined using fluorescence method. The results indicated that the addition of NaCl could increase the hydrophobicity of IgG, which would be the main reason for the selective partition of IgG in top phase. In addition, the effects of NaCl addition on the solubility of IgG were investigated. It was found that the addition of NaCl could increase the precipitation of IgG in the presence of (NH4)2SO4while improve the solubility of IgG in the presence of PEG, which could explain the experimental phenomenon of the difference of IgG recovery in PEG/(NH4)2SO4and PEG/HPS ATPSs. Furthermore, the effect of NaCl on the interaction between IgG and PEG was inveatigated using isothermal titration calorimetry (ITC). It was found that the addition of NaCl might make a shift on the IgG-PEG interaction mode and the hydrophobic interaction of IgG-PEG would increase with high NaCl concentration. This would be the reason that the NaCl addition could improve the solubility of IgG in the presence of PEG, and the possible mechanism was proposed.(3) A two-step extration process with ATPSs were studied to purify monoclonal antibody MAB from chinese hamster ovary (CHO) cell culture supernatant. The effects of NaCl addition, pH and feedstock loading on the extraction of MAB with PEG/HPS ATPS were investigated. The optimal conditions were12%PEG,18%HPS,15%NaCl, pH6.0and6%feedstock loading, and the yield of MAB in top phase was96.7%with the purity of96.0%. PEG/phosphate ATPS was used for the back extractions of MAB, and the optimal conditions were8%phosphate (pH7.0) and the mass ratio of top phase to phosphate stock solution (40%w/w) of2.5:1. After two-step extraction, the purity of MAB could reach97.6±0.5%with the yield of86.8±1.0%. It was found that the purity of MAB obtained with ATPE was comparable to that of Protein A chromatography. The results indicated that the ATPE process could be used as the alternative technique for the purification of monoclonal antibody from cells culture broth.(4) The separation of antibody using ATPS with mixed-mode ligand was studied. Firstly, the ligand-PEG was prapared by coupling mixed-mode ligand onto PEG efficiently. PEG was activated by epichlorohydrin, and the epoxide content of activated PEG could reach460μmol·g-1PEG The PEG-expoxide was then coupled with mixed-mode ligands with above90%coupling rate. The effects of the ligand-PEG on the partition of IgG in ATPS were investigated, including the hydrophobic ligand (MMI-PEG) and the charged ligands (MBA-PEG, AHNSA-PEG, MBIA-PEG and MBIS-PEG). It was found that2-mercapto-5-Benzimidazole-carboxylic acid (MBIA) was a proper ligand for IgG separation. At pH5.0, the addition of MBIA-PEG could make IgG riched in top phase while most of HSA still partitioned in bottom phase. The results indicated that ATPE with MBIA-PEG could be used for the separation of IgG. However, more follow-up research should be done to improve the separation efficency.In this thesis, the separation of IgG and monoclonal antibody with ATPE was focused. In order to improve the separation efficiency, two strategies have been developed:(1) NaCl was added into PEG/HPS ATPS to tranfer antibody from bottom phase into top phase by influencing the phase equilibrium and the hydrophobic property of antibody;(2) The mixed-mode ligands were coupled with PEG to induce antibody into top phase to purify antibody from the impurity proteins by the specific interactions between ligand and antibody. The feasibility of two strategies was verified and the mechanism was analyzed, which provided some new ideas for the development of new techniques for antibody separation.

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