Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts (original) (raw)
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Electronic Journal of Biotechnology, 2013
Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfidebond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain.
2013
Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-...
Microbial Cell Factories, 2013
Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in Abstract Background: Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions.
Production of Antibody Fab Fragments in Escherichia coli
Cell Engineering, 2011
A phage-display library is the most broadly used platform for preparation of recombinant human monoclonal antibody Fab fragments. Panning is effective for the selection of immunoglobulin genes from naïve and immune libraries. However, it is possible to bypass the phage display system if human peripheral lymphocytes are obtained from seropositive patients with infectious diseases as a source of immunoglobulin genes. Direct screening of bacterial colonies producing Fab fragments by colony blotting using filter membranes is practical for the isolation of human Fab fragments to major antigens of pathogens. An oligoclonal culture can also be used, and is a partial application of Epstein-Barr virus transformation of peripheral lymphocytes. Using these procedures, neutralizing antibody Fab fragments to various antigens can be obtained with a sufficient level of cloning efficacy. Chain shuffling and site-directed mutagenesis are also useful ways to improve the quality of the cloned antibody Fab fragments.
Journal of Chemical …, 2012
BACKGROUND: In the commercial-scale purification of immunotherapeutics, Protein A chromatography is employed routinely for its high binding capacity and selectivity. Nevertheless, matrix cost and ligand leaching issues remain and there are many alternatives such as ion-exchange and multi-modal resins that are less expensive. However, binding capacities are lower than Protein A owing to the co-adsorption of protein impurities. One solution involves removing impurities before chromatography by precipitation and a potential approach presented in the literature recently employs a dual-salt precipitation technique. The current study explores the impact of this upon the capture of an antibody fragment by a multimodal cation exchange resin.
Bacterial Expression and Purification of Recombinant Bovine Fab Fragments
Protein Expression and Purification, 2002
We have previously described a recombinant other than mice, including veterinary species (3). This phagemid expression vector, pComBov, designed for is due to the success of the technique being dependent the production of native sequence bovine monoclonal upon the ability to recover the immunoglobulin reperantibodies (mAb) generated by antibody phage display. toire from a source of B lymphocyte RNA and the con-Bovine mAb Fab fragments isolated from libraries construction of representative antibody libraries that can structed using pComBov in Escherichia coli strain XL1be screened against an antigen source, rather than Blue, which is routinely used for antibodies expressed on the availability of stable transformed cell lines or on the surface of phage, were expressed at very low methods to immortalize B lymphocytes. In many speyields. Therefore, a study was undertaken to determine cies, the understanding of the basis of immunoglobulin optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli formation is now sufficiently detailed for the applicastrain, and the temperature and length of the culture tion of antibody phage display and the isolation of growth, we were able to substantially increase the species-specific mAbs, and this technology has been yield of soluble Fab fragments. A high yield of Fab successfully applied to animal species including cattle fragments was found in the culture growth medium, (4), sheep (5, 6), rabbits (7-10), chickens (11, 12), camwhich enabled us to devise a rapid and simple singleels (13), and primates (14-17). The antibodies have step method for the purification of native (nondenabeen produced in both scFv and Fab formats utilizing tured) Fabs based on immobilized metal affinity vectors originally devised for human/murine immunolchromatography against a six-histidine amino acid ogy or expression systems optimized for the species carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to under investigation. The ability to produce mAbs from express and purify antigen-specific bovine Fab fraga range of animal species is expected to benefit veteriments from E. coli.
Rapid Two-Step Purification of a Recombinant Mouse Fab Fragment Expressed in Escherichia coli
Protein Expression and Purification, 2001
system offers the possibility to produce large amounts We report a rapid, large-scale process for the purifiof recombinant antibody fragments (2). These are useful cation of a recombinant Fab fragment specific for the in applications in which only the antigen-binding dotobacco mosaic virus coat protein (Fab57P). The fragmains of the antibody are required (3). Genetic engiment is expressed periplasmically in Escherichia coli. neering also provides the possibility to improve anti-The expression level was optimized in 0.3-L fermentors. body specificity and affinity for a given antigen. In this The highest levels were obtained using the following paper we describe a method for the purification of a conditions: (1) low postinduction temperature (21؇C), recombinant antibody fragment, Fab57P, specific for (2) combined use of two -lactam antibiotics (carbenithe tobacco mosaic virus coat protein (4). cillin and ampicillin), (3) IPTG concentration 0.1 mM,
We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E. coli is possible by the simultaneous secretion of both chains to the periplasm. To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident repressor gene and a filamentous phage origin. We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step. We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary. This modification has no deleterious effect on in vivo transport and folding or antigen affinity.