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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
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