Ionexchange chromatography

Several of the 20 amino acids that constitute the building blocks of proteins exhibit charged side chains. At pH 7.0, aspartic and glutamic acids have overall negatively charged acidic

Figure 6.8 Chromatographic columns. The glass column illustrated in (a) is manufactured by Merck. A wide variety of columns (ranging in size from 1 ml to several litres, and constructed from glass/plastic or stainless steel) are available from this and a number of other manufacturers (e.g. Bio-Rad and Pharmacia Biotech). (b) Process-scale chromatographic system. This particular system is utilized by a UK-based biotech company in the manufacture of a (protein) drug for clinical trials. The actual column is positioned to the left of picture

Figure 6.8 Chromatographic columns. The glass column illustrated in (a) is manufactured by Merck. A wide variety of columns (ranging in size from 1 ml to several litres, and constructed from glass/plastic or stainless steel) are available from this and a number of other manufacturers (e.g. Bio-Rad and Pharmacia Biotech). (b) Process-scale chromatographic system. This particular system is utilized by a UK-based biotech company in the manufacture of a (protein) drug for clinical trials. The actual column is positioned to the left of picture

Fractions (b)

Figure 6.9 The application of gel-filtration chromatography to separate proteins from molecules of much lower molecular weight. The mobile phase (the 'running buffer') will be devoid of the molecular species to be removed from the protein. Highly cross-linked porous beads are used, which exclude all protein molecules. The lower molecular weight substances, however, can enter the beads; therefore, their progress down through the column will be retarded (a and b). The earlier fractions collected will contain the proteins, and the latter fractions will contain the low molecular weight contaminants (c). In practice, this 'group separations' application of gel-filtration chromatography is mainly used to separate proteins from salt (e.g. after an ammonium sulfate precipitation step) or for buffer exchange. Note: in practice, the chromatographic beads are tightly packed in the column. They are separated from each other in this diagram only for the purpose of clarity. Also, the drawing is not to scale; protein molecules are considerably smaller than individual beads side groups, whereas lysine, arginine and histidine have positively charged basic side groups (Figure 6.10). Protein molecules, therefore, possess both positive and negative charges, largely due to the presence of varying amounts of these seven amino acids. (N-terminal amino groups and the C-terminal carboxy groups also contribute to overall protein charge characteristics.) The net charge exhibited by any protein depends on the relative quantities of these amino acids present in the protein, and on the pH of the protein solution. The pH value at which a protein molecule possesses zero overall charge is termed its isoelectric point (pi). At pH values above its pi, a protein will exhibit a net negative charge, whereas proteins will exhibit a net positive charge at pH values below the pi.

ion-exchange chromatography is based upon the principle of reversible electrostatic attraction of a charged molecule to a solid matrix that contains covalently attached side groups of opposite charge (Figure 6.11). Proteins may subsequently be eluted by altering the pH or by increasing the salt concentration of the irrigating buffer. ion-exchange matrices that contain covalently attached positive groups are termed anion exchangers. These will adsorb anionic proteins, e.g. proteins with a net negative charge. Matrices to which negatively charged groups are covalently attached are termed cation exchangers, adsorbing cationic proteins, e.g. positively charged proteins. Positively charged functional groups (anion exchangers) include species such as aminoethyl and diethylaminoethyl groups. Negatively charged groups attached to suitable matrices forming cation exchangers include sulfo- and carboxy-methyl groups (Table 6.3).

ASPARTATE

GLUTAMATE

COO"

COO"

CH2 COO"

COO"

ARGININE

HISTIDINE

LYSINE

COO"

COO"

ch2 ch2

Figure 6.10 Structures of amino acids having overall net charges at pH 7.0. In proteins, the charges associated with the a-amino and a-carboxyl groups in all but the terminal amino acids are not present, as these groups are directly involved in the formation of peptide bonds a a

NH3+

NH3+

nh3+

During the cation-exchange process, positively charged proteins bind to the negatively charged ion-exchange matrix by displacing the counter ion (often H+), which is initially bound to the resin by electrostatic attraction. Elution may be achieved using a salt-containing irrigation buffer. The salt cation, often Na+ of NaCl, in turn displaces the protein from the ion-exchange matrix. In the case of negatively charged proteins, an anion exchanger is obviously employed, with the protein adsorbing to the column by replacing a negatively charged counter ion.

The vast majority of purification procedures employ at least one ion-exchange step; it represents the single most popular chromatographic technique in the context of protein purification. Its popularity is based upon the high level of resolution achievable, its straightforward scale-up (for industrial application), together with its ease of use and ease of column regeneration. In addition,

ion-exchange bead

Figure 6.11 Principle of ion-exchange chromatography, in this case anion exchange chromatography. The chromatographic beads exhibit an overall positive charge. Proteins displaying a nett negative charge at the pH selected for the chromatography will bind to the beads due to electrostatic interactions ion-exchange bead

Figure 6.11 Principle of ion-exchange chromatography, in this case anion exchange chromatography. The chromatographic beads exhibit an overall positive charge. Proteins displaying a nett negative charge at the pH selected for the chromatography will bind to the beads due to electrostatic interactions it leads to a concentration of the protein of interest. It is also one of the least expensive chromato-graphic methods available. At physiological pH values most proteins exhibit a net negative charge. Anion-exchange chromatography, therefore, is most commonly used.

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