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November 2013
Carlos Andrés Martínez Cristancho
Process for continuous purification of single-chain antibody fragments based on Simulated Moving Bed Chromatography
During the last years the market for recombinant antibody fragments has been growing very fast. Small-sized fragments can offer new alternatives to the full-length monoclonal antibodies (mAb) in the field of antibody-based therapeutics. Among them, the single-chain antibody fragments (scFv) are the smallest antibody fragments that still possess a complete antigen-binding site. Hence, they are ideal for research, diagnostic and therapy, which require good tissue penetration. The high titers obtained with recombinant proteins are imposing a tremendous challenge in the development of more efficient continuous purification processes, which can isolate recombinant proteins in an easier way, compared to the established multi-step discontinuous chromatographic scheme.
This work focuses on the theoretical design and experimental validation of a Simulated Moving Bed (SMB) chromatography process using Immobilized Metal Ion Affinity Chromatography (IMAC) for the purification of a single-chain antibody fragment. First, the cell culture supernatant containing the antibody fragment and originating from Bacillus megaterium was characterized using a stepwise pH-gradient batch chromatography. The influence of solvent composition (pH) on the adsorption isotherm parameters of the antibody fragment and its impurities were determined in single-column runs.
Based on the estimated adsorption isotherm parameters a feasible multicolumn open-loop 3-zone pH-gradient SMB process is suggested, which possesses the potential to isolate continuously the single-chain antibody fragment. The design is based on a recursive solution of an equilibrium stage model of an equivalent True Moving Bed (TMB) process. Possible operating conditions were theoretically proposed using the simulations obtained by the model and experimentally realized in a lab-scale SMB unit using a methodological approach.
The observed performance of the continuous process is compared to the corresponding batch process in terms of purity, yield, productivity, and buffer consumption. The potential of this innovative SMB process is clearly demonstrated.
This work focuses on the theoretical design and experimental validation of a Simulated Moving Bed (SMB) chromatography process using Immobilized Metal Ion Affinity Chromatography (IMAC) for the purification of a single-chain antibody fragment. First, the cell culture supernatant containing the antibody fragment and originating from Bacillus megaterium was characterized using a stepwise pH-gradient batch chromatography. The influence of solvent composition (pH) on the adsorption isotherm parameters of the antibody fragment and its impurities were determined in single-column runs.
Based on the estimated adsorption isotherm parameters a feasible multicolumn open-loop 3-zone pH-gradient SMB process is suggested, which possesses the potential to isolate continuously the single-chain antibody fragment. The design is based on a recursive solution of an equilibrium stage model of an equivalent True Moving Bed (TMB) process. Possible operating conditions were theoretically proposed using the simulations obtained by the model and experimentally realized in a lab-scale SMB unit using a methodological approach.
The observed performance of the continuous process is compared to the corresponding batch process in terms of purity, yield, productivity, and buffer consumption. The potential of this innovative SMB process is clearly demonstrated.
Schlagwörter: Single-chain Fragment variable antibodies; Immobilized metal ion affinity chromatography; Simulated Moving Bed Chromatography; Histidine-tagged recombinant protein purification
Forschungsberichte aus dem Max-Planck-Institut für Dynamik komplexer technischer Systeme
Herausgegeben von Prof. Dr. Peter Benner, Prof. Dr.-Ing. Udo Reichl, Prof. Dr.-Ing. Andreas Seidel-Morgenstern und Prof. Dr.-Ing. Kai Sundmacher, Magdeburg
Band 42
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