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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3
Separation and Purification of Biochemicals 663
purity levels of sometimes more than 99% are reached.
In some cases, e.g., in the case of assured virus removal,
a minimum number of steps with a certain performance
(measured in orders of magnitude or “logs”) is required
by the authorities. In a typical downstream process the
techniques are chosen in such an order as to produce a
good selectivity but also recovery for the target product,
in function of its surface properties, and to remove all im-
purities below the level of acceptance. The intricacy of the
process development is to optimize the downstream pro-
cess in such a way that both purification and quality prove
to be satisfactory. In order to meet the needs of industrial
biotechnological processes, purification methods should
be fast and highly specific, and highly cost-effective. The
primary goal is thus finding a suitable combination of dif-
ferent modes that present a balanced compromise between
yield and purity of the target biomolecule in as few steps
FIGURE 8 Size-exclusion chromatography. Molecules larger
than the upper exclusion limit cannot enter the intraparticular void (usually <5) as possible using complementary separation
space and elute first, whereas sufficiently small molecules have mechanisms. The following sequences are often found and
access to all the pores, and elute later. can be considered “classical”: IEC followed by HIC (good
interface, since the separation principles of “charge” and
For sample components of intermediate molecular “hydrophobicity” are orthogonal and the high salt elution
dimensions, the retention volume, V r ,isgivenby buffer typical in IEC is an excellent loading buffer for
HIC) followed by GF for polishing (e.g., removal of ag-
V r = V m + K D · V i , (17)
gregates). Another powerful sequence is presented by AC
where V m is the interstitial volume, K D is the distribution followed by IEC and finally GF.
ratio and V i is the intraparticular void volume. Among other things, the adopted strategy is dictated by
The magnitude of K D is determined by the fraction the composition of the feed. Thus, IEC can be suitable for
of intraparticular volume, which can be entered by the initial cleanup form crude broth, and AC can result in an
molecule of interest. Several models have been put for- initial high purification cum concentration provided foul-
ward to relate K D to the properties of the molecule and ing can be avoided. Within their respective limits, both
the chromatographic matrix. Plots of K D the logarithm of HIC and RPC are powerful techniques for the separation
the molecular mass are generally linear between K D val- of closely related molecules (e.g., the target molecule and
ues of approximately 0.15 and 0.8, and SEC may thus some of its degradation products). GF may be used for
also be used for a rough estimation of the molecular the final polishing step with the aim of removing impuri-
mass of a macromolecule. SEC is commonly carried out ties and contaminants differing from the product mainly
with cross-linked macroporous dextran-based beads such in size (e.g., product monomers from dimers). Tables II
as Sephadex from Pharmacia, or modified agarose and and III detail for each chromatographic mode the suitable
polyacrylamide-based gels, i.e., matrices that do not per- areas of application, i.e., initial capture, intermediate pu-
mit the use of high pressures. However, column packings rification or final polishing, with considerations on mobile
of high mechanical stability, ranging from silica-based and stationary phases in Table II, and on sample character-
materials to macroreticular rigid polymers, are increas- istics in Table III. The most important parameters for the
ingly becoming available for HPLC-SEC. optimization of each mode in regard to its successful ap-
plication in preparative chromatography are summarized
in Table III.
II. PROCESS DESIGN IN Chromatographic separations are traditionally batch
CHROMATOGRAPHY procedures. A certain volume of the mixture to be pro-
cessed (“sample,”“feed”) is introduced at one end of a
Anefficientpurification processfor a given biological usu- column packed with the stationary phase (convention-
ally consists of several consecutive steps, comprising a ally: porous particles) through which the mobile phase
combination of the different chromatographic modes de- is passed. The chromatographic separation of the sample
scribedabove.Onlyasequenceofseparationsaccordingto components takes place along the axis of the column and
different separation principles can assure that the required results from the differences in physical and/or chemical