Introduction
Transgenic tobacco is a novel monoclonal antibody (mAb) expression system that is less susceptible to contamination by animal-borne pathogens, toxins, or viruses than traditional recombinant mammalian or bacterial cell lines, ie, safer, and transgenic tobacco sources. Recombinant proteins have similar glycosylation patterns to mammalian proteins and are relatively simple to construct. However, the antibody titer obtained by transgenic tobacco expression is generally low, and contains various impurities which can be non-specifically adsorbed with the chromatographic medium such as polysaccharide, chlorophyll, alkaloid, cellulose and natural protein, which makes downstream purification difficult to carry out efficiently. In this experiment, a two-step cascade hollow fiber ultrafiltration was used as a concentration and pre-purification step to reduce the chromatographic operation load, thereby improving the overall efficiency of the purification process.
Experiment & result
The transgenic tobacco plants expressing the anti-Pseudomonas aeruginosa antibody were constructed to have an antibody molecular weight of 152.6 kD. After the tobacco leaves are pulverized, the phosphate buffer is added to homogenize, the gauze is filtered to remove large-sized plant impurities, and the filtrate is continuously centrifuged at a high speed to obtain a supernatant of 0.2 μm CellFlo Plus hollow fiber module (product number: C72M-061-01N) Microfiltration to remove smaller particle size plant components (average flow rate 6.4L/m2/h, shear <800S-1), 50kD filtrate (product number: M25S-100-01S, membrane surface area: 1050cm2) and 100kD (product) No.: X2AB-300-02N, membrane surface area: 145cm2) MiniKros hollow fiber module is subjected to two-step cascade ultrafiltration (flow path construction, average flow rate 19L/m2/h, shear <1800 S-1), 8.4 The L solution was concentrated to 0.2 L, and ~97% of plant-derived impurities were successfully removed, and the complete recovery of mAb (~100%) was ensured, and the antibody titer was increased by 32 times. The concentrate was subjected to 10 volumes of washing with a 50 kD module in a sodium phosphate buffer, followed by cation exchange chromatography and Protein A affinity chromatography. The obtained purified mAb was subjected to size exclusion chromatography, SDS-PAGE and ELISA analysis. The obtained recombinant human antibody has a purity higher than 95% and a total recovery of 50%.
Two-step cascade ultrafiltration flow diagram: M1-50kD hollow fiber module; M2-100kD hollow fiber module; Pf-Cole-Parmer Masterflex injection pump; Pp-Thomas Welch vacuum filtrate pump; Pr-Cole-Parmer ISMATEC return pump ; PD-pulse damper for stabilizing the flow in the system; the system simultaneously monitors the inlet and filtrate pressures of each component and sets the diverter valve at different locations.
discuss
The results show that the two-step cascade concentration-washing filtration using 50 and 100 kD hollow fiber modules can significantly reduce the impurity content of the feed liquid, increase the concentration of the product, and reduce the load of the subsequent chromatography steps. Compared to PEG/PEI precipitation, this technology does not introduce additional additives and has high selectivity; compared to direct chromatography, pre-filtration improves the purity and concentration of the chromatographic injection and helps to optimize the chromatographic equipment. Utilization, reduce costs. The method is gentle and easy to process amplify and is suitable for large-scale purification of tobacco-derived antibodies.
The content of this article is the editor's translation. If there is any inconvenience, please forgive me. For details, please refer to the original text.
Original: Mayani, M., Filipe, CDM, Mclean, MD, et al., Purification of transgenic tobacco-derived recombinant human monoclonal antibody. Biochemical Engineering Journal, 2013, 72: 33-41.
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