Page 252 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

660 Separation and Purification of Biochemicals

4. Hydroxyapatite Chromatography centration in the mobile phase. By a different mechanism,
butwithcomparableefficacy,cationswithahighaffinityto
HA is an inorganic material that has been used as the sta-
phosphate, such as Ca 2+ or Mg , will displace basic pro-
2+
tionary phase for biopolymer chromatography since 1956.
teins from the HA surface. Negatively charged (“acidic”)
The early materials were soft powders (Tiselius apatite),
proteins are taken to bind via their carboxyl groups, which
but more recently ceramic HA and also fluoroapatite (FA)
chelate the C-sites at the surface. Consequently, proteins
beads have also become available, which are much more
with clusters of carboxylic groups are especially strongly
suitable to the requirements of chromatography in terms
bound. However, acidic proteins are also repelled by the
of mechanical strength and chemical stability. Both ap-
negatively charged apatite surface and therefore retained
atites are stable at elevated pH, but will dissolve rapidly
more weakly than basic proteins under standard condi-
below a pH of 5.0.
tions. They are readily displaced by phosphate, fluoride,
HA and FA bind both negatively and positively charged
and other anions capable of binding strongly to calcium.
substances, yet a simple ion exchange mechanism does not
The most common protocol in protein separation on HA
account for the observed chromatographic behavior. Two
calls for elution in a gradient of increasing phosphate
types of binding sites are present at the chromatographic
concentration. In this way, all proteins are eluted: first
surface—the calcium ions (C-sites) and the phosphate
the acidic proteins due to a specific complexation of the
groups (P-sites)—which are assumed to interact with the
C-sites by the phosphate ions and subsequently the basic
amino and carboxylic groups of the protein (see Fig. 7).
proteins due to general charge screening. A rather inter-
In contact with the mobile phase at neutral pH and
esting alternative for downstream processing is the double
above, the apatite surface carries a negative net charge as a
gradient method, where first the basic proteins are eluted
result of a surplus of phosphate groups. This is amplified
from the surface in a gradient, e.g., of increasing Cl con-
−
in most HA-chromatographic separations on apatite by
centration, followed by the elution of the acidic ones in a
the use of a phosphate buffer as mobile phase. Positively
phosphate gradient.
charged proteins (“basic” proteins) bind by electrostatic
The different retention mechanisms for acidic, neutral
interactions to this negatively charged surface. Desorption
and basic proteins facilitate the development of group sep-
is brought about by charge screening, i.e., a high salt con-
aration schemes in HA chromatography according to the
isoelectric points of the proteins. In processing products
obtained from mammalian cell cultures, HA chromatog-
raphy has, e.g., been used to separate serum albumin, an
acidic protein, from any more basic protein such as Im-
munoglobulin G (IgG), in a quick and simple fashion.
HA chromatography of proteins has been known to offer
high selectivity and high resolution. HA chromatography
is also used for the purification of nucleic acids.
C. Separation by Affinity (Biospecific
Interactions)
Affinity chromatography (AC) exploits biospecific inter-
actions for separation purposes and has become increas-
ingly popular in the last decade because of the unique
selectivity of the method. The basis for the interaction
in this case is the nearly perfect steric fit between two
molecules, the so-called affinity ligand anchored to the
stationary phase matrix and the target molecule. If this is
possible, a sufficient number of weak, usually noncova-
lent interactions can be realized, with the overall result of a
strong and highly specific retention of the target molecule.
However, affinity ligands used in preparative AC should
to allow for the elution of the product under conditions,
FIGURE 7 Different forces involved in the adsorption of charged which are not too harsh or even destructive. Under such
proteins on hydroxyapatite. circumstances, AC is the most adequate means to capture a