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Process Design Considerations for Large–Scale Chromatography of Biomolecules
Published in Kenneth E. Avis, Vincent L. Wu, Biotechnology and Biopharmaceutical Manufacturing, Processing, and Preservation, 2020
Richard Wisniewski, Egisto Boschetti, Alois Jungbauer
In spite of this unequivocal classification, there are examples of aerogel-xerogel hybrids with limited properties of shrinking-swelling. For more details see the review by Mikes et al. (1976). The chemical constitution of media and, more importantly, their surface characteristics are made to fit with the applications for which these media are designed. The separation of the constituents of complex mixtures is based on the differences in the protein-solid phase interaction of the individual components. Ion exchange, for instance, is one of the most common interactions used in separating proteins, which takes advantage of the difference in the total net charge of proteins at a given pH. Hydrophobic associations between low polar regions of proteins and hydrophobic ligands chemically attached to a polymer also constitute a valuable molecular interaction with the possibility of discriminating between proteins. More specifically, affinity chromatography uses biochemical ligands and is more focused on the biological properties of the proteins to separate than on physicochemical differences. Gel filtration takes advantage of differences between the molecular size of proteins and separates under a real molecular sieve effect. In practice, all of the mechanisms under which chromatographic separation of proteins occurs must be handled following known rules. Table 3.3 shows a summary of common experimental adsorption, elution, and regeneration conditions as a function of the molecular interaction mechanism.
Membrane Chromatography
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
Affinity chromatography is defined by using ligands that have a specific affinity for specific moieties of particular molecules. Immobilized Protein A is the most common use of affinity chromatography and is an excellent example of how this mode of chromatography operates. Protein A is a ligand that has a strong affinity for the fragment crystallizable region (Fc region) of antibodies. Under neutral pH conditions, the Fc regions of antibodies bind to Protein A. The Fc region will disassociate with Protein A when conditions in the mobile phase are changed to acidic (pH 2–4). This is a powerful purification tool when highly purified antibodies are desired and the mobile medium (process feed stream) is a complex mixture of contaminant molecules and target antibody. Contaminants flow through and antibodies bind to Protein A, as the mobile phase passes through the chromatography device. Once the entirety of antibody is bound, it can be stripped off or eluted as a separate fraction. The result is a process intermediate containing high purity antibody.
Chromatography
Published in Pau Loke Show, Chien Wei Ooi, Tau Chuan Ling, Bioprocess Engineering, 2019
Kirupa Sankar Muthuvelu, Senthil Kumar Arumugasamy
Many traditional purification methods have been replaced by affinity chromatography (Cuatrecasas et al., 1968). Among the purification techniques used, affinity chromatography comprises over 60% (Lowe et al., 1992). The powerful feature of any given biomolecule is that it has a unique inherent recognition site for the binding of natural and artificial substrates. Thus, purification in affinity chromatography is based on the specific binding or molecular recognition of an analyte by a ligand bound to a solid matrix. Affinity purification follows three major steps: The crude sample is incubated with the affinity support specific binding of the target molecule to the immobilized ligand.The unbound components are washed away from the support.By altering the buffer condition, the target molecule is eluted from the immobilized ligand (Roque and Lowe, 2008).
p(HEMA)-RR241 hydrogel membranes with micron network for IgG depletion in proteomic studies
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Kevser Kuşat, Selami Bağlamış, Cansu İlke Kuru, Fulden Ulucan, Murat Uygun, Sinan Akgöl
Affinity chromatography is a method based on the specific and reversible adsorption of the target biomolecule by ligands that are immobilized on an insoluble support material (matrix) [7]. Biological interactions between the ligand and the target molecule can occur through electrostatic or hydrophobic interactions, van der Waals, and/or hydrogen bonds. Elution of the target molecule from the medium is achieved either by using a competitive ligand or by changing the pH, ionic strength, or polarity of the medium (mobile phase) [8]. Molecular recognition-based affinity chromatography is a method that enables the separation of proteins according to their biological functions rather than their physical or chemical properties [9]. Affinity chromatography is divided into several sub-branches such as bioaffinity, immunoaffinity, DNA affinity, lectin affinity, boronate affinity, biomimetic affinity, and metal ion affinity. These methods can be used in the separation of many molecules such as proteins, enzymes, carbohydrates, vitamins, and in the purification of antigens and antibodies [10, 11].
Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus
Published in Preparative Biochemistry and Biotechnology, 2019
Jun Zhao, Hai-Yang Yu, Yu Zhao, Feng-Hua Li, Wei Zhou, Bin-Bin Xia, Zhi-Yuan He, Jason Chen, Guo-Tuo Jiang, Ming-Li Wang
Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between a molecular species and its ligand. Nickel columns are used for immobilized metal affinity chromatography (IMAC) for the purification of recombinant proteins with a 6 × His tag on either terminus. A recombinant protein with a 6 × His tag has a high affinity for nickel, whereas most other proteins will either bind with low affinity or not at all. After application of the sample, using a wash buffer with a low concentration of imidazole, which can wash nonspecific binding proteins or those that are weakly bound to the nickel column. The recombinant protein is usually eluted from the Ni column with a high concentration of imidazole.
Recent developments in purification techniques and industrial applications for whey valorization: A review
Published in Chemical Engineering Communications, 2020
Navpreet Kaur, Poorva Sharma, Seema Jaimni, Bababode Adesegun Kehinde, Shubhneet Kaur
Affinity chromatography (or adsorption chromatography) works on the adsorption mechanism which depends on the interaction between proteins and immobilized ligands. This method involves the selective adsorption and elution processes. Urtasun et al. (2017) implemented a novel methodology for the purification of bovine LPO by the application of dye affinity chromatography. Their study involved the comparison of packed-bed column and Red 4-Sepharose modes in terms of purification factor, purification yield and the need for any pretreatment(s). Results obtained showed that the batch mode operation of Red 4-Sepharose matrix showed a higher purification factor and yield at 46.1 ± 1.1 and 86.5 ± 3.8%, respectively without any pretreatment.