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    Affinity Chromatography in Bioprocess

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    Izreen Fara
    Affinity chromatography uses biological affinity between a ligand and target molecule to selectively separate molecules from a mixture. It has high selectivity and capacity depending on the ligand used. While it is effective, affinity chromatography is expensive for large-scale purification due to costly materials. It is commonly used to purify antibodies, antigens, enzymes, recombinant proteins, and other biomolecules by conjugating them to specific ligands like antibodies. Recombinant proteins are often purified using affinity tags like histidine that bind metals, while antibodies can take advantage of their specific binding to antigens.

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    Affinity Chromatography in Bioprocess

    Uploaded by

    Izreen Fara
    54 views3 pages
    Affinity chromatography uses biological affinity between a ligand and target molecule to selectively separate molecules from a mixture. It has high selectivity and capacity depending on the ligand used. While it is effective, affinity chromatography is expensive for large-scale purification due to costly materials. It is commonly used to purify antibodies, antigens, enzymes, recombinant proteins, and other biomolecules by conjugating them to specific ligands like antibodies. Recombinant proteins are often purified using affinity tags like histidine that bind metals, while antibodies can take advantage of their specific binding to antigens.

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    AFFINITY CHROMATOGRAPHY IN BIOPROCESS

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    Affinity chromatography uses biological affinity between a ligand and target molecule to selectively separate molecules from a mixture. It has high selectivity and capacity depending on the ligand used. While it is effective, affinity chromatography is expensive for large-scale purification due to costly materials. It is commonly used to purify antibodies, antigens, enzymes, recombinant proteins, and other biomolecules by conjugating them to specific ligands like antibodies. Recombinant proteins are often purified using affinity tags like histidine that bind metals, while antibodies can take advantage of their specific binding to antigens.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    54 views3 pages

    Affinity Chromatography in Bioprocess

    Uploaded by

    Izreen Fara
    Affinity chromatography uses biological affinity between a ligand and target molecule to selectively separate molecules from a mixture. It has high selectivity and capacity depending on the ligand used. While it is effective, affinity chromatography is expensive for large-scale purification due to costly materials. It is commonly used to purify antibodies, antigens, enzymes, recombinant proteins, and other biomolecules by conjugating them to specific ligands like antibodies. Recombinant proteins are often purified using affinity tags like histidine that bind metals, while antibodies can take advantage of their specific binding to antigens.

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    4.

    1 APPLICATIONS OF AFFINITY CHROMATOGRAPHY

    Affinity chromatography is basically separation based on biological affinity. Affinity


    chromatography can be known by the following characteristics:
     Its selectivity is high;
     The capacity can be very high depending on the ligand;
     High speed separation

    In the development of successful procedure for affinity chromatography purification,


    different classes of affinity targets, as well as different purification goals, which require
    consideration of different priorities that is either high purity or high yield, technical limitations
    and buffer conditions.
    Affinity chromatography is used from small-scale research to large scale
    purification. However, it is not used extensively in the large scale manufacture due to cost
    because the materials are expensive. Thus, it is necessary for choosing the right matrices
    for substance to be purified.

    4.2 APPLICATIONS FOR PURIFICATION

    Affinity chromatography is basically used in biotechnology, genetic engineering, biomedical


    and basic metabolic research at which can be used to purify these bio-molecules
    substances1:
    (i.) Antibodies and antigens
    Antigens are usually a protein substance foreign to the body and because
    being so, it stimulates the body’s immune system to produce the unique
    antibodies that can fight this antigen.
    (ii.) Enzymes and inhibitors
    (iii.) Regulatory enzymes
    (iv.) Recombinant proteins including hormone, enzyme and other binding proteins
    (v.) Receptors
    (vi.) RNA and DNA (genes)
    (vii.) Bacteria
    (viii.) Viruses and phages
    (ix.) Cells
    (x.) Others

    1
    B.Pascal, K.E. George, F. Wen Jian, B. Wolfgang, Affinity Chromatography: Methods and Protocols.
    Affinity chromatography effectively binds specific solutes by taking advantages on
    the solutes biological interactions. Antibodies, antigens, or dyes are conjugated to polymer
    resins for the purpose of binding specific solutes from a mixture. For example, an antibody
    could be raised against a target protein. The antibody then captures the solute out of the
    mixture, and the impurities flow through the column. The solute can be recovered by
    changing the pH, increasing the salt concentration, or adding displacer, that is, a molecule
    that also has some affinity for the antibody, or other binding agent (affinity ligand) on the
    stationary phase.
    Other examples such as isolation of proteins, it is made possible by using the
    special interactions of enzyme-ligand, enzyme-cofactor, receptor-agonist (antagonist). In
    each case, one member of any of these pairs may be immobilized to a resin to isolate the
    desired partner. Affinity chromatography is commonly coupled with cloning techniques to
    synthesize the target molecule with an “epitope” or recognition sequence that can be
    captured by the affinity ligand.
    Below are a few descriptions on application of affinity chromatography:

    4.1.1 Purification of Recombinant Proteins

    Recombinant protein is a protein that carries a code by a recombinant DNA.


    Recombinant DNA means that there are two segments of DNA in a plasmid. Plasmids are
    usually occurring in bacteria. When a recombinant DNA is inserted into bacteria, the bacteria
    will produce protein based on this recombinant DNA. This protein is called recombinant
    protein. The use of bacteria in order to produce recombinant protein has grown in the recent
    years. Using recombinant DNA and inserting it to a plasmid of rapidly reproducing bacteria
    enables the manufacture of recombinant protein. These recombinant proteins can be variety
    of types; they can be antibodies, antigens, hormones and enzymes.
    Recombinant proteins are commonly purified using this method. Proteins with a
    known affinity are protein tagged in order to aid their purification. The protein may have been
    genetically modified so as to allow it to be selected for affinity binding; this is known as a
    fusion protein. Tags include glutathione-S-transferase, hexahistidine (his), and maltose
    binding protein (MBP). His tags have an affinity for nickel or cobalt ions which are coordinate
    covalent bond with a chelator for the purposes of solid medium entrapment. For elution, an
    excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used.
    GST has an affinity for glutathione which is commercially available immobilized as
    glutathione agarose. During elution, excess glutathione is used to displace the tagged
    protein.

    4.1.2 Purification of Immunoglobulin

    Immunoglobulin or antibody is a protein used by the immune system for the purpose
    of defence against foreign substances such as bacteria and or viruses. The antibody
    recognizes the antigen. The antibody contains a paratope that is specific for one antigen,
    thus, aids in high attraction between the antibody and antigen. So, the antibody can tag a
    microbes or infected cell that may attack immune system.
    Due to the variety of antibody-antigen interactions has created many uses for
    antibodies and antibody fragments. They are used for therapeutic and diagnostic
    applications as well as for immunochemical techniques within general research. The use of
    recombinant technology has greatly expanded our ability to manipulate the characteristics of
    these molecules to our advantage. The potential exists to create an infinite number of
    combinations between immunoglobulin and immunoglobulin fragments with tags and other
    selected proteins. A significant advantage for the purification of antibodies and their
    fragments is that a great deal of information is available about the properties of the target
    molecule and the major contaminants, no matter whether the molecule is in its a native state
    or has been genetically engineered and no matter what the source material.

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