Ion Exchange Chromatography

Ion ExchangeChromatographyPrinciples and Methods18-1114-21Edition AA

Ion ExchangeChromatographyPrinciples and MethodsISBN 91 970490-3-4

Contents1. Introduction ............................................................................................92. Ion exchange chromatography .........................................................10The theory of ion exchange.................................................................10The matrix.....................................................................................11Charged groups .............................................................................13Resolution in ion exchange chromatography .................................13Capacity factor ........................................................................15Efficiency.................................................................................16Selectivity.................................................................................17Capacity ........................................................................................183. Product Guide.......................................................................................20MonoBeads ...................................................................................20MiniBeads .....................................................................................20SOURCE .......................................................................................20Sepharose High Performance ion exchangers .................................21Sepharose Fast Flow ion exchangers ..............................................21Sepharose Big Beads ion exchangers...............................................21STREAMLINE ion exchangers ......................................................21DEAE Sepharose CL-6B and CM Sepharose CL-6B .......................22DEAE Sephacel..............................................................................22Sephadex ion exchangers ...............................................................22Bulk quantities...............................................................................22Equipment.....................................................................................224. MonoBeads and MiniBeads ..............................................................23MonoBeads.....................................................................................23Properties ......................................................................................24Chemical stability ....................................................................24Physical stability ......................................................................25Flow rate..................................................................................27Capacity ..................................................................................27Recovery..................................................................................28Reproducibility ........................................................................29Availability ....................................................................................30MiniBeads .......................................................................................30Properties ......................................................................................31Chemical stability ....................................................................31Physical stability ......................................................................32Reproducibility ........................................................................33Availability ....................................................................................332

5. SOURCE................................................................................................34Properties ......................................................................................37Chemical stability ....................................................................37Flow rate..................................................................................38Capacity ..................................................................................40Recovery..................................................................................40Reproducibility ........................................................................41Availability ....................................................................................416. Sepharose based ion exchangers.......................................................42Chemical stability ..........................................................................42Physical stability ............................................................................43Sepharose High Performance ion exchangers.........................43Properties ......................................................................................44Physical stability ......................................................................44Capacity ..................................................................................44Flow rate..................................................................................46Availability ....................................................................................46Sepharose Fast Flow ion exchangers ........................................46Properties ......................................................................................47Physical stability ......................................................................47Capacity ..................................................................................47Flow rate..................................................................................49Availability ....................................................................................50Sepharose Big Beads ion exchangers.........................................50Properties ......................................................................................51Physical properties...................................................................51Capacity ..................................................................................51Flow rate..................................................................................51Availability ....................................................................................51STREAMLINE SP and STREAMLINE DEAE ......................52Properties ......................................................................................53Physical stability ......................................................................53Capacity ..................................................................................53Availability ....................................................................................54DEAE Sepharose CL-6B and CM Sepharose CL-6B.............54Properties ......................................................................................54Physical stability ......................................................................54Capacity ..................................................................................55Flow rate..................................................................................56Availability ....................................................................................577. DEAE Sephacel .....................................................................................58Properties ......................................................................................58Chemical stability ....................................................................58Physical stability ......................................................................593

Capacity ..................................................................................59Flow rate..................................................................................60Availability ....................................................................................608. Sephadex ion exchangers ...................................................................61Properties ......................................................................................61Chemical stability ....................................................................61Physical stability ............................................................................62Swelling ...................................................................................62Ionic strength dependence........................................................62pH dependence ........................................................................62Capacity ..................................................................................62Availability ....................................................................................649. Experimental design ............................................................................65Choice of ion exchanger ..............................................................65Specific requirements of the application .........................................65Column separation, batch separation or...................................65expanded bed adsorptionThe scale of the separation .......................................................65The required resolution............................................................65The required throughput..........................................................66Scaleability...............................................................................66Reproducibility ........................................................................67Economy .................................................................................67The molecular size of the sample components ................................67Choice of exchanger group ............................................................68Determination of starting conditions .......................................69The isoelectric point.................................................................69Test-tube method for selecting starting pH ...............................69Electrophoretic titration curves (ETC) .....................................70Chromatographic titration curves (retention maps) ..................74Choice between strong and weak ion exchangers ...........................76Choice of buffer.............................................................................76Choice of buffer pH and ionic strength.....................................76Choice of buffer substance .......................................................77Test-tube method for selecting starting ionic strengths..............7910. Experimental Technique.....................................................................80Column chromatography............................................................80Choice of column...........................................................................80Column design.........................................................................80Column dimensions .................................................................81Quantity of ion exchanger .............................................................81Preparation of the ion exchanger ...................................................81Pre-swollen ion exchangers ......................................................814

Pre-packed ion exchange media ...............................................81Sephadex ion exchangers .........................................................82Alternative counter-ions...........................................................82Decantation of fines.................................................................82Packing the column........................................................................82Column Packing Video Film.....................................................82Checking the packing...............................................................83Equilibrating the bed ...............................................................84Sample preparation........................................................................85Sample concentration...............................................................85Sample composition.................................................................85Sample volume.........................................................................85Sample viscosity.......................................................................85Sample preparation..................................................................86Sample application ........................................................................87Sample application with an adaptor .........................................87Other methods of sample application.......................................89Sample application onto a drained bed.....................................89Sample application under the eluent .........................................89Elution ..........................................................................................90Change of pH ..........................................................................90Change of ionic strength ..........................................................91Gradient direction....................................................................91Choice of gradient type ............................................................91Resolution using a continuous gradient ....................................93Choice of gradient shape ..........................................................94Sample displacement................................................................95Gradient generation.......................................................................96Gradient formation with two pumps or a single pump .............96in combination with a switch valveGradient Mixer........................................................................96Batch separation............................................................................97Expanded bed adsorption ...........................................................98Expanded bed technology ..............................................................99Basic principle of operation............................................................99STREAMLINE adsorbents ..........................................................100STREAMLINE columns ..............................................................100Auxiliary equipment ....................................................................100Regeneration ................................................................................101Cleaning, sanitization and sterilization procedures ............101Cleaning ......................................................................................101Sanitization .................................................................................101Sterilization .................................................................................101Protocols for cleaning-in-place (CIP),...........................................102sanitization and sterilization5

SOURCE and Sepharose Based ion exchangers......................102MonoBeads and MiniBeads columns......................................102DEAE Sephacel and Sephadex based ion exchangers..............103Storage of gels and columns......................................................103Prevention of microbial growth..............................................103Storage of unused media ........................................................104Storage of used media ............................................................104Storage of packed columns.....................................................104Determination of the available and dynamic capacities.....104Calculation ............................................................................10611. Process considerations .....................................................................107Defining the purpose ..................................................................108The strategic focus ......................................................................109Capture .......................................................................................109Intermediate purification .............................................................111Polishing......................................................................................111Selection of chromatography media .......................................112Base matrix properies and derivitization chemistry.................113Bead size................................................................................113Documentation and technical support....................................113Regulatory support ................................................................113Vendor certification ...............................................................114Delivery capacity ...................................................................114Method design and optimization ............................................114Binding conditions.......................................................................114Elution ........................................................................................115Sample load .................................................................................116Flow rate .....................................................................................117Selecting a column ......................................................................118Aspects of column design.............................................................119Flow distribution system ........................................................119Material resistance and durability ..........................................119Sanitary design.......................................................................119Pressure vessel safety..............................................................120Regulatory support ................................................................120Ergonomics............................................................................120Packing large scale columns......................................................120Column configuration .................................................................120Packing the column......................................................................121Scale-up .........................................................................................1216

12. Applications ........................................................................................124The design of a biochemical separation ........................................124Application examples ..................................................................127Enzymes ................................................................................127Isoenzymes ............................................................................128Immunoglobulins...................................................................129Nucleic acid separation ..........................................................129Polypeptides and polynucleotides...........................................130Antisense phosphorothioate oligonucleotides.........................132Areas of application.....................................................................133Purification of a recombinant Pseudomonas ...............................136aeruginosa exotoxin A, PE553DStrategy..................................................................................14113. Fault-finding chart .............................................................................14314. Ordering information .......................................................................15115. References............................................................................................1557

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1. IntroductionAdsorption chromatography depends upon interactions of different types betweensolute molecules and ligands immobilized on a chromatography matrix. The firsttype of interaction to be successfully employed for the separation of macromoleculeswas that between charged solute molecules and oppositely charged moietiescovalently linked to a chromatography matrix. The technique of ion exchangechromatography is based on this interaction.Ion exchange is probably the most frequently used chromatographic technique forthe separation and purification of proteins, polypeptides, nucleic acids, polynucleotides,and other charged biomolecules (1). The reasons for the success of ionexchange are its widespread applicability, its high resolving power, its high capacity,and the simplicity and controllability of the method.This handbook is designed as an introduction to the principles of ion exchangechromatography and as a practical guide to the use of the media available fromPharmacia Biotech. The handbook is illustrated with examples of different typesof biological molecules which have been separated using ion exchange chromatographyand different ways the technique can be used. For information on specificseparations, the reader is recommended to consult the original literature.9

2. Ion exchange chromatographyThe theory of ion exchangeSeparation in ion exchange chromatography depends upon the reversible adsorptionof charged solute molecules to immobilized ion exchange groups of oppositecharge. Most ion exchange experiments are performed in five main stages. Thesesteps are illustrated schematically below.?W&??W26X? ?W26X? ?W&??*@??.MB1? ?.MB1? W&@??N@??J5? ?J5? ?W.Y@?@?W.Y? ?*U? ?7Y?@?@??W.Y ?N1? ?@@@@?@??7Y? ?/KC5? @?@?e@??V40Y??@ @??@hg?@@@@??@?@ @? @?@??@ @? @?@??@ @? @?@?W2@6X?hf?@ ?@ @? @?@?7

The fourth and fifth stages are the removal from the column of substances not elutedunder the previous experimental conditions and re-equilibration at the startingconditions for the next purification.Separation is obtained since different substances have different degrees of interactionwith the ion exchanger due to differences in their charges, charge densitiesand distribution of charge on their surfaces. These interactions can be controlledby varying conditions such as ionic strength and pH. The differences in chargeproperties of biological compounds are often considerable, and since ion exchangechromatography is capable of separating species with very minor differences inproperties, e.g. two proteins differing by only one charged amino acid, it is a verypowerful separation technique.In ion exchange chromatography one can choose whether to bind the substancesof interest and allow the contaminants to pass through the column, or to bind thecontaminants and allow the substance of interest to pass through. Generally, thefirst method is more useful since it allows a greater degree of fractionation andconcentrates the substances of interest.The conditions under which substances are bound (or free) are discussed in detailin the sections dealing with choice of experimental conditions, Chapter 9. In additionto the ion exchange effect, other types of binding may occur. These effects aresmall and are mainly due to van der Waals forces and non-polar interactions.Ion exchange separations may be carried out in a column, by a batch procedure orby expanded bed adsorption. All three methodologies are performed in the stagesof equilibration, sample adsorption etc. described previously.The matrixAn ion exchanger consists of an insoluble matrix to which charged groups havebeen covalently bound. The charged groups are associated with mobile counterions.These counter-ions can be reversibly exchanged with other ions of the samecharge without altering the matrix.It is possible to have both positively and negatively charged exchangers (Fig. 2).Positively charged exchangers have negatively charged counter-ions (anions) availablefor exchange and are called anion exchangers. Negatively charged exchangershave positively charged counter-ions (cations) and are termed cation exchangers.The matrix may be based on inorganic compounds, synthetic resins or polysaccharides.The characteristics of the matrix determine its chromatographic propertiessuch as efficiency, capacity and recovery as well as its chemical stability, mechanical11

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Charged groupsThe presence of charged groups is a fundamental property of an ion exchanger.The type of group determines the type and strength of the ion exchanger; theirtotal number and availability determines the capacity. There is a variety of groupswhich have been chosen for use in ion exchangers (3); some of these are shown inTable 1.Table 1. Functional groups used on ion exchangers.Anion exchangersFunctional groupDiethylaminoethyl (DEAE) -O-CH 2 -CH 2 -N + H(CH 2 CH 3 ) 2Quaternary aminoethyl (QAE) -O-CH 2 -CH 2 -N + (C 2 H 5 ) 2 -CH 2 -CHOH-CH 3Quaternary ammonium (Q) -O-CH 2 -CHOH-CH 2 -O-CH 2 -CHOH-CH 2 -N + (CH 3 ) 3Cation exchangersFunctional groupCarboxymethyl (CM) -O-CH 2 -COO -Sulphopropyl (SP) -O-CH 2 -CHOH-CH 2 -O-CH 2 -CH 2 -CH 2 SO 3-Methyl sulphonate (S) -O-CH 2 -CHOH-CH 2 -O-CH 2 -CHOH-CH 2 SO 3-Sulphonic and quaternary amino groups are used to form strong ion exchangers;the other groups form weak ion exchangers. The terms strong and weak refer tothe extent of variation of ionization with pH and not the strength of binding.Strong ion exchangers are completely ionized over a wide pH range (see titrationcurves on page 49) whereas with weak ion exchangers, the degree of dissociationand thus exchange capacity varies much more markedly with pH.Some properties of strong ion exchangers are:• Sample loading capacity does not decrease at high or low pH values due to lossof charge from the ion exchanger.• A very simple mechanism of interaction exists between the ion exchanger andthe solute.• Ion exchange experiments are more controllable since the charge characteristicsof the media do not change with changes in pH. This makes strong exchangersideal for working with data derived from electrophoretic titration curves. (seeChapter 9)Resolution in ion exchange chromatographyThis section discusses the main theoretical parameters which affect the separationin ion exchange chromatography. For more in-depth information the reader isreferred to standard works on the subject (4, 5).The result of an ion exchange experiment, as with any other chromatographicseparation, is often expressed as the resolution between the peaks of interest.13

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Selectivity in ion exchange chromatography depends not only on the nature andnumber of the ionic groups on the matrix but also on the experimental conditions,such as pH and ionic strength. It is the ease and predictability with which theseexperimental conditions, and thus the selectivity, can be manipulated which givesion exchange chromatography the potential of extremely high resolution.CapacityThe capacity of an ion exchanger is a quantitative measure of its ability to take upexchangeable counter-ions and is therefore of major importance. The capacitymay be expressed as total ionic capacity, available capacity or dynamic capacity.The total ionic capacity is the number of charged substituent groups per gram dryion exchanger or per ml swollen gel. Total capacity can be measured by titrationwith a strong acid or base.The actual amount of protein which can be bound to an ion exchanger, under definedexperimental conditions, is referred to as the available capacity for the gel. Ifthe defined conditions include the flow rate at which the gel was operated, theamount bound is referred to as the dynamic capacity for the ion exchanger. Availableand dynamic capacities depend upon:The properties of the protein.The properties of the ion exchanger.The chosen experimental conditions.The properties of the protein which determine the available or dynamic capacityon a particular ion exchange matrix are its molecular size and its charge/pH relationship.The capacity of an ion exchanger is thus different for different proteins.On a porous matrix used for ion exchange chromatography, molecules which aresmall enough to enter the pores will exhibit a higher available capacity than thosemolecules which are restricted to the charged substituents on the surface of the gel.Similarly, since the interaction is ionic, the protein’s charge/pH relationship mustbe such that the protein carries the correct net charge, at a sufficiently high surfacecharge density, to be bound to a particular ion exchanger under the chosen bufferconditions.The properties of the ion exchange matrix which determine its available capacityfor a particular protein are the exclusion limit of the matrix, and the type andnumber of the charged substituents. High available capacity is obtained by havinga matrix which is macroporous and highly substituted with ionic groups whichmaintain their charge over a wide range of experimental conditions. Non-porous18

matrices have considerably lower capacity than porous matrices, but higher efficiencydue to shorter diffusion distances.The experimental conditions which affect the observed capacity are pH, the ionicstrength of the buffer, the nature of the counter-ion, the flow rate and the temperature.The flow rate is of particular importance with respect to dynamic capacity,which decreases as the flow rate is increased. These conditions should always betaken into consideration when comparing available capacities for different ionexchangers.Methodologies for determining the available and dynamic capacities for an ionexchanger are given in Chapter 10.19

?@f?@?@?@?/K?@e@@?@?@?@?V4?)X?'@?@?@@?W.? ?@?@)?V'e?@@?.Y????@f?@?@?@?/K??@@?????@e@@?@?@?@?V4@@@@@@6XeW2@@@S,?W&@@X@ ?@@@@@@@U?7>(R@@@I/?@0Y?@@ ?O2@??@@e@?@@@@@U@?? ?@@@@@@6Xe@?@?W?@f?@?@?@?/KS,?J@@@?7?@@?W.?@?@@??@? B@@???@e@@?@?@?@?V4 ?@@?.Y?@?@@?e?@@@@@@@U?7@??J@ ?@??@@e@?@?@??@@?????I/?@@??@@??@@???@f?@?@?@?/K?@e@@?@?@?@?V4@@@@@@6Xe@6T26 ??@?@e?@@?@@??@e?@@?@??@Khf?S,?J@@@R4 @ O2@??@@e@?@@@@@U@???@@?e?@g?@@@@@@@?e?@e@?@@@@@@@U?7@V'9? ?B@@??I/?@@?V4@??? ?@@??@@e@?@?@??@@??@@@@@@6Xe@@@6T???@@?@??@?@@?e???S,?J@@@>@@@@@@@@U?7@(R@>?@?@e?@@ O2@??@@e@?@@@@@U@??I/?@0Y?@0? ?@@????@Khf?@@@@@@@?e@?@?@ B@@???@@e@?@?@??@@???@@?e?@g? O2@???@@e@?@@@@@U@????@?@e?@@ B@@???@@??@Khf?@@e@?@?@??@@????@@@@@@@?e@@?@????@?@e?@@ O2@??@@e@?@@@@@U@??@Khf?@@@@@@@??@f@? B@@??@@e@?@?@??@@??3. Product GuidePharmacia Biotech manufactures a wide range of ion exchange media suitable foranalytical, micropreparative, small scale preparative, and process scale applications.The product range is summarized below.MonoBeads (page 23)Mono Q and Mono S are strong ion exchangers based on MonoBeads, monodisperse10 µm hydrophilic polymer particles. Mono Q and Mono S are the establishedstandards for high performance ion exchange separations and are best suitedfor analytical and small scale preparative applications.MiniBeads (page 30)MiniBeads, a non-porous matrix of monodisperse 3 µm hydrophilic polymer particles,is the base for two strong ion exchangers, Mini Q and Mini S. Both mediaare available pre-packed in Precision Columns 3.2/3, for micropreparative chromatographyin SMART System. With a specially designed column holder, thesecolumns can also be used in FPLC and HPLC systems.?@f?@?@?@?/K?@e@@?@?@?@?V4BioProcess?@??@@@@@@6XfO26S,?W2@@Y@@@@@@@@U?7UI'X@I/?@)?V4@??SOURCE (page 34)SOURCE 15Q, SOURCE 15S, SOURCE 30Q and SOURCE 30S are strong ionexchangers based on the same type of rigid polymer matrix as MonoBeads, polystyrene/divinylbenzene beads. SOURCE 15Q and SOURCE 15S are based on15 µm monodisperse particles while SOURCE 30Q and SOURCE 30S are basedon 30 µm mondisperse particles. SOURCE ion exchange media are designed forhigh performance applications at both research and industrial scales. They providehigh capacity at high flow rates and at a minimum of back-pressure, thus allowingshort cycle times, high productivity and scaleability.SOURCE 15 matrices are ideal for purification when very high resolution (efficiency)is required. SOURCE 30 matrices gives, in comparison with SOURCE 15matrices, slightly less resolution (lower efficiency) but at much lower back-pressure.This makes SOURCE 30 ideal for purification with more complex samples andlarger volumes. Using SOURCE 30, a higher degree of purification can be obtainedwith high productivity. Typically working flow rate ranges for ion exchangersbased on SOURCE 15 and SOURCE 30 are 30-600 cm/h and 300-1000 cm/hrespectively.20

?@f?@?@?@?/K?@e@@?@?@?@?V4?)X?'@?@?@@?W.? ?@?@)?V'e?@@?.Y????@f?@?@?@?/K??@@?????@e@@?@?@?@?V4@@@@@@6XeW2@@@S,?W&@@X@ ?@@@@@@@U?7>(R@@@I/?@0Y?@@ O2@?? ??@@e@?@@@@@U@??@@@@@@6Xe@?@?W?@@?W.?@?@@??@? B@@???@f?@?@?@?/KS,?J@@@?7?@@?.Y?@?@@?e??@e@@?@?@?@?V4?@@@@@@@U?7@??J@@@e@?@?@??@@????I/?@@??@@ @??@f?@?@?@?/K ?@@???@e@@?@?@?@?V4 ???@?@e?@@@@@@@@6Xe@6T26?@@??@e?@@?@?? ?@??S,?J@@@R4 O2@???@@?e?@g?@Khf?@@@@@@@?e?@e@?@@@@@@@U?7@V'9?@@e@?@@@@@U@???I/?@@?V4@ B@@??@@e@?@?@??@@?? ?@@????@@@@@@6Xe@@@6TS,?J@@@>@?@@?@??@?@@?e??@@@@@@@U?7@(R@> O2@??I/?@0Y?@0???@?@e?@@@@e@?@@@@@U@???@Khf? ?@@??@@@@@@@?e@?@?@?B@@??@@e@?@?@??@@????@@?e?@g???O2@??@@e@?@@@@@U@???B@@???@?@e?@@@Khf? ?@@????@@e@?@?@??@@??@@@@@@@?e@@?@????@?@e?@@ O2@??@@e@?@@@@@U@??@Khf?@@@@@@@??@f@ B@@??@@e@?@?@??@@?? ??@f?@?@?@?/K?@e@@?@?@?@?V4?)X?'@?@?@@?W.? ?@?@)?V'e?@@?.Y????@f?@?@?@?/K??@@?????@e@@?@?@?@?V4@@@@@@6XeW2@@@S,?W&@@X@ ?@@@@@@@U?7>(R@@@I/?@0Y?@@ O2@?? ??@@e@?@@@@@U@??@@@@@@6Xe@?@?W?@@?W.?@?@@??@? B@@???@f?@?@?@?/KS,?J@@@?7?@@?.Y?@?@@?e??@e@@?@?@?@?V4?@@@@@@@U?7@??J@@@e@?@?@??@@????I/?@@??@@ @??@f?@?@?@?/K ?@@???@e@@?@?@?@?V4 ???@?@e?@@@@@@@@6Xe@6T26?@@??@e?@@?@?? ?@??S,?J@@@R4 O2@???@@?e?@g?@Khf?@@@@@@@?e?@e@?@@@@@@@U?7@V'9?@@e@?@@@@@U@???I/?@@?V4@ B@@??@@e@?@?@??@@?? ?@@????@@@@@@6Xe@@@6TS,?J@@@>@?@@?@??@?@@?e??@@@@@@@U?7@(R@> O2@??I/?@0Y?@0???@?@e?@@@@e@?@@@@@U@???@Khf? ?@@??@@@@@@@?e@?@?@?B@@??@@e@?@?@??@@????@@?e?@g???O2@??@@e@?@@@@@U@???B@@???@?@e?@@@Khf? ?@@????@@e@?@?@??@@??@@@@@@@?e@@?@????@?@e?@@ O2@??@@e@?@@@@@U@??@Khf?@@@@@@@??@f@ B@@??@@e@?@?@??@@?? ??@f?@?@?@?/K?@e@@?@?@?@?V4?)X?'@?@?@@?W.? ?@?@)?V'e?@@?.Y????@f?@?@?@?/K??@@?????@e@@?@?@?@?V4@@@@@@6XeW2@@@S,?W&@@X@ ?@@@@@@@U?7>(R@@@I/?@0Y?@@ ?O2@??@@e@?@@@@@U@?? ?@@@@@@6Xe@?@?W?@f?@?@?@?/KS,?J@@@?7?@@?W.?@?@@??@? B@@???@e@@?@?@?@?V4 ?@@?.Y?@?@@?e?@@@@@@@U?7@??J@ ?@??@@e@?@?@??@@?????I/?@@??@@??@@???@f?@?@?@?/K?@e@@?@?@?@?V4@@@@@@6Xe@6T26 ??@?@e?@@?@@??@e?@@?@??@Khf?S,?J@@@R4 @ O2@??@@e@?@@@@@U@???@@?e?@g?@@@@@@@?e?@e@?@@@@@@@U?7@V'9? ?B@@??I/?@@?V4@??? ?@@??@@e@?@?@??@@??@@@@@@6Xe@@@6T???@@?@??@?@@?e???S,?J@@@>@@@@@@@@U?7@(R@>?@?@e?@@ O2@??@@e@?@@@@@U@??I/?@0Y?@0? ?@@????@Khf?@@@@@@@?e@?@?@ B@@???@@e@?@?@??@@???@@?e?@g? O2@???@@e@?@@@@@U@????@?@e?@@ B@@???@@??@Khf?@@e@?@?@??@@????@@@@@@@?e@@?@????@?@e?@@ O2@??@@e@?@@@@@U@??@Khf?@@@@@@@??@f@? B@@??@@e@?@?@??@@???@f?@?@?@?/K?@e@@?@?@?@?V4?)X?'@?@?@@?W.? ?@?@)?V'e?@@?.Y????@f?@?@?@?/K??@@?????@e@@?@?@?@?V4@@@@@@6XeW2@@@S,?W&@@X@ ?@@@@@@@U?7>(R@@@I/?@0Y?@@ ?O2@??@@e@?@@@@@U@?? ?@@@@@@6Xe@?@?W?@f?@?@?@?/KS,?J@@@?7?@@?W.?@?@@??@? B@@???@e@@?@?@?@?V4 ?@@?.Y?@?@@?e?@@@@@@@U?7@??J@ ?@??@@e@?@?@??@@?????I/?@@??@@??@@???@f?@?@?@?/K?@e@@?@?@?@?V4@@@@@@6Xe@6T26 ??@?@e?@@?@@??@e?@@?@??@Khf?S,?J@@@R4 @ O2@??@@e@?@@@@@U@???@@?e?@g?@@@@@@@?e?@e@?@@@@@@@U?7@V'9? ?B@@??I/?@@?V4@??? ?@@??@@e@?@?@??@@??@@@@@@6Xe@@@6T???@@?@??@?@@?e???S,?J@@@>@@@@@@@@U?7@(R@>?@?@e?@@ O2@??@@e@?@@@@@U@??I/?@0Y?@0? ?@@????@Khf?@@@@@@@?e@?@?@ B@@???@@e@?@?@??@@???@@?e?@g? O2@???@@e@?@@@@@U@????@?@e?@@ B@@???@@??@Khf?@@e@?@?@??@@????@@@@@@@?e@@?@????@?@e?@@ O2@??@@e@?@@@@@U@??@Khf?@@@@@@@??@f@? B@@??@@e@?@?@??@@???@f?@?@?@?/K?@e@@?@?@?@?V4BioProcess?@??@@@@@@6XfO26S,?W2@@Y@@@@@@@@U?7UI'X@I/?@)?V4@??Sepharose High Performance ion exchangers(page 43)Q and SP Sepharose High Performance are strong ion exchangers based on a 34µm highly cross-linked agarose matrix, providing high physical and chemical stability.These media are ideal for intermediate and final purification. They shouldbe used when resolution is the main objective. As resolution and efficiency aremaintained with increasing column diameter and sample load, separations usingthese media are easy to scale up. Typical working flow rates are 50-150 cm/h.?@f?@?@?@?/K?@e@@?@?@?@?V4BioProcess?@??@@@@@@6XfO26S,?W2@@Y@@@@@@@@U?7UI'X@I/?@)?V4@??Sepharose Fast Flow ion exchangers (page 46)Sepharose Fast Flow ion exchangers are based on 90 µm highly cross-linked 6%agarose beads of high chemical and physical stability. The range consists of theweak exchangers DEAE Sepharose Fast Flow and CM Sepharose Fast Flow as wellas the strong exchangers Q Sepharose Fast Flow and SP Sepharose Fast Flow. Theexceptional flow characteristics make these ion exchangers the first choice forseparating crude mixtures early in a purification scheme. Here, fast removal and acombination of good resolution and high flow rate are essential. Typical workingflow rates for these media are 100-300 cm/h. Sepharose Fast Flow ion exchangersare ideal for purifications with high demands on productivity.?@f?@?@?@?/K?@e@@?@?@?@?V4BioProcess?@??@@@@@@6XfO26S,?W2@@Y@@@@@@@@U?7UI'X@I/?@)?V4@??Sepharose Big Beads ion exchangers (page 50)Q and SP Sepharose Big Beads are strong ion exchangers designed for industrialapplications. They are based on 100-300 µm highly cross-linked 6% agarosebeads. The large particle size combined with high physical stability of the basematrix ensures rapid processing, even for viscous samples. Sepharose Big Beads istherefore the choice at the beginning of a purification scheme, where viscosity andback-pressure may limit the throughput attainable with ion exchangers based onsmaller bead sizes, such as Sepharose Fast Flow ion exchangers. The mediumshould be chosen when large volumes are handled and fast adsorption is requiredand when resolution is of less importance.?@f?@?@?@?/K?@e@@?@?@?@?V4BioProcess?@??@@@@@@6XfO26S,?W2@@Y@@@@@@@@U?7UI'X@I/?@)?V4@??STREAMLINE ion exchangers (page 52)STREAMLINE adsorbents, available as STREAMLINE DEAE andSTREAMLINE SP, are specially designed for use in STREAMLINE columns forexpanded bed adsorption. Together they enable the high flow rates needed forhigh productivity in industrial applications of fluidized beds. STREAMLINEadsorbents, based on cross-linked 6% agarose beads with a mean particle size of200 µm, are designed to handle samples directly from both fermentation homogenatesand crude samples from cell culture/fermentation at working flow rates oftypically 200-400 cm/h.21

DEAE Sepharose CL-6B and CM Sepharose CL-6B(page 54)DEAE and CM Sepharose CL-6B ion exchangers are based on 90 µm cross-linked6% agarose beads. These two gels are the traditional agarose based ion exchangersfrom Pharmacia Biotech. Their performance has been demonstrated in severalhundred applications for the separation of proteins, polysaccharides, nucleicacids, membrane components and other high molecular weight substances.Sepharose CL-6B based ion exchangers are typically used with working flow ratesof up to 60 cm/h.DEAE Sephacel (page 58)DEAE Sephacel is a beaded cellulose ion exchanger for separations over a widemolecular weight range (up to l x 10 6 for globular proteins). DEAE Sephacel is themedium of choice when a cellulose ion exchanger is needed for standard chromatographyof proteins, nucleic acids or other biopolymers.Sephadex ion exchangers (page 61)Sephadex ion exchangers are bead-formed media based on cross-linked dextran.They are available as strong and weak ion exchangers covering the pH range 2-10.Sephadex ion exchanger are suitable for batch-type applications.Bulk quantitiesAll Pharmacia Biotech ion exchangers are available in larger pack sizes or largerpre-packed columns. Contact your local Pharmacia Biotech supplier for furtherinformation.EquipmentPharmacia Biotech also supply a full range of equipment for operating all of theion exchangers covered in this handbook. Information regarding specific equipmentis available upon request from Pharmacia Biotech.22

?W&?W26X?W26X??*@?7

The name MonoBeads is derived from the unique monodisperse nature of thematrix. This monodispersity (Fig. 8) was accomplished through a process developedby Professor John Ugelstad of SINTEF, Trondheim, Norway.Fig. 8. An electron micrograph of MonoBeads showing their distinct monodispersity.The resolution which can be achieved on any chromatographic matrix is a resultof a combination of the efficiency and selectivity of the system. Maximum efficiencyis obtained through the use of small, perfectly spherical, monodisperse particles,optimally packed in a well designed column. All pre-packed MonoBeadscolumns have efficiencies at about 25 000 plates per metre. High efficiency, coupledwith the excellent selectivity of the Q and S substituents, results in high resolutionseparations.Scale-up to SOURCE Q and S (see Chapter 5), Q and SP Sepharose HighPerformance and Q and SP Sepharose Fast Flow (see Chapter 6) is simple, sincethese gels have similar selectivities to MonoBeads based media.PropertiesChemical stabilityThe gels are stable for continuous use in the pH range 2-12, although pH values ashigh as 14 can be used during cleaning and sanitizing procedures. MonoBeads canbe used with solutions of most buffers used in biochemical separations of biomoleculesand in water-alcohol (C1 - C4) and acetonitrile-water solutions.The resistance of the MonoBeads matrix to organic solvents allows complete cleaningand the use of conditions necessary for the solubilization of very hydrophobicsamples. An example of the use of MonoBeads with organic solvents is given inFigure 9, which shows the analysis of the peptide bacitracin on Mono S usinglithium chlorate as the eluting salt and 90% methanol as the liquid phase (7).24

W-X?7R1??J@?@L?7@@@)X??@e?@)KO-X?W-K??@e?(R4@?,?7R@@@@@@@@@??@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@S@U?@?@@?@@?@?@??@KO&?,?3T@@?@@?@?@??@@0R+Y?V+R'?@@?@?@??@?@?@?@?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?he?@@?heJ@@?he7@@?he@@@?he@@@?he@@@?he@@?J@?he@@ O2@@@@@@@@@@@@@@e?@?7@?he@@ O2@@@@0M?@?@@?he@@ O20M?@?@@?he@@ W20M?@?@@?he@@ ?O.M?@?@@?he@@ ?W20Y??@?@@?he@@ O.M??@?@@?he@@ W20Y?@?@@?he@@ ?W.M?@?@@?he@@ W.Y??@?@@?he@@ ?W.Y?@?@@?he@@ W.Y??@?@@?he@@ ?W.Y?@?@@?he@@ W.Y??@?@@?he@@ ?W.Y?@?@@?he@@ W.Y??@?@@?he@@ ?W.Y?@?@@?he@@L? W.Y??@?@?@?@?@?@?@?@?@?@?@?@?@?@@Lhe@V1?hf?W.Y?@V1he@?@?hfW.Y??@?@he@?@?he?W.Y?@?@he@?@?heW.Y??@?@he@?@?h?W.Y?@?@he@?@?hW.Y??@?@he@?@?g?O.Y?@?@he@?@?f?W20Y??@?@he@?@?fW.M??@?@he@?@?e?W.Y?@?@he@?@?eW.Y??@?@he@?@??W.Y?@?@he@?@?W.Y? @??@?@?@he@?@W.Y @??@?@?@he@?@(Y? @L?@?@?@he@?@H ?J@1?@?@?@he@@@? ?7Y@?@?@?@h?J@?@? ?@?@?@?@?@hW&5?@? ?@?@?@?@?@g?W&@H?@? ?@?@?@?@?@gW.Y@e@? ?@?3L??@?@?@f?W.Y?@e@? ?@?N1??@?@?@fW.Y??@e@? ?@e@??@?@?@f7He?@e@? ?@e@??@?@?@e?J5?e?@e@? ?@e@??@?@?@eW.Y?e?@e@? J5e@??@J5?@?W.Yf?@e@? 7He3L?@7H?@W.Y?f?@e@? @?eN1?@@??@(Yg?@e@? @?e?@?@@?J@H?g?@e@? @?e?@?@@?7@h?@e@? @?e?@?@@W@@h?@e@? @?e?@?@@(Y@h?@e@? @?e?3L??@?J@H?@h?@e@? @?e?N1??@W&@??@h?@e@? @?f@??@?W.Y@??@h?@e@? @?f@??@W.Y?@??@h?@e@? @?f@??@?W.Ye@??@h?@e@? @?f3L?@?7H?e@??@h?@e@? @?fN1?@J5f@??@h?@e@? ?J5?f?@?@?W.Yf@??@h?@e@? ?7H?f?@?@W.Y?f@??@h?@e@? ?@g?@?@@?f?W.Yg@??@h?@e@? ?@g?@?@@?fW.Y?g@??@h?@e@? ?@g?3L??@@?e?W.Yh@??@h?@e@? ?@g?N1??@@?e?7H?h@??@h?@e@? ?@h@??@@?eJ5he@??@hJ5e@? ?@h@??@?J@L?W.Yhe@??@h7He@? ?@h@??@?7R)T.Y?he@??@h@?e3L ?@h@??@?@?@(Yhf@??@h@?eN1 ?@h3L?@?@?@H?hf@??@h@?e?@ ?@hN1?@?@@@hf?J5??@h@?e?@ J5h?@?@J@?@hf?7H??@h@?e?@ 7Hh?3L??@7@?@hf?@e?@h@?e?@ @?h?N1??@?J@5?@hf?@e?@h@?e?@ @?he3Le?@hg?@W&@H?3L?he?@e?3L?g@?e?3L??)X? @?heN1e?@hg?@?W.Y@??N1?he?@e?N1?f?J5?e?N1?J@1? ?J5?he?3L??@L?hf?@W.Y?@?e@?he?@f3Lf?7H?f@?7R'L O.Y?he?N1??@1?hf?@?W.Ye@?e@?heJ5fN1f?@g3T5?N1 ?W2@0Y 3L?@@?hf?@?7U??J5?e3LfW-X?e7Hf?3=?eC5gV+Y??3L?hfO.M? N1?@@Lhf?@J@)X?7H?eN1?)X?7R1?e@?f?V46T20Yhe?V/XheO20Y ?3X@V1hf?@7@V/X@f?3T@)T5?3T-X@?g?I+M V/K?fO2@@0M ?V4@?3L?he?@@5?V4@f?V+MI+Y?V+R4@? ?V4@@@@@0M ?N1?he?@?O2@@@0Y 3Lhe?@N)X?h?@hO2@@@@@@@@@0M?@??@)Kh?@@@@@@@@@0Mhe?J5??7H? I4@@@@@?e?@J5?@?W.Y?@?@?@?@?@?@?@?@?@@@@@@@@@@@@@@@0Y?@?@?W.YW.Y?7H?J5?O.Y??7H?J5?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@@@@@?hg?@e@?@@@@@?W26Xf?@e@?@?@?@?7YV1f?@e@?@?@?@?@@@@f?@e@?@?@?@?3Xg?@e@?@?@?@?V4@?fFig. 9. Separation of the peptidebacitracin on Mono S.(Work by PharmaciaBiotech, Uppsala, Sweden.)Dimethylsulphoxide (DMSO) and similar solvents can be used, but will change theseparation properties of the gels. Aqueous solutions of urea, ethylene glycol andsimilar compounds can be used but will increase the back-pressures due to theirhigher viscosities. Non-ionic detergents, zwitterionic detergents or detergents withthe same charge as the ion exchange groups may be used. Oxidizing agents shouldbe avoided.Physical stabilityMonoBeads are based on highly rigid beads which means that they can be used athigh flow rates. As a consequence of the monodisperse nature of the matrix thesehigh flow rates do not result in high back-pressures. For example, an HR 5/5column (5 mm inner diameter and 50 mm bed height) packed with a MonoBeadsmatrix normally generates a back-pressure of 1.0-1.5 MPa (10-15 bar) when operatedat a flow rate of 1 ml/min (300 cm/h).Note: These back-pressures are beyond the operating limits of standard laboratoryperistaltic pumps.25

A summary of the characteristics of MonoBeads is shown in Table 2.Table 2. Characteristics of MonoBeads.Properties Mono Q Mono SType of gel strong anion exchanger strong cation exchangerCharged group -O-CH 2 -CHOH-CH 2 -O- -O-CH 2 -CHOH-CH 2 -O--CH 2 -CHOH-CH 2 -N + (CH 3 ) 3 -CH 2 -CHOH-CH 2 SO - 3Total ionic capacity (µmoles/ml gel) 270-370 140-180Total protein binding capacity (mg/ml gel)Thyroglobulin (MW 669 000) 25 N.D.HSA (MW 68 000) 65 N.D.a-lactalbumin (MW 14 300) 80 N.D.IgG (MW 150 000) N.D. 75Ribonuclease (MW 13 700) N.D. 75Typical protein recoveries (%) 90-100 90-100Typical enzyme activity recoveries (%) >80 >80Average particle size (µm) 10 ±0.5 10 ±0.5MW range (proteins) up to 10 7 up to 10 7working pH range* 3-11 3-11pH stability**long term 2-12 2-12short term 2-14 2-14N.D. = Not determinedSolvent restrictions: The ion exchangers are stable in alcohol/water solutions (C1-C4). 100% dimethylsulphoxide, dimethylformamide, and formic acid can change the separation properties of the gel.Avoid oxidizing and reactive reagents. Detergents can be used if they are non-ionic or have the samecharge as the gel.* working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.26

Flow rateThe rigid monodisperse nature of the media enables high flow rates to be used onMonoBeads columns. Normal recommended flow rates for high resolution separationsare in the range 150 to 600 cm/h for HR 5/5 columns. Higher flow ratescan be used during column washing and regeneration.In addition, the absence of buffering capacity means that buffer exchange andre-equilibration can be executed quickly and with small amounts of buffer. Detailsof the recommended flow rates to be used on the different columns are given inTable 3.CapacityThe high substitution levels coupled with the large pore size of the matrix, theexclusion limit for globular proteins is 10 7 , give MonoBeads exchangers highcapacities for large proteins as well as for smaller polypeptides and peptides.Typical saturation capacities are in the range of 60 mg protein per ml of gel andtypical sample loading capacities are in the region of 25 mg of protein per ml ofgel. Data on the saturation capacities for some specific proteins are given inTable 2.Table 3. Chromatographic properties of pre-packed columns of MonoBeads.Properties PC 1.6/5 HR 5/5 HR 10/10 HR 16/10 BioPilot Column35/100 60/100Column volume (ml) 0.1 1 8 20 100 300Column dimensions 1.6x50 5x50 10x100 16x100 35x100 60x100i.d. x bed height (mm)Recommended workingflow rate range (ml/min) 0.01-0.40 0.5-2.0 up to 6 up to 10 up to 32 up to 94Max operating 5 5 4 3 2 2pressure (MPa)Number of theoreticalplates per meter (N/m) 25 000 25 000 25 000 25 000 25 000 25 000Normal separationtimes (min) 5-20 5-20 40 40 60-90 60-9027

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AvailabilityMono Q and Mono S are available pre-packed in columns HR 5/5, HR 10/10and HR 16/10, containing 1, 8 and 20 ml of gel respectively. The media are alsoavailable in BioPilot Columns 35/100 and 60/100 containing respectively 100 and300 ml bed volumes, for chromatography at BioPilot scale. MonoBeads ionexchangers are also available as Mono Q PC 1.6/5 and Mono S PC 1.6/5, prepackedcolumns specially designed for micropurification on SMART System.These columns can also be used in other high performance chromatography systemsby using a column holder for Precision Columns. For further informationabout the column holder, please contact your Pharmacia Biotech representative.For ordering information, see Chapter 14.MiniBeadsMiniBeads is the base matrix for 3 µm high resolution ion exchange media. Thisnon-porous matrix, consisting of monodisperse hydrophilic polymer beads is substitutedwith Q and S functional groups to give Mini Q and Mini S. Both mediaare packed in Precision Columns 3.2/3 (3.2 mm inner diameter and 30 mm bedheight) for micropreparative chromatography on SMART System. With a speciallydesigned column holder, these columns can also be used in FPLC and HPLC systems.MiniBeads are highly efficient pH-stable adsorbents designed for high performancemicropurification of proteins, peptides and polynucleotides. The monodispersitypermits the use of high flow rates at relatively low back-pressures. The mainproperties are listed in Table 5.Due to the smaller particle size, Mini Q and Mini S give faster separations withhigher resolution of peaks than ion exchangers based on MonoBeads, seeFigure 12. This resolution is crucial for success when separating complex samplesin the pg to µg micropreparative scale.30

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Physical stabilityThe combination, non-porous and monodisperse beads gives MiniBeads very highphysical stability. It has better flow kinetics and can withstand higher back-pressure(up to 10 MPa) than MonoBeads.Note: These back-pressures are beyond the operating limits of standard laboratoryperistaltic pumps.Table. 5. Main properties of Mini Q PC 3.2/3 and Mini S PC 3.2/3.Properties Mini Q PC 3.2/3 Mini S PC 3.2/3Type of gel strong anion exchanger strong cation exchangerCharged group -O-CH 2 -CHOH-CH 2 -O- -O-CH 2 -CHOH-CH 2 -O--CH 2 -CHOH-CH 2 -N + (CH 3 ) 3 -CH 2 -CHOH-CH 2 SO - 3Total ionic capacity (µmoles/ml gel) 60-90 16-30Column dimensions i.d. x bed height (mm) 3.2 x 30 3.2 x 30Column volume (ml) 0.24 0.24Average particle size (µm) 3 µm 3 µmBinding capacity (mg/column)a-amylas (MW 49 000) Å 1.4 N.D.Trypsin inhibitor (MW 20 100) Å 1.4 N.D.Ribonuclease (MW 13 700) N.D. Å 1.3Lysozyme (MW 14 300) N.D. Å 1.3Max loading capacity (mg/column) 1-1.5 1-1.5Practical loading capacity (µg/column) ²200 ²200Typical protein recoveries (%) 70-90 70-90working pH range* 3-11 3-11pH stability**long term 3-11 3-11short term 1-14 1-14Maximum flow rate (ml/min) 1.0 1.0Operational pressure limit (MPa) 10 10Normal separation times (min) 5-20 5-20N.D. = Not determined* working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.32

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Table 6. Characteristics of SOURCE 15Q and 15S, and SOURCE 30Q and 30S.Properties SOURCE 15Q SOURCE 30Q SOURCE 15S SOURCE 30SType of gel strong anion exchangers strong cation exchangersCharged group -O-CH 2 -CHOH-CH 2 -O- -O-CH 2 -CHOH-CH 2 -O--CH 2 -CHOH-CH 2 -N + (CH 3 ) 3 -CH 2 -CHOH-CH 2 SO - 3MatrixPolystyrene/divinyl benzeneBead formRigid, spherical, porous monodisperseMean particle size (µm) 15 30 15 30Dynamic bindingcapacity* (mg/ml gel)Lysozyme (MW 14 500) N.D. N.D. 80 80BSA (MW 67 000) 45 45 N.D. N.D.MW range (proteins) up to 10 7 up to 10 7 up to 10 7 up to 10 7working pH range** 2-12 2-12 2-12 2-12pH stability***long term 2-12 2-12 2-12 2-12short term 1-14 1-14 1-14 1-14Maximum flow rate (cm/h) 1800 1 2000 2 1800 1 2000 2Recommended working 30-600 300-1000 30-600 300-1000flow rate range (cm/h)Operating temperature (°C) 4-40 4-40 4-40 4-40N.D. = Not determinedSolvent restrictions: SOURCE is stable in alcohol/water solutions (C1-C4). 100% dimethyl sulphoxide,dimethylformamide, and formic acid can change the separation properties of the gel. Avoid oxidizingand reactive reagents. Detergents can be used if they are non-ionic or have the same charge as the gel.* Determined by frontal analysis at a flow rate of 300 cm/h, using a 5.0 mg/ml solution of protein in 20mM sodium phosphate buffer, pH 6.8 (lysozyme) and 20 mM BIS TRIS PROPANE buffer, pH 7.0(BSA).** working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.*** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.1) Maximum flow rate to be applied, will depend on the pressure specification of the chromatographicsystem used. A linear flow rate of 1800 cm/h will give a pressure drop of approximately 10 MPa at abed height of 3 cm.2) Maximum flow rate to be applied, will depend on the pressure specification of the chromatographicsystem used. A linear flow rate of 2000 cm/h will give a pressure drop of approximately 10 MPa at abed height of 10 cm.A uniquely wide pore size distribution and a large specific surface area offer excellentresolution and capacity for a wide range of molecules; from small peptidesand oligonucleotides up to large proteins. The performance is well maintained athigh flow rates and high sample loads. This is illustrated in Figure 16 and 17,which show separations of model protein mixtures on SOURCE 30Q.35

Fig. 16. The influence of increasing flow rate on resolution. (Work by Pharmacia Biotech,Uppsala, Sweden.)Fig. 17. The influence of increasing sample load on resolution. (Work by PharmaciaBiotech, Uppsala, Sweden.)SOURCE 15 and SOURCE 30 ion exchangers are designed for research andindustrial applications, with emphasis on high performance, high capacity, highreproducibility, and easy scaleability.In comparison with media based on SOURCE 15 matrix, SOURCE 30 givesslightly less resolution (lower efficiency) but at much lower back-pressure. Thismakes SOURCE 30 ideal for purifications with more complex samples and largervolumes. Using SOURCE 30, a high degree of purification can be obtained with36W-X?@?e@?7R1?@?e@??J@?@?@?e@??7@@@@@?e@??@e?@@=?C5??@e?(R4@0Y?Column: SOURCE 30Q, 10 mm i.d. x 50 mm (4 ml)Sample: Mixture of lactoglobulin B and amyloglucosidaseSample load: 1 mg/ml bed volumeEluent A: 20 mM BIS-TRIS PROPANE, pH 7.0Eluent B: 0.5 M sodium chloride, 20 mM BIS-TRIS PROPANE, pH 7.0Flow rate: a) 4 ml/min (300 cm/h)b) 13 ml/min (1000 cm/h)W-X?@?e@?7R1?@?e@?Gradient: 0-100% B, 20 column volumes?J@?@?@?e@??7@@@@@?e@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@? @? ?@@?@? @? ?@@?@? @? ?@@?@L @? ?@@??J@1 @? ?@@??7Y@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? ?@@??@?@ @? @? ?@@??@?@ @? @? ?@@??@hf?@?@ ?J@L @? ?@@??@hf?@?@ ?7R1 @? ?@@??@hf?@?@ ?@?@ @? ?@@??@hf?@?@ ?@?@ @? ?@@??@e?@@=?C5??@e?(R4@0Y?J@L?he?@?@ ?@?@ @? ?@ ?@@?7R1?he?@?@ ?@?@ @? ?@ ?@@?@?@?he?@?@ J5?3L? @? ?@ ?@@?@?@?he?@?3L? 7H?N1? @? ?@ J@L?@?@?@?he?@?N1? @?e@? @? ?@ 7R1?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e@? @? ?@ @?@?@?@?@?he?@e@? @?e3L @? ?@ ?J5?@?@?@?@?he?@e@? @?eN1 @? ?@ ?7H?3L@?@?@?he?@e@? @?e?@ @? ?@ ?@eN1@?@?@?he?@e@? @?e?@ @? ?@ ?@e?@@?@?@?he?@e@? ?J5?e?@ @? ?@ ?@e?@@?@?@?he?@e@? ?7H?e?@ @? ?@ ?@e?@@?@?@?heJ5e@? ?@f?@ @? ?@ ?@e?@@?@?@?he7He@? ?@f?@ @? ?@ ?@e?@@?@?@?he@?e@? ?@f?@ @? ?@ ?@e?@@?@?@?he@?e@? ?@f?@ @? ?@ @?he?@e?@@??J5?@?he@?e@? ?@f?@ @? ?@ @Lhe?@e?@@??7H?@?he@?e@? ?@f?@ @? ?@ ?J@1he?@e?@@??@e@?he@?e@? ?@f?@ @? ?@ ?7Y@he?@e?@@??@e@?he@?e@? ?@f?@ @? ?@ ?@?@heJ5e?@@??@e@?he@?e@? ?@f?@ @? ?@ ?@?@he7He?@@??@e@?he@?e@? ?@f?@ @? ?@ ?@?@he@?e?3L?@??@e@?he@?e@? ?@f?@ @? ?@ J5?@he@?e?N1?@??@e@?he@?e@? ?@f?@ @? ?@ 7H?@he@?f@?@??@e3Lhe@?e@? ?@f?3L? @? ?@ @??@he@?f@?@??@eN1he@?e@? ?@f?N1? @? ?@ @??@he@?f@?@??@e?@he@?e@? ?@g@? @? ?@ @??@he@?f@?@??@e?@he@?e@? ?@g@? @? ?@ @??3L?h@?f@?@??@e?@he@?e3L ?@g@? @? ?@ @??N1?h@?f@?@??@e?@he@?eN1 ?@g@? @? ?@ ?J5?e@?h@?f@?@??@e?@he@?e?@ ?@g@? @? ?@ ?@e?7H?e@?h@?f@?@??@e?@he@?e?@ ?@g@? @? ?@ ?@L??@f@?g?J5?f@?@??@e?@he@?e?@ ?@g@? @? ?@ ?@1??@f@?g?7H?f@?@??@e?@he@?e?@ ?@g@? @? ?@ J@@L?@f@?g?@g@?@??@e?@he@?e?@ J5g@? @? ?@ 7

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I4@@0Y?@M??@@@@@@@@@@@@?I4@0?4@@@??@6. Sepharose based ionexchangersPharmacia Biotech offers a range of ion exchange media based on agarose, whichis cross-linked to produce Sepharose High Performance, Sepharose Fast Flow,Sepharose Big Beads, Sepharose CL-6B and STREAMLINE base matrices.Exchanger groups are attached to the gels by stable ether linkages to the monosaccharideunits to give the final ion exchange gels.The polysaccharide chains of Sepharose based ion exchangers are arranged inbundles (Fig. 24). These bundles are further strengthened by different degrees ofintra-chain cross-linking which provide a high matrix rigidity. The resulting structureis macroporous and the capacity of the gels very good for globular proteinsup to 10 6 in molecular weight.The gels, particularly the most highlycross-linked forms, e.g. Sepharose FastFlow and Sepharose High Performance ionexchangers, have good flow properties andstable bed volumes that are largely insensitiveto changes in ionic strength and pH.They also show extremely low non-specificadsorption of macromolecules (8).Fig. 24. Structure of cross-linked agarose gels.Chemical stabilitySepharose ion exchangers tolerate extreme working conditions of temperature,pH and chemical agents. They are stable in water, salt solutions, and organic solvents.Details on the pH stability range for each gel is given in the appropriate sectionbelow.The ion exchangers can be used in solutions of non-ionic detergents such asTriton X-100 ® and with strongly dissociating solvents such as 8 M urea and 6 Mguanidine hydrochloride (9). The Sepharose based ion exchangers also tolerate1 M acetic acid, 30% isopropanol, 30% acetonitrile, 70% ethanol and 1 MNaOH. Under oxidizing conditions, limited hydrolysis of the polysaccharidechains may occur.The gel-forming fibres of agarose are stiff bundles of polysaccharide chains ratherthan flexible single chains (10). For this reason the water in the gel can be replacedby other solvents with relatively little effect on pore size. Sepharose ion exchangersTriton ® is a registred trademark of Rohm and Haas Co.42

can be used in polar organic solvents and in aqueous/organic mixtures. The gelmatrix is stable in a wide variety of solvents including ethanol, dimethylformamide,tetrahydrofuran, acetone, dimethylsulphoxide, chloroform, dichloromethane,dichloroethane and dichloroethane/pyridine (50:50).Sepharose is very resistant to microbial attack due to the presence of the unusualsugar, 3,6-anhydro-L-galactose. However, most buffers can support bacterialgrowth and so a antimicrobial agent should be used in storage (see page 103).Physical stabilityThe highly cross-linked structure of the modern Sepharose based ion exchangers,e.g. Sepharose Fast Flow and Sepharose High Performance, not only gives themincreased chemical stability but also results in improved physical stability. Thisimproves flow properties enormously compared to Sepharose CL-6B gels. Crosslinkingprevents fluctuations in bed volume under conditions of increasing ionicstrength. Thus Sepharose ion exchangers can be regenerated and re-equilibratedrepeatedly in the column. Repacking between experiments is thus eliminated,improving reproducibility.Sepharose ion exchangers can be used at temperatures up to 70 °C and can be sterilizedrepeatedly in the salt form by autoclaving at 121 °C, pH 7, for 30 minutes.Sepharose High Performance ionexchangersSepharose High Performance ion exchange media are based on 34 µm highlycross-linked agarose beads. The small bead size gives the media high efficiency,which in combination with high selectivity offers the possibility of high resolutionstandard chromatography separations.In addition, the use of identical functional groups to those used in Q andSP Sepharose Fast Flow, Mono Q and Mono S and SOURCE Q and SOURCE Smedia, at comparable substitution levels (i.e. same selectivity), simplifies scalingup from FPLC and up to BioPilot and BioProcess scales.43

PropertiesPhysical stabilityThe high degree of cross-linking of Sepharose High Performance renders themedia extremely stable physically. The high rigidity of the cross-linked agarosematrix eliminates volume variations due to changes in pH or ionic strength.CapacityAs is the case with all ion exchangers, the capacity depends upon the accessibilityof the charged groups and their number. Sepharose High Performance gels havean exclusion limit of approximately 4 x 10 6 and are highly substituted with strongion exchange groups. Thus they remain charged and maintain consistently highcapacities for proteins over a broad working pH range (Table 7). This allows theselection of a pH value and buffer that best suit the properties of the sample.Titration curves for Q and SP Sepharose High Performance are similar to thosefor Q and SP Sepharose Fast Flow, which are shown in Figure 26.Capacity varies from case to case depending on protein properties and flow rate.As an example, the dynamic capacity for human serum albumin onQ Sepharose High Performance is approximately 70 mg per ml gel at 150 cm/h(start buffer 20 mM Tris, pH 8.2). The dynamic capacity for ribonuclease onSP Sepharose High Performance has been estimated to 55 mg/ml gel at 150 cm/h(start buffer 0.1 M sodium acetate, pH 6.0). The characteristics of the media areshown in Table 7. Characteristics specific for HiLoad, HiTrap and BioPilotcolumns pre-packed with Q and SP Sepharose High Performance are shown inTable 8.44

Table 7. Characteristics of Q and SP Sepharose High Performance.Product Q Sepharose SP SepharoseHigh Performance High PerformanceType of gel strong anion strong cationTotal ionic capacity (µmol/ml gel) 140-200 140-200Dynamic binding capacity* (mg/ml gel)BSA (MW 67 000) 70 N.D.Ribonuclease (MW 13 700) N.D. 55Recommended working up to 150 up to 150flow rate range (cm/h)Approx. mean particle size (µm) 34 34Particle size range (µm) 24-44 24-44working pH range** 2-12 4-13pH stability***short term 1-14 3-14long term 2-12 4-13N.D. = Not determined* The dynamic binding capacity was determined at a linear flow rate of 150 cm/h using a 10.0 mg/mlsolution of BSA in 20 mM Tris buffer, pH 8.2 and a 5.0 mg/ml solution of ribonuclease in 100 mMsodium acetate, pH 6.0.** working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.*** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.Table 8. Characteristics of HiLoad, HiTrap and BioPilot columns pre-packed with Q andSP Sepharose High Performance.Product HiLoad HiTrap BioPilot Column16/10 26/10 1 ml 5 ml 35/100 60/100Column dimensions (mm) 16x100 26x100 7x25 16x25 35x100 60x100(inner diameter x bed height)Bed volume (ml) 20-22 53-58 1 5 100 300Maximum flow rate* (ml/min) 5 13 4 20 25 70Recommended working up to 5 up to 13 1 5 up to 24 (for SP) up to 70 (for SP)flow rate range (ml/min) up to 16 (for Q) up to 47 (for Q)Max pressure* (MPa) 0.3 0.3 0.3 0.31.1 1.1Number of theoretical >12 000 >12 000 N.D. N.D. >10 000 >10 000plates per meter** (N/m)* Max. pressures and flow rates should not be used routinely.** Determined with acetone.45

?W2@@@?*U?@@@@@@@?he@@e?@@@@@?@@? ?V4@@@@@?@@@@6X?@@@@@?e@@e@@@@@?hW2@6X?he?@@?@@ @@L??@@?e?@@? @@?@@??@1?@@f?J@@L?@@hf7@V4)?he?@@??W2@6Xh?W2@@6X?@@e@@fW2@@6X?@@??@@?e?@6X?@@?@@g@@e@@?@@??@@?@@f?7@@1?@@hf3@e?O2@@6X?@@@6X@@@6X?@@6X?@@@@@@6X?W2@@@@@@6X?e@@f@@6X?W2@@@@@@??@@?e?@@W2@@@@6?2@?@@W&(?')X?g?7@?I4)?@@e@@f7@?I4)?@@??@@?e?@@1?@@?h@@e@@?@@??@@?@@@@@??@@@@?@@@@@?hV4@@@@@??@1?@@?@@@@?@1e?@1?@@@@e@1?*@??@@??@1?e@@@@@?e@1?*@?e@@H??@@@@@?@@@@??@@@@@@?@5@@@@@@@?@@?@@@@?@@W2@@@?@@@@e@@?V4@@@@@@@@?e@@e?W2@@@?V4@6X@@e?@@?e?@@@@??@@@@@@@@H 7@H?N@1?g?@@?f@@e@@f@@f?@@??@@?e?@@@?@@?h@@e@@?@@??@@?@@fJ@@@@?@@@@e?@@?g?@@?f@@@@@@f@@f?@@@@@@?e?@@@@@@?h@@@@@@?@@??@5?@@e?O&@e@@@@he?'6Ke@@@?f@@?@@@@?@@@@@@@?@@@@e@5f@@@?g@@e?*@@@@f@@@@L??@@?e?@@@@??@@@@@@@@??@@@@?e3@L?J@5?@@@@e?@@?f@@e@@W26X@@f?@@??@@W26X@@?@@@??@@@@?e@@e@@?@@@@0Y?@@@@@@@@e@@@@@@@@g?V4@@@0?4@@@@?@@@0?4@?@0?4@@@?@0?4@@0Y?@@@0?4@@@@?e@@e?V4@@@?@@@0MI4@??@@?e?@0?4@@0MI40?4@?@@@@?O20M??O20M??O2@

stability allows the gels to be used at the higher flow rates required for modernlaboratory separations as well as meeting the throughput and cleaning-in-placerequirements of process scale chromatography.To give a complete range of ion exchange media Sepharose Fast Flow is availablewith the weak exchanger groups, DEAE and CM and the strong exchanger groupsQ and SP. Figure 25 shows the partial structures of these media. Characteristics ofthe different ion exchangers based on Sepharose Fast Flow is listed in Table 9.Table 9. Characteristics of Q, SP, DEAE and CM Sepharose Fast Flow.Product Q Sepharose SP Sepharose DEAE Sepharose CM SepharoseFast Flow Fast Flow Fast Flow Fast FlowType of gel strong anion strong cation weak anion weak cationTotal ionic capacity 180-250 180-250 110-160 90-130(µmol/ml gel)Recommended 100-300 100-300 100-300 100-300working flow raterange (cm/h)Approx. meanparticle size (µm) 90 90 90 90Particle size range (µm) 45-165 45-165 45-165 45-165working pH range* 2-12 4-13 2-9 6-10pH stability**short term 1-14 3-14 1-14 2-14long term 2-12 4-13 2-13 4-13* working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.PropertiesPhysical stabilitySepharose Fast Flow ion exchangers are supplied pre-swollen and ready for packingor in pre-packed columns. The highly cross-linked nature of the matrix meansthat the bead size and bed volumes do not change with changes in ionic strengthor pH.CapacityAs is the case with all ion exchangers the capacity is dependent upon the accessibilityof the charged groups and their number. Sepharose Fast Flow ion exchangersare highly substituted and have an exclusion limit of approximately 4 x 10 6 giving47

high capacity for proteins. Capacity data for Fast Flow ion exchange media aregiven in Table 10. Characteristics specific for pre-packed columns with Q and SPSepharose Fast Flow, HiLoad columns, are shown in Table 11.Table 10. Capacity data for Sepharose Fast Flow ion exchangers.Ion ExchangerQ Sepharose SP Sepharose DEAE Sepharose CM SepharoseFast Flow Fast Flow Fast Flow Fast FlowTotal ionic capacity(µmol/ml gel) 180-250 180-250 110-160 90-130Dynamic binding capacity*(mg/ml gel)Thyroglobulin (MW 669 000) 3 N.D. 3.1 N.D.HSA (MW 68 000) 120 N.D. 110 N.D.a-lactalbumin (MW 14 300) 110 N.D. 100 N.D.IgG (MW 160 000) N.D. 50 N.D. 15Bovine COHb (MW 69 000) N.D. 50 N.D. 30Ribonuclease (MW 13 700) N.D. 70 N.D. 50N.D. = Not determined*Capacities were determined using the method described in Chapter 10 at a flow rate of75 cm/h. For anion exchangers (DEAE and Q) the starting buffer was 0.05 M Tris, pH 8.3and for cation exchangers (CM and S) 0.1 M acetate buffer, pH 5.0. Limit buffers were therespective start buffers containing 2.0 M NaCl.Table 11. Characteristics of HiLoad columns pre-packed with Q andSP Sepharose Fast Flow.Product HiLoad 16/10 HiLoad 26/10Bed volume (ml) 20-22 53-58Maximum flow rate* (ml/min) 10 26Recommended working up to 7 up to 18flow rate range (ml/min)Max pressure* (MPa) 0.3 0.3Number of theoreticalplates per meter** (N/m) >3000 >3000* Max. pressures and flow rates should not be used routinely.** Determined with acetone.Q Sepharose Fast Flow and SP Sepharose Fast Flow are highly substituted withstrong ion exchange groups. These groups remain charged and maintain consistentlyhigh capacities over broad working pH ranges of 2-12 and 4-13 respectively.This allows the selection of a pH value and buffer that best suit the propertiesof the sample. Titration curves for both gels are shown in Figure 26. The workingpH ranges for DEAE Sepharose Fast Flow and CM Sepharose Fast Flow are 2-9and 6-10 respectively.48

?@e?@?@@6X@e?@?@?B@@@@@@?@e@@e?@?@?C@@e?@?@@0R'e?@?@?@?W&?W26X?*@?.MB1?N@?e?@@?W.f?@@@@@@?7Yh?@@?@@@@g?@Fig. 26. Titration curves; approximately 5 ml Q and SP Sepharose Fast Flow in50 ml 1 M KCl. (Work by Pharmacia Biotech, Uppsala, Sweden.)Flow rateThe optimal cross-linking of Sepharose Fast Flow confers excellent flow propertieson the matrix. This is illustrated in Figure 27 which shows the relationship betweenflow rate and operating pressure for DEAE Sepharose Fast Flow.W& @K?@@@@@?@e?@?W26X?@?@?@??@@??'6T&@W26Xe?W26T2@@@@f@@@??@e?@?7

Flow rates achievable with Fast Flow media are above 300 cm/h at 0.1 MPa(1 bar) in an XK 50/30 column packed with a 15 cm high bed of gel. In laboratoryseparations where the best possible separation is frequently a major considerationthis flow rate is frequently traded off against improved resolution. In industrialprocessing, the high throughput properties of Sepharose Fast Flow ion exchangersgive significantly reduced cycle times and improved productivity.AvailabilityDEAE and CM Sepharose Fast Flow are available in packs of 500 ml, 10 and 60litres. Q and SP Sepharose Fast Flow are available in packs of 25 ml, 300 ml, 5and 60 litres and are also available pre-packed in HiLoad 16/10 and 26/10columns. Q, DEAE and CM Sepharose Fast Flow are supplied in 20% ethanol andSP Sepharose Fast Flow in 20% ethanol with 0.2 M sodium acetate. For orderinginformation, please refer to Chapter 14.Sepharose Big Beads ion exchangersSepharose Big Beads ion exchangers are based on 100-300 µm agarose beads. Ahigher degree of cross-linking, compared to Sepharose CL-6B based ion exchangers,is used to give the media greatly improved physical and chemical stability.Due to its excellent physical stability and large bead size, Sepharose Big Beads canbe run at high flow rates even with viscous samples. Table 12, gives the characteristicsof Q and SP Sepharose Big Beads.Table 12. Characteristics of Q and SP Sepharose Big Beads.Product Q Sepharose Big Beads SP Sepharose Big BeadsType of gel strong anion strong cationTotal ionic capacity (µmol/ml gel) 180-250 180-250Recommended workingflow rate range (cm/h) 1200-1800 1200-1800Approx. mean particle size (µm) 200 200Particle size range (µm) 100-300 100-300working pH range* 2-12 4-13pH stability**short term 2-14 3-14long term 2-12 4-13* working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.50

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STREAMLINE SP andSTREAMLINE DEAESTREAMLINE adsorbents are specifically developed for expanded bed adsorption,see page 98 for details, and are based on a modified Sepharose matrix, crosslinked6% agarose.STREAMLINE adsorbents have been designed with a well-defined density distribution,which is required in expanded bed adsorption. This is achieved throughinclusion of inert crystalline quartz material in the base matrix. Mean particledensity is approximately 1.2 g/ml drained gel. The porosity is comparable toSepharose Fast Flow ion exchangers, with an exclusion limit of 4 x 10 6 forglobular proteins.Table 13. Characteristics of STREAMLINE ion exchange adsorbents.Product STREAMLINE SP STREAMLINE DEAEType of gel strong cation weak anionTotal ionic capacity (µmol/ml gel) 170-240 130-210Dynamic binding capacity* (mg/ml gel)Lysozyme (MW 14 500) 70 N.D.BSA (MW 67 000) N.D. 55Recommended working flow rate range (cm/h)- sample application 200-400 200-400- elution 50-150 50-150Approx. mean particle size (µm) 200 200Particle size range (µm) 100-300 100-300working pH range** 4-13 2-9pH stability***short term 3-14 1-14long term 4-13 2-13N.D. = Not determined*The binding capacity was determined in STREAMLINE 50 column at a linear flow rate of 300 cm/husing a 2.0 mg/ml solution of protein in phosphate buffer, pH 7.5 (lysozyme) and 50 mM Tris-HClbuffer, pH 7.5 (BSA).** working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.*** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.52

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?@@@@6X?@@@@@?e@@e?@@@@@f@@?W2@@6X?he@@?@@??@1?@@f?J@@L??@@?f?J@5?7@?I4)Khe@@???W2@@6X?f@@e@@g??7@?I4)?f@@e@@g??@@?h@@e@@g??@@?h@@@@@@g??@@?h@@e@@g??3@?O26T26X?@@e@@W2@@e??V4@@0R+MS1?@@e@@@Yf?O.hW&@?f?I4@6Xe?O20Yg?W&YheS,e?O20Mh?&@@@?g@@0Ye??W20M?O.M ??@@??O20Y? ??@@???@@@@@@?he?O20M???@@?he?O20M???@@?h?W20M??O.M??O20Y?O20M?W20M?.MW2@@6X?@@?fW2@@6X?@@@@6?2@?heW2@6X?h?W2@@6?2@??@@?f?W2@@6X?@@e@@f@6X?@@h?@@??@@?hf?W2@@6X?@@?@@@@?g?7@?I4)?@@?f7@?I4)?@@??@@@@Lh?W&(?')Xh?7@?I4@@@??@@?f?7@?I4)?@@e@@f@@1?@@h?@@??@@?hf?7@?I4)?@@hf??@@??@@?@@f?7@@1??@@?f?7@H?3@?eV@@@6?2@@6X?@@@6X?@@@6?2@@@@@@6X?W2@@@@@@6X?e@@f?@@?f@@f?@@??@(Y@1h?7@H?N@1h?@@?e?@@??@@?f?@@?f@@e@@f@@@?@@h?@@??@@?hf?@@?f@@hf??@@??@@?@@@@@??@@@@??@@@@@e?@@??V4@@@@@e@@@@?@1?@@?@1?e?@@@@@@@e@1?*@??@@??@1?e@@f?@@?f@@@@6X?@@@@@e@@h?@@?e@@h?@@?e?@@@@@@?f?@@?f@@@@@@f@@@@@@h?@@@@@@?hf?@@?f@@hf??@@??@@?@@fJ@@@@L?@@?f?@@?f?@@@@@@@@@?@@?@@?@@W2@@@@@@?@@e@@?V4@@@@@@@@?e@@f?@@??@@@@@e@1?@@??@1?@@e@@@@e?3@L?J@5e@@@@e?@@?e?@@??@@?f?@@?f@@e@@f@@?@@@e@@@@e?@@??@@?hf?@@?f@@hf??@@??@5?@@e?O&@e@1?@@?f?@@?'6K??@@@f@@?@5?@@?@@@@?@@@@@?3@e@5f@@@?g3@?O2(?@@?e?I'@e@5?@@??@5?@@h?V')?&(Yh?3@?O2@@@??@@?W26X?3@?O2(?@@e@@@6X?@@?3@@h?@@??@@?hf?3@?O2(?@@hf??@@@@0Y?@@@@@@@@e@@?@@@@@@??3@LV4@@@0?4@@@@@@@0Y?@@?@0?4@@@@@@?V4@@0Y?@@@0?4@@@@?eV4@@0Y?@@@@@eV4@@0Y?@@@@0YJ@5heV4@0Y?h?V4@@0?4@??@@?.MS1?V4@@0Y?@@e@0MS1?@@?V4@h?@@??@@?hf?V4@@0Y?@@hf??7@H ?W&@ W&@? ??N@1hf@@?@@? W&Y?hf?W&Y ?@@hf@@?&@@@hf?&@@@?g/K?V46X?I/K???V46K???I46X???I/K?V46X?I/K???V46K???I46X???I/K?V46X?I/K???V46K??I46X??W2@@6X?f@@e@@g??I/??7@?I4)?f@@e@@g??@@?h@@e@@g??@@?h@@@@@@g??@@?e?O26X?@@e@@W2@@e??3@?O2@0MS1?@@e@@@Yf??V4@@0M?W&@?@@e@@@@6Xe??W&YheS,e??&@@@?g@@0Ye?????????????????????????@@? ??@@@@???@@?W2@@6X?@@?f?W2@@6?2@@@6X?@@heW2@6X?h?W2@@6X?@@e@@f?W2@@6X??W2@6X ?@6X?@@?e?@@@@@@? ??W2@@6X?@@@??@@@e@@?W2@@6X?he?@@?7@?I4)?@@?f?7@?I4@@@??@1?3@L?g?W&(?')Xh?7@?I4)?@@e@@f?7@?I4)?W&(?')X? ?@@1?@@?f?@@? ??7@?I4)?@@@??@@@?J@5?7@?I4)?he?@@??@@@?@@?@@@6T@@? ??@@?f@@@@@@@@?7@H?3@?e?W2@@6?2@@6X?@@@6T2@@6X?@@@@@@6X?W2@@@@@@6X?e@@f?@@?f?@@?e?@@??@5?N@1?g?7@H?N@1h?@@?f@@e@@f?@@?f7@H?N@1??@@@@@@?e?@@< ??@@?f@@@@@@@@?@@??V4@@6?&@??@@@@?@1?@@?@@Y??@1?@@@@e@1?*@??@@??@1?e@@f?@@?f?@@@@6X@@@@@e?@@?g?@@?e@@h?@@?f@@@@@@f?@@?f@@e?@@??@@?@@@W2@@@@? ??@@?f@@@@@@@@?@@?f?@@@@@@@@@@?@@?@@?@@@@@@@?@@@@e@@?V4@@@@@@@@?e@@f?@@?e@@@@@??@@@@??@1??@@??@@@@??3@L?J@5e@@@@e?@@?f@@e@@W26X?@@?f3@L?J@@@6X?@@?3@@@@?@@@? ??3@?O2(?@@?@@?@@?@@?'6K??@@@@?e?@@?@5?@@?@@@?@@@?@@@@e@5f@@@?g3@?O2(?@@?fI'@??@@@@??@5??@@?g?V')?&(Yh?3@?O2(?@@e@@(MS1?3@?O2(?V')?&@0MS1?@@?V40?4@@@@? ??V4@@0Y?@@?@@?@@?3@LV4@@@0MI4@@@@@@@0Y?@@?@@@@@@@?@0?4@@0Y?@@@0?4@@@@?eV4@@0Y?@@@@@e?V4@@0?4@@@0Y?J@5?hV4@0Y?h?V4@@0Y?@@e@0YW&@?V4@@0Y??V4@0M?W&@7@H? W&Y? W&Y? ??N@1hf?@@?@@ &@@@ &@@@ ??????@@hf?@@?AvailabilitySTREAMLINE SP and DEAE are available in packs of 300 ml and 7.7 litres.For ordering information, please refer to Chapter 14.DEAE Sepharose CL-6Band CM Sepharose CL-6BDEAE Sepharose CL-6B and CM Sepharose CL-6B are macroporous bead-formed(mean particle size of 90 µm diameter) ion exchangers derived from the crosslinkedagarose gel Sepharose CL-6B. DEAE or CM groups are then attached to thegel by ether linkages to the monosaccharide units to give the final ion exchange gel(Fig. 30).DEAE Sepharose CL-6B and CM Sepharose CL-6B have good chemical and physicalstability and can be used to advantage in the ion exchange chromatography ofproteins, polysaccharides, nucleic acids, membrane components and other highmolecular weight substances.Fig. 30. Partial structure of Sepharose CL-6B ion exchangers.PropertiesPhysical stabilityDEAE and CM Sepharose CL-6B are supplied pre-swollen and ready for packing.As stated earlier, the cross-linked nature of the matrix means that the bed volumechanges very little with changes in ionic strength or pH (approximately 2% changewhen the pH is reduced from 10 to 4).54

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Table 14. Characteristics of Sepharose CL-6B ion exchangers.DEAE Sepharose CL-6BCM Sepharose CL-6BTotal ionic capacity (µmol/ml gel) 130-170 100-140Dynamic binding capacity* (mg/ml gel)Thyroglobulin (MW 669 000) 2.0 N.D.IgG (160 000) N.D. 9.5Bovine COHb (MW 69 000) N.D. 75HSA (MW 68 000) 170 N.D.a-lactalbumin (MW 14 300) 150 N.D.Ribonuclease (MW 13 700) N.D. 120Recommended working flow rate range (cm/h) up to 60 up to 60Approx. mean particle size (µm) 90 90Particle size range (µm) 45-165 45-165working pH range** 2-9 6-10pH stability***short term 2-14 2-14long term 3-12 4-13N.D. = Not determined*Capacities were determined using the method described in Chapter 10 at a flow rate of 75 cm/h. Forthe anion exchanger (DEAE) the starting buffer was 0.05M Tris, pH 8.3 and for the cation exchangers(CM) 0.1 M acetate buffer, pH 5.0. Limit buffers were the respective start buffers containing 2.0 MNaCl.** working pH range refers to the pH range over which the ion exchange groups remain charged andmaintain consistently high capacity.*** pH stability, long term refers to the pH interval where the gel is stable over a long period of timewithout adverse effects on its subsequent chromatographic performance.pH stability, short term refers to the pH interval for regeneration and cleaning procedures.Flow rateThe cross-linked structure of Sepharose CL-6B ion exchangers allows flow rates ofup to 100 cm/h to be used. Figure 32 illustrates the variation of flow rate withpressure drop for DEAE and CM Sepharose CL-6B.56

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8. Sephadex ion exchangersSephadex ion exchangers are produced by introducing functional groups ontoSephadex, a cross-linked dextran matrix. These groups are attached to glucoseunits in the matrix by stable ether linkages.Sephadex is suitable as a base for an ion exchanger matrix since it is hydrophilicand shows very low non-specific adsorption.Sephadex ion exchangers are derived from either Sephadex G-25 orSephadex G-50 and swell readily in aqueous solutions. Ion exchangers based onSephadex G-25 are more tightly cross-linked than those based on Sephadex G-50and therefore swell less and have greater rigidity. Ion exchangers based onSephadex G-50 are more porous than those based on Sephadex G-25 and thereforehave a better capacity for molecules with molecular weights larger than 30 000.The full range of Sephadex ion exchangers is shown in Table 16. Anion and cationexchangers are designated as A-25 or A-50 and C-25 or C-50, respectively, dependingon the matrix porosity.Table 16. Sephadex ion exchangers.Types Description Functional groups Counter ionDEAE A-25 Weakly basic Diethylaminoethyl ChlorideSephadex A-50 anion exchangerQAE A-25 Strongly basic Diethyl-(2-hydroxy- ChlorideSephadex A-50 anion exchanger propyl)aminoethylCM C-25 Weakly acidic Carboxymethyl SodiumSephadex C-50 cation exchangerSP C-25 Strongly acidic Sulphopropyl SodiumSephadex C-50 cation exchangerPropertiesChemical stabilitySephadex ion exchangers are insoluble in all solvents. They are stable in water, saltsolutions, organic solvents, alkaline and weakly acidic solutions. In strongly acidicsolutions, hydrolysis of the glycosidic linkages may occur and thus pH valuesbelow 2 should be avoided, particularly at elevated temperatures. Sephadex ionexchangers can also be used in the presence of denaturing solvents which can beimportant when substances are to be separated on the basis of their electrostaticproperties alone (12, 13, 14).Exposure to strong oxidizing agents or dextranases should be avoided. Duringregeneration, the ion exchanger can be exposed to 0.2 M NaOH for a short time61

without appreciable hydrolysis. Sephadex ion exchangers are susceptible to attackby dextranases and should be stored in the presence of an antimicrobial agent (seepage 103).Physical stabilitySwollen Sephadex ion exchangers can be sterilized by autoclaving for up to 30 minat 121 °C, at neutral pH in the salt form. During autoclaving, minute quantities ofcarbohydrate are released; these can be washed away with sterile buffer.SwellingThe swelling properties of Sephadex ion exchangers are related to those of theparent Sephadex G-types, those based on 50-types swelling more than those basedon 25-types. Due to the presence of charged groups in the matrix, the swellingvaries with ionic strength and pH.Ionic strength dependenceAt low ionic strengths, repulsion between groups carrying the same charge on thematrix is maximal, and swelling of the gel is at its greatest. The degree of swellingdecreases with increasing ionic strength.Note: Sephadex ion exchangers should not be swollen in distilled water since thebead structure may be broken down due to strong ionic interactions.pH dependenceThe degree of dissociation and hence the extent to which an ion exchanger is chargedis dependent on pH. Repulsion between charged groups is greatest at pH valueswhere the ion exchanger is fully dissociated, and decreases at pH values close tothe pK of the charged groups.Note: QAE Sephadex and SP Sephadex have swelling properties quite independentof pH since they are charged over a very wide pH range.CapacityDue to differences in swelling characteristics, ion exchangers based on SephadexG-25 have a much higher ionic capacity per ml gel than those based on SephadexG-50. (Table 17).62

?W&?W26X?*@?.MB1?@e?@?@@6X@e?@?@?B@@@@@@?@e@@e?@?@?C@@e?@?@@0R'e?@?@?@?N@?e?@f@@@@@?W.h?@@?7Yh?@@?@@@@g?@W&?W26X?*@?.MB1?@?e@?@@6X@?e@?@?B@@@@@@?@??@@?e@?@?C@@?e@?@@0R'?e@?@?@?N@f@?e?@@@@@?@?W.?h?@?@?7Y?h?@?@?@@@@?g?@O2@@@@@@@@@@@@@@@@@@@@@@@@@? ?@ O2@@@@@@@@@@@@@@@@@@ @@@@@@@@@@@@@? ? ?@?????????????????O2@@@@@@@0M ?@ O2@@@@@@@@0M?@O2@@@@@@@0M? ?@ O2@@@@0M?@O2@@@@0M ?@ O2@@0M?@O20M ?@ ?O2@0M?@O20M ?@ ?O2@@0M??@O20M ?@ ?O20M??@O20M ?@ ?W20M??@O20M ?@ ?7

If working with larger molecules (MW > 100 000), a higher available capacity isfrequently observed with the A-25 and C-25 types since at these molecular weightsbinding is only occurring on the bead surface and the higher ionic capacity can beused to advantage.As capacity also depends upon the number of substituent groups which are chargedunder given buffer conditions, it will also vary with pH. The variation of thecharge on Sephadex ion exchangers with pH is illustrated by their titration curves(Fig. 36).Dynamic capacity data for Sephadex ion exchangers are given in Table 18.Table 18. Dynamic capacity (mg/ml wet gel) data for Sephadex ion exchangersProtein Thyroglobulin HSA a-lactalbumin IgG Bovine COHb(MW) (669 000) (68 000) (14 300) (160 000) (69 000)Ion exchangerDEAE A-25 1.0 30.0 140.0 N.D. N.D.Sephadex A-50 2.0 110.0 50.0 N.D. N.D.QAE A-25 1.5 10.0 110.0 N.D. N.D.Sephadex A-50 1.2 80.0 30.0 N.D. N.D.CM C-25 N.D. N.D. N.D. 1.6 70.0Sephadex C-50 N.D. N.D. N.D. 7.0 140.0SP C-25 N.D. N.D. N.D. 1.1 70.Sephadex C-50 N.D. N.D. N.D. 8.0 110.0N.D. = Not determinedCapacities were determined using the method described in Chapter 10 at a flow rate of 75 cm/h. Foranion exchangers (DEAE and QAE) the starting buffer was 0.05M Tris, pH 8.3 and for cationexchangers (CM and SP) 0.1 M acetate buffer, pH 5.0. Limit buffers were the respective start bufferscontaining 2.0 M NaCl.AvailabilitySephadex ion exchangers are supplied as dry powders in packs of 100 g and 500 g.Bulk quantities of 5 kg or more are available on request. For ordering information,please refer to Chapter 14.64

9. Experimental designChoice of ion exchangerNo single ion exchanger is best for every separation. The choice of matrix andionic substituent depends on:1. The specific requirements of the application2. The molecular size of the sample components3. The isoelectric points of the sample componentsSpecific requirements of the applicationColumn separation, batch separation or expanded bed adsorptionIf the separation is to be carried out using a batch separation technique ratherthan column chromatography, the flow and packing characteristics of the matrixare of minor importance. The economy and high capacity of Sephadex based ionexchangers make them a natural choice.In large scale applications, capturing proteins from crude samples containing particulatematter, expanded bed adsorption using STREAMLINE has proven to beeffective and cost efficient.The scale of the separationThe amount of sample to be processed is an important parameter when choosingan ion exchange medium. For laboratory scale separations, any of the PharmaciaBiotech range of ion exchangers can be used. However for large scale separations,which must satisfy the throughput and cleaning-in-place (CIP) requirements ofindustry, the choice of a BioProcess Media such as SOURCE, Sepharose High Performance,Sepharose Fast Flow, Sepharose Big Beads or STREAMLINE adsorbentis indicated.The same reasoning applies to experiments designed as method scouting for eventualscale-up since such procedures should be developed using the gel which willeventually be used at the larger scale. SOURCE media and Sepharose Fast Flowbased exchangers are extremely well suited to this type of method optimization aswell as routine laboratory separations.The required resolutionWhen choosing an ion exchanger it is important to decide the degree of resolutionrequired from the separation. Normally analytical or semi-analytical separations65

place high demands on resolution. In contrast, resolution is frequently traded offagainst capacity and speed in the case of preparative work.Resolution in ion exchange chromatography depends upon the selectivity and efficiencyof the media. Maximum selectivity is often obtained by choosing one of thegels carrying the strong exchanger groups Q or S/SP, since strong ion exchangerscan be used at any pH tolerated by the sample molecules.Maximum efficiency is obtained by choosing a gel based on a small particle sizematrix. The media in order of their particle sizes and potential efficiencies areMiniBeads (3 µm) > MonoBeads (10 µm) > SOURCE 15 (15 µm) > SOURCE 30(30 µm)> Sepharose High Performance (34 µm) > Sepharose Fast Flow/ SepharoseCL-6B/ Sephacel (90 µm) > Sephadex (40-125 µm in dry form) > STREAMLINEadsorbents/Sepharose Big Beads (200 µm).The media thus offering the highest degree of resolution are MiniBeads,MonoBeads and SOURCE 15 exchangers for high resolution in SMART, FPLCand HPLC systems and SOURCE 30 and Sepharose High Performance exchangersfor high resolution standard chromatography.The required throughputHow much material which can be processed in a defined time is determinedamongst other things by the capacity, the flow characteristics of the media and thesize of the column. All of the ion exchangers available from Pharmacia Biotechhave high capacities for macromolecules but differ considerably in their flow properties.The media which have optimal flow characteristics are MiniBeads formicropreparative chromotography in SMART System, MonoBeads and SOURCE15 for high performance, FPLC separations, and SOURCE 30, Sepharose HighPerformance, Sepharose Fast Flow and Sepharose Big Beads media for laboratoryand process scale preparative separations.The uniform size distribution of beads in SOURCE media provides comparativelylow pressure drops over packed beds and thus makes SOURCE ion exchangersalso ideal for scaling up in industrial applications such as the separation of closelyrelated product variants.ScaleabilityFrequently ion exchange separations are carried out initially on a small scale tooptimize conditions before committing the sample to full scale separations. It isthus important to choose an ion exchanger which will allow simple and convenientscale up so that methods established on a small column can be applied more orless directly to the larger column. Detailed information on scaling up ion exchangeseparations is given in Chapter 11.66

ReproducibilityReproducibility is obtained when the characteristics of the chromatography bedremain unchanged during the course of the separation and during regeneration ofthe column. The more rigid varieties of Pharmacia Biotech ion exchangers, such asMiniBeads, MonoBeads, SOURCE, Sepharose High Performance, Sepharose FastFlow and Sepharose Big Beads, show no changes in bed size with changes in pHand ionic strength and can thus be washed and regenerated in the columns providingadditional reproducibility.The use of media which are supplied pre-packed and tested, such as MiniBeads,MonoBeads, RESOURCE, HiTrap columns pre-packed with Sepharose High Performanceand HiLoad columns pre-packed with Sepharose High Performance orSepharose Fast Flow assures reproducibility since variability in column packing iseliminated.EconomyColumn or batch procedures in which the ion exchanger is used once and thrownaway, as well as applications requiring large amounts of gel, may make economy amajor consideration. Sephadex A-50 and C-50 ion exchangers are the least expensivein terms of bed volume, followed by Sephadex A-25 and C-25 ion exchangers.Using expanded bed adsorption, STREAMLINE product line, reduces the numberof operations in a process by fusing the function clarification, concentration andadsorption into one operation. It offers process developers the selectivity affordedby chromatography, the throughput of ultra-filtration and the convenience ofsmall scale centrifugation.The molecular size of the sample componentsThe accessibility of the sample components to the charged groups will determinethe available capacity of the ion exchanger for those particular substances. All ofthe ion exchange media supplied by Pharmacia Biotech, with the exception ofSephadex based media, have exclusion limits for globular proteins in excess of1 x 10 6 .Steric factors only affect the separation of charged solutes via their influence onthe available capacity for each substance. When choosing ion exchangers it isunnecessary to consider the possibility of gel filtration effects on the sample. Samplemolecules, although always larger than those of the eluent buffer, cannotmigrate ahead of the eluting buffer since they then encounter conditions whichfavour their re-binding to the matrix. Only uncharged solutes will be fractionatedaccording to size as in gel filtration. These uncharged molecules will normally beremoved during the initial isocratic elution phase which proceeds the applicationof the gradient.67

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Methods for determining the optimum pH and corresponding ion exchanger typeare discussed later in this chapter.Many biological macromolecules become denatured or lose activity outside a certainpH range and thus the choice of ion exchanger may be limited by the stabilityof the sample. This is illustrated in Figure 37. Below its isoelectric point a proteinhas a net positive charge and can therefore adsorb to cation exchangers. Above itspI the protein has a net negative charge and can be adsorbed to anion exchangers.However, it is only stable in the range pH 5-8 and so an anion exchanger has to beused.In summary:1. If the sample components are most stable below their pI’s, a cation exchangershould be used.2. If they are most stable above their pI’s, an anion exchanger should be used.3. If stability is high over a wide pH range on both sides of pI, either type of ionexchanger can be used.Determination of starting conditionsThe isoelectric pointThe starting buffer pH is chosen so that substances to be bound to the exchangerare charged. The starting pH should be at least 1 pH unit above the isoelectricpoint for anion exchangers or at least 1 pH unit below the isoelectric point forcation exchangers to facilitate adequate binding. Substances begin to dissociatefrom ion exchangers about 0.5 pH units from their isoelectric points at ionicstrength 0.1 M (15).There are comprehensive lists of isoelectric points determined for proteins (16, 17)which can be useful in the design of ion exchange experiments.If the isoelectric point of the sample is unknown, a simple test can be performed todetermine which starting pH can be used.Test-tube method for selecting starting pH1. Set up a series of 10 test-tubes (15 ml).2. Add 0.1 g Sephadex ion exchanger or 1.5 ml Sepharose or Sephacel ionexchanger to each tube.3. Equilibrate the gel in each tube to a different pH by washing 10 times with10 ml of 0.5 M buffer (see page 78 for choice of buffers for ion exchange). Use arange of pH 5-9 for anion and pH 4-8 for cation exchangers, with 0.5 pH unitintervals between tubes.69

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sample application. A schematicdescription of the various steps isshown in Fig. 40. A detailed descriptionof the method using PhastSystem electrophoresissystems is available uponrequest from Pharmacia Biotech.Fig. 39. The electrophoretic titration curveof chicken breast muscle. (19)?@@@@??@@??@e?O2@6KO26X?@e@?W2@@@?@@6X?W2@@??@@@@@

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Since the lines on the ETC reflect the degree of charge of the components at differentpH’s, the curves may be used to predict the order in which the componentswill be eluted from the column. The molecular species with the lowest electrophoreticmobility at a certain pH has logically the lowest charge at that particular pHand should be the first substance eluted from the column in the gradient. Similarlythe species showing highest electrophoretic mobility will be the most stronglyretained on a column of opposite charge and should be eluted last. The order inwhich solutes are eluted cannot be predicted with 100% certainty from the titrationcurve since electrophoretic mobility depends on the total net charge on amolecule and ion exchange chromatography depends on the net charge on thesolutes surface.Chromatographic titration curves (retention maps)For those ion exchanger types which allow rapid separations, optimal starting pHand choice of anion or cation exchanger can be determined using chromatographictitration curves (18).In a specified salt gradient, the retention of particular molecular species is dependentupon the molecules net charge and charge density. These in turn depend uponthe pH of the eluent. The chromatographic titration curve is based on this relationshipbetween retention and buffer pH.The methodology is extremely simple. The sample is analysed in a series of rapidseparations, carried out on a cation exchanger (Mono S) or an anion exchanger(Mono Q), using the same salt gradient but over a range of pH’s.A plot is then made, for each separated peak, of elution salt concentration, elutiontime or elution volume against pH. This will produce a series of curves as illustratedin Fig. 43.An analysis of this composite plot for the point of maximum separation will indicateat what pH and on which type of exchanger maximum resolution betweenany two or more components can be expected.The conditions used (column type, buffers, pH, etc.) during the rapid runs for thegeneration of the chromatographic titration curves can be directly applied whendeveloping the final optimized procedure.74

@@@@@@eW.e/Xe7HeN1e@?e?@e3=eC5eV4@@0Ye?W2@@@e?7@X?@e?(R)T5e?@@Ue@@@@@,e?W(Ye?W20Y?eW&Y?f&@@@@@e@@@@@@e@@@XfI46XeS,eO20Ye@@@Yf@@@@@@e?@@@@@e?@g?@@@@@e?@M?f@@@@@@eW.e/Xe7HeN1e@?e?@e3=eC5eV4@@0Ye?W2@@@e?7@X?@e?(R)T5e?@@Ue@@@@@,e?W(Ye?W20Y?eW&Y?f&@@@@@e@@@@@@e@@@XfI46XeS,eO20Ye@@@Yf@@@@@@e?@@@@@e?@g?@@@@@e?@M?fA 280nm a)A 280nm b)A 280nmMono SMono SpH 3.0pH 5.0BCBBACACf)Mono QpH 11.0AElution ionicstrengthData from seven chromatograms plotted as chromatographictitration curves - A, B, C.Elution conc.aABbMono ScC3 4 5 69 10 11 12pHdeMono QfFig. 43. Chromatographic titration curves.75

Choice between strong and weak ion exchangersHaving selected a suitable starting pH to use on a cation or anion exchanger, it isnecessary to choose between a strong and weak ion exchange group. In those caseswhere maximum resolution occurs at an extreme of pH and the molecules of interestare stable at that pH, the choice is clearly to use a strong exchanger. The majorityof proteins however, have isoelectric points which lie within the range 5.5 to7.5 and can thus be separated on both strong and weak ion exchangers. Someadvantages in using a strong ion exchanger are discussed in Chapter 2.Choice of bufferAs with the choice of ion exchanger, there are a number of variables which have tobe considered. These include:1. The choice of buffer pH and ionic strength.2. The choice of buffering substance.3. The price of the buffer if it is to be used in production process.Choice of buffer pH and ionic strengthThe choice of buffer pH has been discussed in the previous section. It should bepointed out, however, that in many applications the optimum separation may beachieved by choosing conditions so that major and troublesome contaminants arebound to the exchanger while the substance of interest is eluted during the washphase (21). This procedure is sometimes referred to as “starting state elution”.Note: Concentration of sample does not occur with starting state elution.The highest ionic strength which permits binding of the selected substances andthe lowest ionic strength that causes their elution should normally be used as thestarting and final ionic strengths in subsequent column experiments (i.e. the startingand limiting buffers for gradient elution). A third and higher ionic strengthbuffer is frequently employed as a wash step before column regeneration andre-use.The required concentration of the start buffer will vary depending on the nature ofthe buffering substance. A list of some suitable buffers and suggested start concentrationsis shown in Table 19. In the majority of cases a starting ionic strength of atleast 10 mM is required to ensure adequate buffering capacity.Salts also play a role in stabilizing protein structures in solution and so it is importantthat the ionic strength should not be so low that protein denaturation or precipitationoccurs. A major advantage of using Pharmacia Biotech ion exchangers isthat they have excellent capacities and so the initial ionic strength of the buffer canbe quite high without significantly affecting capacity for sample.76

In the case of pre-packed ion exchangers and columns which can be run convenientlyquickly, trial experiments using salt gradients will allow the determination ofan optimal starting ionic strength.In the case of Sephadex based exchangers for batch applications or where columnrunning times are prohibitively long, a simple test-tube technique is recommendedas a test for a suitable ionic strength.Choice of buffer substanceIf the buffering ions carry a charge opposite to that of the functional groups of theion exchanger they will take part in the ion exchange process and cause local disturbancesin pH. It is preferable, therefore, to use buffering ions with the samecharge sign as the substituent groups on the ion exchanger. There are of courseexceptions to this rule as illustrated by the frequency with which phosphate buffersare cited in the literature in connection with anion exchangers. In thoseinstances when a buffering ion which interacts with the ionic groups on the matrixis used, extra care must be taken to ensure that the system has come to equilibriumbefore application of sample.In cases where substances purified by ion exchange chromatography have to befreeze dried it is advantageous to use volatile buffer systems. Examples of such systemsare shown in Table 20.77

Table 19. Buffer tables.Buffer substances for cation exchange chromatographypKa pH Substance Conc. dpKa/ Counter-ion Comments(25°C) interval (mM)dT (°C)2.00 1.5-2.5 Maleic acid 20 Na + Dicarboxylic acid2.88 2.38-3.38 Malonic acid 20 Na + /Li + Dicarboxylic acid3.13 2.63-3.63 Citric acid 20 -0.0024 Na + Dicarboxylic acid3.81 3.6-4.3 Lactic acid 50 Na +*3.75 3.8-4.3 Formic acid 50 +0.0002 Na + /Li +*4.21 4.3-4.8 Butanedioic acid 50 -0.0018 Na +*4.76 4.8-5.2 Acetic acid 50 +0.0002 Na + /Li +*5.68 5.0-6.0 Malonic acid 50 Na + /Li + Dicarboxylic acid*7.20 6.7-7.6 Phosphate 50 -0.0028 Na + Often needspurificationbefore use*7.55 7.6-8.2 HEPES 50 -0.0140 Na + /Li + Zwitterionic*8.35 8.2-8.7 BICINE 50 -0.0180 Na + ZwitterionicBuffer substances for anion exchange chromatographypKa pH Substance Conc. dpKa/ Counter-ion Comments(25°C) interval (mM)dT (°C)*4.75 4.5-5.0 N-methyl 20 -0.015 Cl -piperazine*5.68 5.0-6.0 Piperazine 20 -0.015 Cl - /HCOO -*5.96 5.5-6.0 L-histidine 20 Cl -*6.46 5.8-6.4 bis-Tris 20 -0.017 Cl -*6.80 6.4-7.3 bis-Tris propane 20 Cl -*7.76 7.3-7.7 Triethanolamine 20 -0.020 Cl - /CH 3 COO -*8.06 7.6-8.0 Tris 20 -0.028 Cl - Often needspurificationbefore use and*8.52 8.0-8.5 N-methyl- 50 -0.028 SO - 2 /Cl - / especiallydiethanolamine CH 3 COO - sensitive totemperature*8.88 8.4-8.8 Diethanolamine 20 at pH 8.4 -0.025 Cl - change.50 at pH 8.8*8.64 8.5-9.0 1,3-diamino- 20 -0.031 Cl -propane*9.50 9.0-9.5 Ethanolamine 20 -0.029 Cl -*9.73 9.5-9.8 Piperazine 20 -0.026 Cl -*10.47 9.8-10.3 1,3-diamino- 20 -0.026 Cl -propane11.12 10.6-11.6 Piperadine 20 -0.031 Cl -12.33 11.8-12.0 Phosphate 20 -0.026 Cl -* Recommended on the basis of experiments performed in our laboratories.78

Table 20. Volatile buffer systems.pH Substance Counter-ion2.0 Formic acid H +2.3-3.5 Pyridine/formic acid HCOO -3.0-5.0 Trimethylamine/formic acid HCOO -3.0-6.0 Pyridine/acetic acid CH 3 OO -4.0-6.0 Trimethylamine/acetic acid CH 3 COO -6.8-8.8 Trimethylamine/HCl Cl -7.0-8.5 Ammonia/formic acid HCOO -8.5-10.0 Ammonia/acid CH 3 COO -7.0-12.0 Trimethylamine/CO 2 CO - 37.0-12.0 Triethylamine/CO 2 CO - 37.9 Ammonium bicarbonate HCO - 38.0-9.5 Ammonium carbonate/ammonia CO - 38.5-10.5 Ethanolamine/HCl Cl -8.9 Ammonium carbonate CO - 3Test-tube method for selecting starting ionic strengths1. Set up a series of tubes with ion exchanger as detailed on page 69.2. Equilibrate the gel in each tube with 0.5 M buffer at the selectedstarting pH (10 x 10 ml washes).3. Equilibrate the gel in each tube to a different ionic strength, at constantpH, using a range from 0.05 M to 0.5 M NaCl for Sephadex ionexchangers and from 0.01 M to 0.3 M NaCl for Sephacel andSepharose ion exchangers. This will require 5 x 10 ml washes. Intervalsof 0.05 M NaCl are sufficient.4. Add sample, mix and assay the supernatant to determine the maximumionic strength which permits binding of the substance of interest and theminimum ionic strength required for complete desorption.In the hypothetical example shown in Figure 38 (b) the ionic strength for samplebinding (start buffer) would be at most 0.15 M and for elution at least 0.3 M.79

10. Experimental TechniqueThere are three ways of performing an ion exchange separation: by column chromatography,by batch methods, and by expended bed adsorption. This section willmostly deal with column chromatography.Column chromatographyChoice of columnGood results in column chromatography are not solely dependent on the correctchoice of gel media. The design of the column and good packing technique are alsoimportant in realising the full separation potential of any gel. These factors arebuilt into the pre-packed columns supplied by Pharmacia Biotech and should beconsidered before packing a chromatography column in the laboratory.Column designThe material used in the construction of the column should be chosen to preventdestruction of labile biological substances and minimize non-specific binding toexposed surfaces. The bed support should be designed so it is easily exchangeableto restore column performance whenever contamination and/or blockage in thecolumn occurs. Bed supports made from coarse sintered glass or glass wool cannotbe recommended because they soon become clogged, are difficult to clean andcause artifacts (22). Dead spaces must be kept to a minimum to prevent re-mixingof separated zones.The pressure specifications of the column have to match the back-pressure generatedin the packed bed when run at optimal flow rate. This is particularly importantwhen using high performance media with small bead size.Pharmacia Biotech has developed a series of standard columns suitable for ionexchange chromatography. All are easy to dismantle and reassemble to allow thoroughcleaning, which is a particularly important aspect when handling biologicalsamples.Further information on the full range of Pharmacia Biotech chromatographycolumns are available upon request.Larger chromatography columns, specially designed for pilot and process scalechromatography are also available. Some aspects regarding process scale columnsare described in Chapter 11.80

Column dimensionsAs for most adsorptive, high selectivity techniques, ion exchange chromatographyis normally carried out in short columns. A typical ion exchange column is packedto a bed height of 5-15 cm. Once the separation parameters have been determined,scale-up is easily achieved by increasing the column diameter.Quantity of ion exchangerThe amount of ion exchanger required for a given experiment depends on theamount of sample to be chromatographed and on the available or dynamic capacityof the ion exchanger for the sample substances. For the best resolution in ionexchange chromatography, it is not usually advisable to use more than 10-20% ofthis capacity, although this value can be exceeded if resolution is adequate. Thecapacity data given for each specific ion exchanger in respective product Chapterserves as a guideline for calculating the required amount of ion exchanger neededfor a given experiment.Preparation of the ion exchangerHaving chosen the appropriate ion exchanger and starting buffer it is essentialthat the exchanger is brought to equilibrium with start buffer before sample application.Preparation of Sephadex ion exchangers, which are supplied as powders,differs somewhat from the other ion exchangers available from Pharmacia Biotech,which are supplied pre-swollen and/or pre-packed.Pre-swollen ion exchangersSOURCE, Sepharose based, and DEAE Sephacel ion exchange media are suppliedready to use. To prepare the gel, the supernatant is decanted and replaced withstarting buffer to a ratio of approximately 75% settled gel to 25% buffer.If large amounts of ion exchangers are to be equilibrated with a weak buffer, theion exchanger should first be equilibrated with a 10 times concentrated buffersolution at the correct pH, and then with a few volumes of starting buffer.Pre-packed ion exchange mediaMiniBeads and MonoBeads are supplied pre-packed in PC and HR columnsrespectively. SOURCE 15 are available pre-packed and ready to use inRESOURCE columns, 1 or 6 ml. Pre-packed HiLoad columns are XK Columnspre-packed with Sepharose Fast Flow and Sepharose High Performance ionexchangers. Sepharose High Performance ion exchangers are also available prepackedin HiTrap columns, 1 and 5 ml. For pilot scale applications, Mono Q,Mono S and Q Sepharose High Performance are available in pre-packed BioPilot81

Columns of 100 ml(35/100) and 300 ml (60/100). SP Sepharose High Performancepre-packed in BioPilot Columns are available on request. For the above mentionedpre-packed columns, the preparation consists of washing out the 20% ethanolpacking solution with 5 column volumes of start buffer.Sephadex ion exchangersSephadex ion exchangers should be swollen at the pH to be used in the experiment.Complete swelling takes 1-2 days at room temperature or 2 hours (at pH 7)in a boiling water bath. Swelling at high temperature also serves to de-aerate thegel. Vigorous stirring (e.g. with a magnetic stirrer) and swelling in distilled watershould be avoided due to the risk of damaging the beads.The required amount of ion exchanger should be stirred into an excess of startingbuffer. Remove the supernatant and replace with fresh buffer several times duringthe swelling period. Instead of decantation, the ion exchanger can be washedextensively on a Büchner funnel after the initial swelling.Alternative counter-ionsIf ion exchangers are to be used with counter-ions other than those supplied (i.e.other than sodium or chloride) then the following procedure should be used.Suspend the required amount of ion exchanger in and excess of 0.5-1.0 M solutionof a salt of the new counter-ion. After sedimentation and decantation, re-suspendthe ion exchanger in the buffer to be used in the experiment. Decant and re-suspendthe ion exchanger in this buffer several times.Decantation of finesDecantation of fines is not necessary with any Pharmacia Biotech ion exchangers.Packing the columnAs with any other chromatographic technique, packing is a very critical stage in anion exchange experiment. A poorly packed column gives rise to poor and unevenflow, zone broadening, and loss of resolution.Detailed packing instructions are to be found in the instructions supplied withrespective media.Column Packing Video FilmA video film describing the correct methodologies for packing laboratory columnsis available and can be ordered from your local distributor of Pharmacia Biotechproducts.82

Checking the packingThe bed should be inspected for irregularities or air bubbles using transmittedlight from a lamp held behind the column. Be careful in the choice of any dye substancesused for checking beds as many of them are strongly charged. For example,Blue Dextran 2000 binds strongly to anion exchangers.Testing the bed is easily done by injecting a test substance on the column and calculatingthe number of theoretical plates (N) or the height equivalent to a theoreticalplate (H).Choose a test substance which shows no interaction with the media and which hasa low molecular weight, to give full access to the interior of the beads.Acetone at a concentration of 1% (v/v) can be used with all kinds of chromatographicmedia and is easily detected by UV-absorption. Keep the sample volumesmall to ensure a narrow zone when the sample enters the top of the column. Foroptimal results, the sample volume should be ²0.5% of the column volume for acolumn packed with a medium of approximately 30 µm bead diameter and ² 2%of the column for a column packed with a medium of approximately 100 µm beaddiameter. Keep the linear flow rate low to reduce zone spreading due to non-equilibriumat the front and rear of the zone. For 30 µm media the flow rate should bebetween 30-60 cm/h and for 100 µm media, 15-30 cm/h. Use the following equationsto calculate the number of theoretical plates (N) and the hight equivalent to atheoretical plate (H).2V RN = 5.54 x(w h )H = L/NwhereV R is the volume eluted from the start of sample application to the peak maximumand w h is the peak width measured as the width of the recorded peak at half of thepeak height, see Figure 44. L is the height of the packed bed.Measurements of V R and w h can be made in distance (mm) or volume (ml) butboth parameters must be expressed in the same unit.83

?@?@J@L?7@1?3@5?N@H??@?@?@?O@K?@e?@?3L?J5?N1?7H@?@?3T@T-XV+R@@)?3X??V/?@?@??O26K?@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??I40M?@??@?I@M@??@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@@??J@L?7@1J@@@L?7Y@V1??J5?@?3L?7H?@?N1?@e@??@?@e@??@?@e@??@J5e@??3L?7He@??N1?@?e@?e@?@?e@?e@?@?e@?e@?@?e@?e@?@?e@?e@?@?e@?e3L?J5?e@?eN1?7H?e@?e?@?@f@?e?@?@f@?e?@?@f@?e?@?@f@?e?3L??@f@?e?N1?J5f@?f@?7Hf@?f@?@?f@?f@?@?f@?f@?@?f@?f@?@?f@?f3L@?f@?fN1?J5?f@?f?@?7H?f@?f?@?@g@?f?@?@g@?f?@?@g@?f?@?@g@?f?3L??@g@?f?N1??@g@?g@?J5g@?g@?7Hg@?g@?@?g@?g@?@?g@?g3L@?g@?gN1@?g@?g?@@?g@?g?@?J5?g@?g?@?7YO@Kf@?eO2@6X@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@?I@Mf@?eI40MI'X?@?e@?g?@h@?g?N1? @@?@3L?J5?g?@h@?h@? ?J@@?@N1?7H?g?@h@?h@? ?@@@?@?@?@?@K?fJ5h@?h@? ?@?@?3T5?@@@f7Hh@?h@? ?@?@?V+Y?@?@f@?h@?h@? ?@?@?@?@f@?h@?h3L ?@?@?@?@f@?h@?hN1 ?@?@@?h@?h?@ ?@?@@?h@?h?@ ?@?@@?h@?h?@ ?@?@?J5?h@?h?@ ?@?@?7H?h@?h?3L? ?@?@?@he@?h?N1? ?@?@?@he@?he@? ?@?@?@he@?he@? ?@?@?@he@?he@? ?@?@?@he@?he@? ?@?@J5he@?he@? ?@eW2@6T26X?@7Hhe@?he3L ?@e7@@>@)Xhe?@@?he@?he?@ ?@fC@@=C5e?J@@?,he?@@?he@?he?@ ?@e?@0MI40Ye?.MI+Yhe?@?J5?he@?he?@ ?@?@?7H?he@?he?3L? ?@?@?@hf@?he?N1? ?@?@?@hf@?hf@? ?@?@J5hf@?f@Kg@? ?@?@7He?'6Xg@?f@@6Xf@? ?@?@@?e?S@1g@?f@?B1f@? ?@?@?J5?e@(Y@g@?f@??@f3L ?@?@?7H?e3U?@g@?f@?C5fN1 ?@?@J5fV4@@g@?f@@0Yf?@ ?@?@7H @?hf?3L? ?@?@?J5? @?hf?N1? ?@?@?7H? @? 3L ?@?@J5hf@Ke@?he@KeN1 ?@?@7H?@6Kh@@@?@?@6K?g@@@??3=? ?@?@?@?J5??@@@@@@@@@@@@@@?@?@@@@@@@@@@@@@??N@@@@@@@@@@@@@@@@@@@@@@@@?7H? @? 3X ?@f@@fW&?W26X?he?@J5 @? N1 ?@e?J@@f*@?7)X?e?@O.Y? @? V46XheJ@f7@f?@?3=C5?eJ@@?,?e?@O20Y @? I/K?h7@f@@f?@?V40Y?e.MI+Y?e?@O20M @? ?V46K?g@@?@?O2@0M @? ?I46K??@?O2@@@@@Y? @? ?V@@@@@@@6K??@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@??@?@?@?@?@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@?@?@K@@@?@?e@Ke@?3L?J@@6X@?@?@??@@@@@?W26X?N1?7@?B@@?@?@??@?@?@?7YV1??@?@@??@@?@?@??@?@?@?@@@@??3T@@=C@@?@?@??@?@?@?3X??V+MI40R'?@@@??@?@?@?V4@Fig 44. A UV trace for acetonein a typical test chromatogramshowing the HETP and A svalue calculations.As a general rule of thumb, a good H value is about two to three times the meanbead diameter of the gel being packed. For a 90 µm particle packing, this meansan H value of 0.018-0.027 cm.Another useful parameter for testing the packed bed is the symmetry factor (A s )bA s =awherea = 1st half peak width at 10% of peak height. (see Figure 44)b = 2nd half peak width at 10% of peak height. (see Figure 44)A s should be as close as possible to 1. A reasonable A s value for a short columnsuch as an IEX column is 0.80-1.80. (For longer gel filtration columns it will probablyfall within 0.70-1.30).An extensive leading edge is usually a sign that the gel has been packed too tightlyand extensive tailing is usually a sign that the gel has been packed too loosely.Equilibrating the bedRun at least two bed volumes of buffer through the ion exchange bed to allow thesystem to reach equilibrium. Counter-ion concentration, conductivity, and pH ofthe eluent should be checked against the ingoing solution. It is often sufficient justto measure the pH of the effluent.84

Sample preparationSample concentrationThe amount of sample which can be applied to a column depends on the dynamiccapacity of the ion exchanger and the degree of resolution required. For the bestresolution it is not usually advisable to use more than 10-20% of this capacity(23). Information on the available capacities for the different exchangers is givenin the relevant product sections. Methods for determining available and dynamiccapacities are given later in this chapter.Sample compositionThe ionic composition should be the same as that of the starting buffer. If it is not,it can be changed by gel filtration on Sephadex G-25 using e.g. Pharmacia BiotechDisposable Column PD-10, Fast Desalting Column HR 10/10 or HiTrap DesaltingColumns, dialysis, diafiltration or possibly by addition of concentrated start buffer.Sample volumeIf the ion exchanger is to be developed with the starting buffer (isocratic elution),the sample volume is important and should be limited to between 1 and 5% of thebed volume. If however, the ion exchanger is to be developed with a gradient, startingconditions are normally chosen so that all important substances are adsorbedat the top of the bed. In this case, the sample mass applied is of far greater importancethan the sample volume. This means that large volumes of dilute solutions,such as pooled fractions from a preceding gel filtration step or a cell culture supernatantcan be applied directly to the ion exchanger without prior concentration.Ion exchange thus serves as a useful means of concentrating a sample in additionto fractionating it.If contaminants are to be adsorbed, and the component of interest is allowed topass straight through, then the sample volume is less important than the amountof contaminant which is present. Under these conditions there will be no concentrationof the purified component, rather some degree of dilution due to diffusion.Sample viscosityThe viscosity may limit the quantity of sample that can be applied to a column. Ahigh sample viscosity causes instability of the zone and an irregular flow pattern.The critical variable is the viscosity of the sample relative to the eluent. A rule ofthumb is to use 4 cP as the maximum sample viscosity. This corresponds to a proteinconcentration of approximately 5%. Approximate relative viscosities can bequickly estimated by comparing emptying times from a pipette.85

If the sample is too viscous, due to high solute concentration, it can be dilutedwith start buffer. High viscosity due to nucleic acid contaminants can be alleviatedby precipitation with a poly-cationic macromolecule such as polyethyleneimine orprotamine sulphate. Nucleic acid viscosity can also be reduced by digestion withendonuclease. Such additives may however be less attractive in an industrial processsince they will have to be proven absent from the final product.Sample preparationIn all forms of chromatography, good resolution and long column life time dependon the sample being free from particulate matter. It is important that “dirty” samplesare cleaned by filtration or centrifugation before being applied to the column.This requirement is particularly crucial when working with small particle matrices,such as MiniBeads (3µm), MonoBeads (10 µm), SOURCE (15 and 30 µm) andSepharose High Performance (34µm).The “grade” of filter required for sample preparation depends on the particle sizeof the ion exchange matrix which will be used. Samples which are to be separatedon a 90 µm medium can be filtered using a 1 µm filter. For 3, 10, 15, 30 and 34 µmmedia, samples should be filtered through a 0.45 µm filter. When sterile filtrationor extra clean samples are required, a 0.22 µm filter is appropriate.Samples should be clear after filtration and free from visible contamination bylipids. If turbid solutions are injected onto the column, the column lifetime, resolutionand capacity can be reduced. Centrifugation at 10 000 g for 15 minutes canalso be used to prepare samples. This is not the ideal method of sample preparationbut may be appropriate if samples are of very small volume or adsorb nonspecificallyto filters.Note: The latter may indicate that the substance in question may also adsorbstrongly to chromatography matrices. Care should therefore be taken and perhapsa buffer additive such as glycerol or a detergent used.Crude samples containing lipids, salts, etc. can be passed through a suitably sizedcolumn of Sephadex G-25 e.g. Pharmacia Biotech Desalting Column PD-10, FastDesalting Column HR 10/10 or HiTrap Desalting Column. Preliminary sampleclean-up can be achieved simultaneously in this way.In expanded bed adsorption sample preparation is not as crucial as for columnchromatography. Samples can be applied directly to the expanded bed withoutprior sample preparation, e.g. filtration, centrifugation etc. (Expanded bedadsorption is described in detail on page 98.)86

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Superloops can be used together with the manual valves V-7 and IV-7 or the motorisedvalves MV-7 and IMV-7 when larger volumes of sample have to be applied(Fig. 47). Superloops are available with capacities of 10, 50 and 150 ml. (The 150ml Superloop is most often used in BioPilot System.)Fig. 47. Sample application using a Superloop.Syringe method (Fig. 48). The valves LV-3 and LV-4 can be used as syringe holdersto give a very simple method for the application of small samples in standard chromatography.Using this method the sample is allowed to run onto the columnunder gravity.88?W2@V'@?@??@@6KO26X?@@@?W26X?W26T2@6X? ??7U??V@?@??@?B@@YV1?@?@?7

Sample reservoir (Fig. 49). In a similar way, a sample reservoir (e.g. R9, RK 16/26)can be connected via a 3-way valve to apply larger samples.?W2@@@@@@@@@@@@6X??7@@@@@@@@@@@@@@1??@@@@@@@@@@@@@@@@??@@??@f?@@@g@??@f?@@@f?J@??@?@fI@?@?@?@@?fW2@6X?@@e?@@?e?W&??@hf?N@?f7

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Continuous ionic strength gradients are the most frequently used type of elution inion exchange chromatography. They are easy to prepare and very reproducible.Two buffers of differing ionic strength, the start and limit buffers, are mixed togetherand if the volume ratio is changed linearly, the ionic strength changes linearly.The limit buffer may be of the same buffer salt and pH as the start buffer, but athigher concentration, or the start buffer containing additional salt e.g. NaCl.Gradient elution generally leads to improved resolution since zone sharpeningoccurs during elution. In all forms of isocratic elution, a limiting factor withregards to achievable resolution is zone broadening as a result of longitudinal diffusion.In gradient elution, the leading edge of a peak is retarded if it advancesahead of the salt concentration or pH required to elute it. In contrast the trailingedge of the peak is exposed to continuously increasing eluting power. Thus thetrailing edge of the peak has a relatively higher speed of migration, resulting inzone sharpening, narrower peaks and better resolution.Gradient elution also reduces zone broadening by diminishing peak tailing due tonon-linear adsorption isotherms.Resolution using a continuous gradientTo optimize a separation it is important first to consider the objectives of the experiment,since the desired features of a separation i.e. speed, resolution and capacity,are often mutually exclusive. In the case of ion exchange separations the speedof separation is not solely related to the flow rate used in the experiment but alsoto the steepness or slope of the gradient applied.Novotny (27) has shown that the retention of charged molecules on an ionexchange column is related to the volume of the column and the molarity differenceacross it. This means that long shallow gradients will give maximum separationbetween peaks but that the separation time will be longer and peak broadeninglarger. In contrast short steep gradients will give faster separations and sharperpeaks but the retention differences between peaks will be reduced. The effect ofgradient slope on resolution is illustrated in Figure 53.It should be remembered that the sample loading also has a major influence onresolution since the width of the peaks is directly related to the amount of substancepresent.In practice it is recommended that trial experiments be carried out to allow theselection of optimal run parameters in terms of gradient shape and length.As a general rule a gradient of 0.05 to 0.5 M salt over a volume of 10 to 20column volumes at the flow rate recommended for the medium (see individualmedia sections) can be used for initial investigative separations.93

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????@e?@??@@6X@e?@??@?B@@@@@@??@e@@e?@??@?C@@e?@??@@0R'e?@??@??@??????W&?W26Xe?W26X???*@?7

When working with batch ion exchange, the starting conditions are selected in thesame way as in column chromatography, i.e. choose buffer pH an ionic strength tobind the substance of interest but to prevent as many contaminants as possiblefrom binding.To maximize recovery, the starting conditions should be selected so that the proteinof interest binds much stronger than is usual in column chromatography.Unless the proteins adsorbs to 100%, losses during subsequent washing will beinevitable, especially if the volume of liquid is large compared to the volume ofadsorbent. To keep recovery high, the pH in a batch experiment may have to beseveral units away from the isoelectric point of the protein.Batch separation is carried out by stirring the ion exchanger previously equilibratedin the appropriate buffer with the solution to be treated until the mixture hasreached equilibrium. This usually takes about one hour. The slurry is then filteredand washed with the buffer solution. In cases of incomplete adsorption this procedureshould be repeated on the filtrate with a new batch of ion exchanger. Thenelution buffer is added (1-2 times the volume of the sedimented gel) and stirreduntil desorption is complete, which can take up to 30 minutes or more. Finally,suction is used to filter the buffer containing the desorbed product of interest fromthe adsorbent. The gel can also be packed in a column after the washing step andbe eluted stepwise in the same way as during normal column chromatography.Resolution will however be lower for such a combined batch and column procedurecompared with a normal column procedure, since the sample is bound uniformlythroughout the gel slurry and the subsequent chromatographic bed.Under these conditions stepwise elution is recommended since gradient elutionwill give broad bands and poor resolution.Batch chromatography is very useful for concentrating dilute solutions and separatingthe substances of interest from gross contaminants during the initial stagesof a purification scheme.Note: Fines will be generated if the ion exchangers are stirred too vigorously. Thiswill increase the time required for filtration.Expanded bed adsorptionExpanded bed adsorption is a unit operation that uses STREAMLINE adsorbentsand columns for recovering proteins directly from crude samples. Proteins arerecovered in a single pass without the need for prior clarification. STREAMLINEhas proven effective in purification proteins from fermentation or cell culture inextracellular processes, and has demonstrated its suitability when used with brothfrom cell lysis and homogenization in intracellular processes with soluble proteins.STREAMLINE reduces the number of operations in a process by fusing the functionsof clarification, concentration and capture (see page 109) in one operation.98

It offers the selectivity afforded by chromatography, the throughput of ultra-filtrationand the convenience of small scale centrifugation.Crude feed from the fermentor containing the desired product and undesired cells,cell debris and particulates is applied to the expanded bed. Target products arebound by the adsorbent while particulates and contaminants pass through unhindered.The desired molecule is then eluted as in packed bed chromatography.Expanded bed technologyExpanded bed adsorption is based on fluidization. The sedimented bed begins toexpand as the adsorbent particles are raised by an upward liquid flow. The differencebetween a fluidized bed and expanded bed is that in an expanded bed theadsorbent particles display very little back-mixing. This is achieved through theunique design of the column and the adsorbents. The column has a special flowdistributor at the bottom, the adsorbent particles have a well-defined size and densitydistribution. The particles are kept in suspension by the balance betweenupward flow rate and particle sedimentation velocity. As the bed expands with theupward liquid flow, the movement of any given particle is very small. This createsa stable, homogeneous expanded bed and a liquid flow which is characterized by aconstant velocity profile, i.e. plug flow. The stability of the expanded bed giveSTREAMLINE characteristics that are similar to those of a packed bed inchromatography.Basic principle of operation1. STREAMLINE adsorbent is poured into STREAMLINE column and allowedto sediment (Fig. 57 a).2. An upward liquid flow of equilibration buffer is applied to the column andSTREAMLINE adsorbent particles are suspended in the flow, creating a stablefluidized bed (Fig. 57 b).3. The sample, a mixture of soluble proteins, contaminants, cells, or cell debris,is passed upwards through the expanded bed. The target proteins are boundon STREAMLINE adsorbent while particulates and contaminants passthrough the expanded bed unrestricted. Loosely bound material is washed outwith the upward flow of the buffer (Fig. 57 c).4. The liquid flow is reversed to downward flow. By using suitable bufferconditions, the bound proteins are eluted from STREAMLINE adsorbent in asedimented bed mode. The eluate contains the target proteins, increased in concentration, free from particulates and ready for further purification (Fig. 57 d).99

?@@@ ?@@@?@@@@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@e@? ?@?@ ?@?@?@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@?@e@??@?@ ?@?@ ?@@@@??@e@??@?@ ?@?@ ?@e@??@e@??@?@ ?@?@ ?@e@??@e@??@?@ ?@?@ ?@e@??@e@??@?@ ?@?@ ?@e@??@e@??@?@ ?@?@ ?@e@??@e@??@?@ ?@?@ ?@e@??@e@?O2@@?@@@6K O2@@?@@6K? O2@@e@@6KO2@@e@@6KO2@@@@0M?@?@eI4@@@6K? O2@@@@0M?@?@?I4@@@6K O2@@@@0M?@e@?I4@@@6K?hg?O2@@@0M?@e@?I4@@@6K??O2@@@0Mg?@?@g?I4@@@6K ?O2@@@0Mg?@?@gI4@@@@6K ?O2@@@0Mg?@e@?f?I4@@@6KheO2@@@0M?f?@e@?f?I4@@@6K?O2@@0M?he?@?@hfI4@@6K ?O2@@0M?he?@?@hfI4@6K? ?O2@@0M?he?@e@?heI4@@6KgO2@@0Mhe?@e@?heI4@@6K?@e@? I46Khf?W20M? ?@?@ I46Khf?O20M? ?@?@ ?I46K?he?W20M? ?@e@? I46KfO20M?@e@? I46XheW.M? ?@?@ I46Xhe@(M? ?@?@ ?I46X?hW.M? ?@e@? I46XeW20M?@e@? I/X?g?W.Y ?@?@ I/X?g?J(Y ?@?@ ?I/Xg?W.Y ?@e@? I/X??W.M?@e@? ?S1?g?7U? ?@?@ ?S1?g?7H? ?@?@ S1g?7U? ?@e@? ?S1??7U??@e@? ?7@?g?@1? ?@?@ ?7@?g?@L? ?@?@ 7@g?@1? ?@e@? ?7@??@1??@e@? C@@?g?@@= ?@?@ C@@?g?@)X ?@?@ ?J@@g?@@= ?@e@? C@@??@@=?@e@? O20Y@?g?@V46K ?@?@ O20Y@?g?@@)K? ?@?@ O.Y@g?@V46K ?@e@? O20Y@??@V46K?@e@? O20Me@?g?@eI46K ?@?@ O20Me@?g?@?I46K? ?@?@ O20Y?@g?@eI46K ?@e@? O20Me@??@eI46K?@fI4@@6Khe?@e@?heO2@@0Mf@?g?@fI4@6K?he?@?@hfO2@@0Mf@?g?@e?I4@@6K?he?@?@hfO2@@0Me?@g?@fI4@6K?he?@e@?heO2@@0Mf@??@hI4@@@6K?f?@e@?f?O2@@@0Mh@?g?@g?I4@@@@6K?f?@?@g?O2@@@0Mh@?g?@g?I4@@@@6K?f?@?@g?O2@@@0Mg?@g?@g?I4@@@@6K?f?@e@?f?O2@@@0Mh@??@hf?I4@@@6K?@e@?O2@@@0M?hf@?g?@hf?I4@@@6K?@?@?O2@@@@0M?hf@?g?@hf?I4@@@6K?@?@?O2@@@@0M?he?@g?@hf?I4@@@6K?@?J@W2@@@@0M?hf@?S@@@e@@@U @?g?@ V@@@?@@@U? @?g?@ V@@@?@@@U? ?@g?@ I4@@?@@(M? @??@?O&@@@e@@@)K? @?g?@ O2@@@@?@@@)K @?g?@ O2@@@@?@@@)K ?@g?@ ?@?N@H @??@?@e@? @??@h?O2@@@@@@0M??@e@??I4@@@@@@6K?h@?g?@hO2@@@@@@0Me?@?@eI4@@@@@@6Khe@?g?@hO2@@@@@@0Me?@?@eI4@@@@@@6Kh?@g?@?@e@? @??@fO2@@@0M?h?@e@?h?I4@@@6Kf@?g?@e?O2@@@0Mhe?@?@heI4@@@@6Kf@?g?@e?O2@@@0Mhe?@?@heI4@@@6K?e?@g?@?@e@? I46Ke@?g?@?W20M? ?@?@ I46Ke@?g?@?O20M? ?@?@ ?I46K??@g?@ ?@e@? @??@eO20M?@e@? I46X@?g?@W.M? ?@?@ I46X@?g?@@(M? ?@?@ ?I46X@g?@ ?@e@? @??@W20M?@e@? B@@?g?@@H ?@?@ B@@?g?@(Y ?@?@ ?B@@g?@ ?@e@? @??@@

RegenerationAfter each cycle, bound substances must be washed out from the column to restorethe original function of the media. Ion exchange adsorbents can normally beregenerated after each run by washing with a salt solution until an ionic strengthof about 2 M has been reached. This should remove any substances bound byionic forces. The salt should contain the counter-ion to the ion exchanger to facilitateequilibration.To prevent a slow build up of contaminants on the column over time, more rigorouscleaning protocols may have to be applied on a regular basis, see below.Cleaning, sanitization andsterilization proceduresCleaningCleaning-in-place (CIP) is the removal from the purification system of very tightlybound, precipitated or denatured substances generated in previous purificationcycles. In some applications, substances such as lipids or denatured proteins mayremain in the column bed instead of being eluted by the regeneration procedure. Ifcontaminants accumulate on the column over a number of purification cycles,they may affect the chromatographic properties of the column. If fouling is severe,it may also block the column, increasing the back-pressure and reducing the flowrate.A specific CIP protocol should be designed according to the type of contaminantsthat are known to be present in the sample. NaOH is a very efficient cleaningagent that can be used for solubilizing irreversibly precipitated proteins and lipids.NaOH can effectively be combined with solvent or detergent based cleaning methods.SanitizationSanitization is the inactivation of microbial populations. When a packed columnis washed with a sanitizing agent, the risk of contaminating the purified productwith viable micro-organisms is reduced. The most commonly used sanitizationmethod in chromatography today is to wash the column with NaOH. NaOH hasa very good sanitizing effect and also has the addition advantage of cleaning thecolumn.SterilizationSterilization, which is not synonymous with sanitization, is the destruction or eliminationof all forms of microbial life in the system.101

Protocols for cleaning-in-place (CIP),sanitization and sterilization.Suggested protocols for cleaning-in-place (CIP), sanitization and sterilization thatcan be applied to each specific ion exchanger from Pharmacia Biotech aresummerized below.SOURCE and Sepharose based ion exchangersCIP, sanitization and sterilization protocols for SOURCE and Sepharose based ionexchangers media are summarized in Table 22.Table 22. Suggested CIP, sanitization and sterilization protocols for SOURCE 15 and 30,and Sepharose based ion exchangers media from Pharmacia Biotech.PurposeRemoval of precipitated proteinsRemoval of strongly boundhydrophobic proteins, lipoproteinsand lipidsProcedure4 bed volumes of 0.5-1.0 M NaOH at 40 cm/hfollowed by 2-3 bed volumes of water.4-10 bed volumes of up to 70% ethanol or30% isopropanol followed by 3-4 bed volumesof water.or1-2 bed volumes of 0.5% non-ionic detergent(e.g. in 1 M acetic acid) followed by 5 bedvolumes of 70% ethanol to remove the detergent,and 3-4 bed volumes of water.Sanitization 0.5-1.0 M NaOH with a contact time of 30-60min.SterilizationAutoclave the medium at 121 °C for 15 min.MonoBeads and MiniBeads columnsDue to the small particle size of MonoBeads and MiniBeads, they are more sensitiveto particulate matter such as precipitated proteins from the sample or buffersolutions than the larger bead size matrices. Preventative measures to ensure cleanlinessof the sample and buffers are essential to ensure long column life. Samplepreparation procedures are described earlier in this Chapter. Should precipitatedmaterial be present, as indicated by a decrease in performance or an increase inback-pressure, the columns may be cleaned using the detailed instructions includedwith the column.102

DEAE Sephacel and Sephadex based ion exchangersDue to the relatively large volume changes of Sephadex based gels in different solvents,we recommend cleaning and washing with organic solvents on a Büchnerfunnel, since the gel needs to be repacked after such treatment.Remove ionically bound proteins by washing the column with 0.5-1 bed volumeof a 2 M NaCl solution.Remove precipitated proteins, hydrophobically bound proteins and lipoproteinsby washing the column with 0.1 M NaOH solution, contact time 1-2 hours, followedby binding buffer until free from alkali. Alternatively, wash the column with2 bed volumes of 6 M guanidine hydrochloride.Strongly hydrophobically bound proteins, lipoproteins and lipids can be removedby washing the gel with up to 70% ethanol or 30% isopropanol. Alternatively,wash the gel with 2 bed volumes of a non-ionic detergent in a basic or acidic solution.Use for example, 0.1-0.5% non-ionic detergent (e.g. Triton X-100) in 0.1 Macetic acid. After treatment with detergent always remove residual detergents bywashing with 5 bed volumes of 70% ethanol.Re-equilibrate the ion exchanger with starting buffer.Storage of gels and columnsPrevention of microbial growthAs well as endangering the sample, bacterial and microbial growth can seriouslyinterfere with the chromatographic properties of ion exchange columns and mayobstruct the flow through the bed. During storage an antimicrobial agent shouldalways be added to the ion exchanger. Antimicrobial agents may be eluted fromthe columns during equilibration before starting a run.Recommended antimicrobial agents for anion exchangers:Equilibrate the column with 20% ethanol in 0.2 M acetate.Recommended antimicrobial agents for cation exchangers:Equilibrate the column with 20% ethanol or 0.01 M NaOH.103

Storage of unused mediaUnused media should be stored in closed containers at +4 °C to +25 °C. Note thatit is important that the media are not allowed to freeze as the structure of thebeads may be disrupted by ice crystals. This disruption will generate fines.Storage of used mediaUsed media should be stored at a temperature of +4 °C to +8 °C in the presence ofan antimicrobial agent, e.g. 0.01 M NaOH or 20% ethanol according to therecommendation given above. Note that it is important that the media are notallowed to freeze as the structure of the beads may be disrupted by ice crystals.This disruption will generate fines.Storage of packed columnsPacked columns should be stored at a temperature of +4 °C to +8 °C in the presenceof an antimicrobial agent, e.g. 0.01 M NaOH or 20% ethanol according to therecommendation given above. For long-term storage, the packed column shouldbe thoroughly cleaned before equilibration with the storage solution. Recyclingthe storage solution through the column or flushing the column once a week withfresh storage solution is recommended to prevent bacterial growth.Determination of the available anddynamic capacitiesThe available capacity of an ion exchanger can be determined by a batch test-tubemethod similar to that used for the determination of suitable buffer pH and bindingand elution ionic strengths, see page 70 and Figure 38. In this case a series ofsolutions with different concentrations of the protein are added to a known quantityof ion exchanger, equilibrated at a suitable binding pH and ionic strength.Assaying the supernatants after mixing will show the maximum protein concentrationwhich can be bound per ml of ion exchanger.For a more realistic and useful measurement of the available capacity of an ionexchanger, a dynamic method is recommended (see page 18 for definition of availableand dynamic capacity). The type of equipment necessary for this determinationis shown in Figure 58. FPLC System can also be used for this determination.104

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11. Process considerationsWhen an ion exchange step is to be part of a purification sequence for a manufacturingprocess (in contrast to analytical chromatography or small scale preparativeapplications), method development work has to find conditions which give thehighest throughput with the highest yield and the lowest possible cost.”What kind of application will the product be used in?”What are the purity issues in relation to the source material and intendeduse of final product? What has to be removed?”What kind of starting material do I have?”What are the major ”Headaches”?”What final scale am I thinking of?”What consequences will this have for the technical approach?”What is my purification strategy?”First, CAPTUREWhat will be the major purpose for the initial chromatographic step? Is myproposal rational?Then, INTERMEDIATE PURIFICATIONWhat will be the major purpose with each subsequent chromatographic step?Finally, POLISHINGWhat will be the major purpose of the polishing stage? Looking back upstream, doesthe overall balance and sequence of techniques appear logical?”How do I get the most out of my process?”Will my process be more productive, safe, robust, economic and easier to use thanone which our competitors could do?Will I arrive at the final process faster than our competitors?Fig. 60. Questions that must be addressed to assure a rational process designThe design must ensure that the purity requirements of the final product are met,and also considering the special safety issues involved in production of biopharmaceuticals,such as infectious agents, pyrogens, immunogenic contaminants andtumorigenic hazards. In general, the purity issues must be addressed in relation tothe nature of the source material and the intended use of the final product. It isimportant to define the impurities and contaminants which have to be removedfrom the source material during downstream processing.107

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?O2@@@@@@@@@6K???O2@@@@@@@@@@@@@@@6K?W2@@@@@@@@@@@@@@@@@@@@@6X???O&@@@@@@@@@@@@@@@@@@@@@@@)K??W2@@@@@@@@@@@@@@@@@@@@@@@@@@@6X?O&@@?@@@@@@@@@@@@@@@@@@@@@@@@@@)K??W2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6X???W&@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X??7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1?J@@X@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L???W&@V@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X??7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1?J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L???W&@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X??7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L??7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@???J@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L??7@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1?W2@@@@6X?O2@@@@@@@@@@@@@@@@@@@@6X ??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@W&@@@@@@)K??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@YgV@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?g@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@V'@@@@@@(?4@@@@@@@@@@@@@@@@@@@@0M???3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5?V4@@@@0Y???N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5??N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H???@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5??V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H???3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5??V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@H???3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@5??V'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(Y?V4@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y??I'@@@@@@@@@@@@@@@@@@@@@@@@@@@@@(M???V4@@@@@@@@@@@@@@@@@@@@@@@@@@@0Y??I'@@@@@@@@@@@@@@@@@@@@@@@(M?V4@@@@@@@@@@@@@@@@@@@@@@H??@@@@@@@@@@@@@@@@@@@@L??@?I4@@@@@@@@@@@@@@@@1???J5?hf@@@@@@@L?W.Y?hf3@@@@@@)X?N@@@@@@@1? ?7H?@@@@@@@@L ??J5??@@@@@@@@)X? ??7H??3@@@@@@@@1? ?J5?N@@@@@@@@@L ?7H@@@@@@@@@)X? ??J5?3@@@@@@@@@1? ??7H?N@@@@@@@@@@L ?J5?@@@@@@@@@@)X? ??W.Y?3@@@@@@@@@@1? ??7H??N@@@@@@@@@@@L ?J5@@@@@@@@@@@)X? ?7H@@@@@@@@@@@@1? ??J5?3@@@@@@@@@@@@L ??7H?N@@@@@@@@@@@@)X? ?J5?@@@@@@@@@@@@@1? ?7H?3@@@@@@@@@@@@@L ??J5??N@@@@@@@@@@@@@)X? ?W.Y?@@@@@@@@@@@@@@1? ?7H3@@@@@@@@@@@@@@L ??J5?N@@@@@@@@@@@@@@)X? ??7H??@@@@@@@@@@@@@@@1? ?J5?@@@@@@@@@@@@@@@@L ?7H?3@@@@@@@@@@@@@@@)X? ??J5??N@@@@@@@@@@@@@@@@1? ??7H?@@@@@@@@@@@@@@@@@L ?J53@@@@@@@@@@@@@@@@)X? ?7HN@@@@@@@@@@@@@@@@@1? ??J5??@@@@@@@@@@@@@@@@@@L ?W.Y??@@@@@@@@@@@@@@@@@@1 ?7H?3@@@@@@@@@@@@@@@@@@L? ??J5??N@@@@@@@@@@@@@@@@@@)X ??7H?@@@@@@@@@@@@@@@@@@@1 ?J5@@@@@@@@@@@@@@@@@@@@L? ?7H3@@@@@@@@@@@@@@@@@@@)X ??J5?N@@@@@@@@@@@@@@@@@@@@1 ??7H??@@@@@@@@@@@@@@@@@@@@@L? ?J5?3@@@@@@@@@@@@@@@@@@@@)X ??W.Y?N@@@@@@@@@@@@@@@@@@@@@1 ??7H?@@@@@@@@@@@@@@@@@@@@@@L? ?J53@@@@@@@@@@@@@@@@@@@@@)X ?7HN@@@@@@@@@@@@@@@@@@@@@@1 ??J5??@@@@@@@@@@@@@@@@@@@@@@@L? ??7H??@@@@@@@@@@@@@@@@@@@@@@@)X ?J5?3@@@@@@@@@@@@@@@@@@@@@@@1 ?7H?N@@@@@@@@@@@@@@@@@@@@@@@@L? ??J5?@@@@@@@@@@@@@@@@@@@@@@@@)X ?W.Y?3@@@@@@@@@@@@@@@@@@@@@@@@1 ?7HN@@@@@@@@@@@@@@@@@@@@@@@@@L? ??J5??@@@@@@@@@@@@@@@@@@@@@@@@@)X ??7H??@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?J5?3@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?7H?N@@@@@@@@@@@@@@@@@@@@@@@@@@)X ??J5?@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ??7H?3@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?J5N@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ??W.Y?@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ??7H??3@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?J5?N@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?7H@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ??J5?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ??7H?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?J5N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?7H?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ??J5??3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ?W.Y??N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?7H@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ??J5?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)X ??7H?3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1 ?J5?O@?h7H N@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@L? ?W2@6X??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@)Xg?W2@6X ?@?W-Xg?7U?I/?@@6KO26X?W26KO2@@?g?J5??3@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1g?7

The number of separation media on the market is quite enormous and it will notbe possible to include all different alternatives in the initial media screening. Thenumber of media have to be reduced to cut down time and effort spent on themethod scouting phase of process development. Only those media supporting theissues of scaleability and suitability for the different stages in a process, listed insidethe filter in Figure 66, should be considered for testing.Base matrix properties and derivitization chemistryBase matrix properties and derivitization chemistry govern the chemical and physicalstability of the chromatography media and are very closely related to the scaleabilityof the complete process. The design in of scaleability by selection of suitablemedia will assure that the media can be packed in large scale columns withoutchange in performance and flow/pressure characteristics and that efficient maintenanceprocedures can be applied to secure a long media life time. The possibility ofany toxic substances leaking from the media into the product stream is also closelyrelated to the properties of the base matrix and the chemistry used for couplingspacers and ligands to the matrix.Bead sizeThe particle size and the range of particle size distribution may also have animpact on scaleability in the sense that particle size is closely related to the backpressuregenerated in the column during a chromatographic run. The optimalbead size in any particular chromatographic step will depend on the characteristicsof the feed and the degree of purification required in this step. In a final polishingstep, for instance, there will be a need for smaller beads, i.e. high efficiency, toaccomplish separation of closely related compounds. In such a step, media with anarrow particle size distribution will help to give a lower back-pressure at a givenbed height and flow rate compared to media with a wider particle sizedistribution.Documentation and technical supportComprehensive documentation is required for chromatography media to be usedin industrial processes, to facilitate the work with setting up and validating thecomplete process. Chromatography media used in production processes are treatedas raw materials. As with any type of raw material, acceptance criteria have tobe established and every new batch of media has to be subjected to tests before itcan be brought into production.Regulatory supportInformation on possible extractable compounds and what kind of methods to useto quantify these compounds should be provided by the vendor. Leakage data onthe most relevant extractable compounds should be available.113

Vendor certificationVendor certification programmes should be initiated for all vendors of criticalchemicals or materials and should certainly include the chromatography mediasupplier. Such programmes should be implemented to assure a long term, reliablesupply of chromatography media of high quality and consistency.Delivery capacityThe delivery capacity of the vendor is an important issue during the vendor certificationphase to secure timely deliveries of large quantities of media when the purificationprocess is scaled up. The largest batch size that the vendor can provideshould be discussed and put in relation to the column size that will be used in thefinal production scale. The stock situation and lead times should be discussed toestimate the consequences for continuity in production in case of an urgent needfor a new batch of media.Long-term contracts, based on forecasts provided by the user, should be discussedto secure future timely deliveries. Long-term delivery guarantees should also bediscussed to assure that the same quality of chromatography media can be deliveredduring the entire life cycle of the product to be purified.Method design and optimizationThe main purpose of optimising a chromatographic step is to reach the pre-definedpurity level with highest possible product recovery by choosing the most suitablecombination of the critical chromatographic parameters. In process chromatography,in contrast to analytical or small scale preparative chromatography, thisalso has to be accomplished as quickly, cheaply and easily as possible. The methodmust be designed carefully to be robust despite variations in feed stock and otherconditions in the production hall.The following sections will give some guidelines for optimising the critical operationalparameters which affect the maximum utilization of an ion exchange chromatographystep to be used in a production process.Binding conditionsSelectivity during adsorption to an ion exchanger is optimized by careful selectionof pH and ionic strength of the start buffer. A pH far away from the isoelectricpoint of the molecule of interest will give stronger binding and increased capacitybut may also have a negative impact on selectivity due to increased binding of contaminatingmolecules. If retention of the molecule of interest is low at selectedstart conditions, due to the pH being very close to the isoelectric point, it will startto elute from the column during sample application (isocratic elution) when thesample volume is increased in a preparative situation. The choice of optimal pH114

will always be a balance between selectivity and capacity which in turn dependson the purpose and strategic focus of any particular chromatographic step.The buffer system should be selected to give maximum buffering power at theleast possible ionic strength to ensure high binding capacity of the molecule ofinterest. To achieve this, the pKa of the buffer should ideally not be more than0.5 pH units away from the pH being used. Generally, 10 mM of a buffer is theminimum desirable level. Ideally, one of the buffering species should also beuncharged, and so not contribute to the ionic strength. In large scale applications,economic considerations often limit the choice to acetate, citrate, phosphate, orother inexpensive components.The pH and conductivity in the binding buffer can sometimes cause aggregation/precipitation in the sample when it has been equilibrated to start conditions. Ifaggregates are formed they may be excluded by the beads and lost in the flowthrough fraction with loss of recovery as a consequence. The extent of aggregates/precipitateformation depends on the pre-column residence time, after samplehas been transferred to start conditions. This problem is often recognized as ascale-up problem since the pre-column residence time may increase considerablyupon scale up.ElutionElution from ion exchangers is usually accomplished by applying a continuos orstepwise increase of the ionic strength of the eluting buffer, thereby weakening theelectrostatic interaction between the bound molecule and the adsorbent.Depending on the purpose and strategic focus, as previously outlined, differentdesorption principles can be applied to achieve the objectives of any particularchromatographic step in the most optimal way.• Stepwise elution• Gradient elution• Isocratic elutionStepwise elution is often preferred in large scale applications since it is technicallymore simple than elution with continuos gradients. Stepwise elution will alsodecrease buffer consumption, shorten cycle times and allow the molecule of interestto be eluted in a more concentrated form.Single-step elution and two-step elution can be characterized as being a ’’groupseparation” technique. This type of elution is usually applied in initial chromatographicsteps (capture) where the purpose is to remove bulk impurities and substancesdiffering greatly from the product. In a large scale initial chromatographicstep, using a crude feed material, media with a large bead size are favoured toavoid problems with high back-pressure and reduced media life time due to highviscosity and severe fouling during sample application. In such an application itwill be very difficult, unless the selectivity is extremely high, to resolve closely rela-115

ted contaminants from the molecule of interest, even when applying very shallowgradients. The strategy will be to resolve the ”group” of substances that the moleculeof interest belongs to from ”group(s)” containing the contaminating substances.This can most conveniently be achieved by eluting one ”group” at a time byapplying one or several steps with increasing eluting strength.In later purification steps however, applying feed material that has been partlypurified and using chromatography media with higher resolving power, it will beeasier to resolve closely related substances by applying multi-step or gradient elutiontechniques. Resolution is maximized by working on the shape or slope of agradient or the eluting strength of different steps in a multi-step procedure. Sucheluting techniques can be characterized as being ”fine separation” techniques asopposed to the ”group separation” refered to above.In final purification steps (polishing), where the main focus will be to reach thepredefined purity of the molecule of interest, resolution is maximized by applyingshallow gradients or even isocratic elution using high resolution media with smallbead size.When stepwise elution is applied, one has to keep in mind the danger of gettingartefact peaks when a subsequent step is administered too early after a tailingpeak. For this reason it is recommended to use continuos gradients in the initialexperiments to characterize the sample and its chromatographic behaviour.Elution by pH gradients is not generally applied. This is because, changing the pHby applying a pH gradient is frustrated by the buffering power of the moleculesadsorbed on the column and, in case of weak ion exchangers, the buffering of theadsorbent groups themselves. For stepwise applications, pH elution can be quitesuccessful. The pH change will be delayed compared with the new buffer frontbecause of these titrations, but eventually the bound molecule is desorbed, coincidentwith a rapid pH change.Sample loadWhen the selectivity parameters have been defined to achieve the most optimalbalance between resolution, capacity, speed and recovery, in ion exchange chromatography,as for most other adsorption techniques, there are then basically twoalternative routes to follow for optimization of sample load and flow rate to achievehighest possible productivity in the system.I. In a typical capture situation the sample will be applied to the column, nonboundsubstances will be washed out from the column and the compound of interestwill be eluted from the column with a simple step elution procedure. The differencein eluting strength, between the different steps will usually be large, i.e. itwill be possible to elute one group of compounds while the others are still retainedon the column. In this mode, the entire bed volume can be utilized for sample bin-116

ding and the prime consideration when optimising for highest possible productivityis to define the highest possible sample load over the shortest possible sampleapplication time with acceptable loss in yield.The dynamic binding capacity for the protein of interest should be determined byfrontal analysis, i.e. by continuously applying sample on the column up to thepoint where the compound of interest starts leaking off at the column outlet.PAGE, ELISA or other appropriate techniques are used for the determination ofthe break-through profile of the compound of interest.II. In many intermediate purification steps, and always in a polishing step, therequirements for resolution will set the limit for the amount of sample that can beapplied to the column. Sample is mainly bound in the upper part of the bed sincethere will be a need for a certain bed height to achieve separation between closelyrelated substances moving down the column with different velocities in a shallowgradient of elution buffer.Maximum sample loading is defined by running a series of experiments with graduallyincreased sample load. Optimal conditions will be the maximum sampleload that provides a resolution still high enough to meet the pre-defined purityrequirements.Flow rateThe maximum flow rate that can be applied in any particular ion exchange chromatographystep will differ between different parts of the chromatographic cycle.Since low molecular weight substances show high diffusion rates, i.e. are transportedrapidly between the mobile phase and stationary phase, the flow rate duringequilibration, washing and regeneration procedures is limited primarily by therigidity of the chromatography media and by system constraints regarding pressurespecification. Larger molecules, i.e. the substances to be separated during thechromatographic run, show a lower diffusion rate which will limit the flow ratethat can be applied during sample adsorption and desorption.In a typical capture situation, the flow rate during sample application has to becontrolled so that the residence time in the column allows for a complete bindingwithout leakage in the flow through fraction. Maximum flow rate is defined byrunning the frontal analysis test (break-through) refered to above at a number ofdifferent flow rates. Optimal conditions will depend on the requirements for speedand capacity in the system. If speed, i.e. sample application time, is critical due toproteolysis or other detrimental effects in the feed material, a higher flow rate mayhave to be used on the expense of the binding capacity in terms of amount of samplethat can be applied per volume of media. If speed is not a big issue, bindingcapacity can be increased on the expense of flow rate which will reduce the scaleof work in the final production process. Occasionally, high back-pressure, due to117

??@@h@@f?@@? ??W2@@6?2@?h@@?@@? ??7@?I4@@@?h@@L?hf@@??3@?e?@@@@@6?2@@@@@@?@@@@6X?@@@6X@@@@@6?2@@@@@@6X?@@???V4@@6X@@?@@@@@@(Y@@H?@@@@@1e@@@@@@@@@@@@@@@@@@@1?@@???@@@@@@@@@@@H?@@L?@@@@@@?'@@@@@@@@e?@@?@@@@@@?@@???@@@@0?4@@@@@@@@e@@@?@@@@@@?V4@@@@@@@@@@@@?@@@@@@?@@?????@??@??@??@??@??@??@?@@ @? @@@6X? ?@@? ??@@@6Xh@@?W2@@?h@@?@@?@@ @@@@@? @@?@1? ?@@? ??@@?@)K?hO&@?he@@N@@@H? @@?@@W2@6?2@@@6T2@@6X?@@6KO2@@@W2@@@hg??@@?@@@@@@?@@@@@@@@@@@@6T2@@6X@@@@@@@6X?@@6XeW2@@@?@@@@6KO2@@6?2@6X?@@@ @@?@@@@@@@@@@@@@Y@@@1?@@@@@@?@@@@@@?hg??@@@0Y@@@@?@@@@@?@@@@@V4@U@@@@@@?@@@?@1?@@@1e*@@@e@@@@@@@@?@@@@@@)?3@5 @@?@@@@?e@@@@@@@@@@@?@@@@@@?@@?@@@,hg??@@?e3@@@?@@?@@?@@@@@eS@@@@@@@?@@@?@5?@@@@e?@@@(?@@@@@@@@?@@@@??N@H @@@0MI4@@@@@@@@0?4@@@?@@@0?4@@@@@@0Yhg??@@?eV4@@?@@?@@?@@@@@@@0R4@@@@@@@@@@0Y?@@@@e@@@0Y?@@@@@0?4@@@V4@@@@? ??@5??@@@0Y??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@ ?@??@ ?@??@he@??@?@f?@K?eW2@? ?W&?hg?@?@? ?@he@??@?@?W2@?@@@e*@@?W-X?@@@@@??@@?)X?*@W)Xhf?W26Xh?W-XfO@@? ?@he@@@@?@?7Y@?@?@eN@@?7R1?@@@@@??@HJ@1?N@@@)hf?7

?@@@@@@@@@@@@@@@@@@@@@@@?@?@?@@??@@??@@?J@@?7@@?@@@?@@@?@@@?@@@?@@@?@@@?@@@?@@@?@@?W.?W.Y?@?@@hf?W.Y@@@@hfW.Y?@@@@hf7H@??@he?J5?@??@heO&Y?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?h@??@hW(M? @?h@?@??@h.Y @?h@?@??@ @?h@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?h@?h@?@?h@?@?@?h?O@K @?h@??@@@@@@@@@@@@@@@@@ @?h@??I@Mhe?@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@?@? ?@ @?h?@ @?h@??@ @?h@??@ @?h@??@ @?h@??@ @?h@?J@L? @?h@?7@@? @?h@?3@H? @?h@?N@ @?h@??@ @?h@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?f?O2@@?e?O20M?@??O20M?e@W20M?f@(M?g?O2@@Hh?O20M?@?h?O20M?e@?h?O20M?f@?h?O20M?g@?h?W20M?h@?h?.M?he@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?h@?hO26Khf?O2@6K O26K @?h@?@?eO2@@@@@@6Khe?O2@0MI46Kh?O20M?I46KheO20MI46Khe?O2@@6K?g?J@?h@?O20MgI4@6K?fO2@0M?fI4@6K?e?O20M?fI46Kf?O2@0MfI4@6K?e?O2@@0M??I46K?fO&@?h@@0Mhe?I46K?O20Mhe?I46KO20M?hI46K?O20M?h?I46KO20M?g?I4@6KO20Mhe?I4@0M ?I40M?hfI4@0M?hf?I40M?hfI40Mhf@MAspects of column designFlow distribution systemThe most important factor in process column construction is to design the flowdistribution system to give as even a flow distribution as possible at the columninlet and outlet. The aim is to retain HETP values of the same order as in the laboratorycolumn. This is usually achieved by a construction where the radial backpressureis negligible compared to the axial back-pressure at the column inlet, seeFigure 68. The simplest method is to place a coarse mesh net between the columnend piece and the finer mesh net retaining the bed, to create channels for radialdistribution. This may be combined with multiple inlet/outlet ports depending oncolumn diameter. Depth filters are more easily clogged due to the relatively largefilter surface. This may be a severe disadvantage in continuous operation.Column inletColumn liddPdrdPdLPacked bedFig. 68. Radial and axial backpressurein a column distributionsystem.Material resistance and durabilityWetted components must be constructed from materials with high chemical resistanceagainst the harsh chemicals which are frequently used for cleaning-in-place(CIP) and sanitization procedures such as 1 M NaOH, salts, acids, etc.Stainless steel of high grades is most commonly used for very large columns.However, stainless steel does not always have sufficient corrosion resistance whenhigh salt concentrations in acidic solution are used. In this case fluoroplastic coatedstainless steel is recommended. Materials should be chosen to minimize leakageand be tested for toxicity.Sanitary designEffective cleaning and sanitizing of packed columns depends on the total columndesign, including the absence of threaded fittings and the smoothness of wettedsurfaces. It is important to minimize dead volumes in the column to hinder bacterialattachment and facilitate cleaning. Columns constructed from calibratedborosilicate glass allow the use of thin O-rings in the adaptor and end piece and119

give minimum dead volumes. Borosilicate also has a smooth and durable surfacewhich facilitates cleaning. Plastic columns are usually less expensive but mostplastics do not meet pharmaceutical industry demands for chemical resistance,hygienic design and in-line cleaning. They may however be well suited for scale-upexperiments.Pressure vessel safetyLarge scale columns should be regarded as pressure vessels even if the actual workingpressures usually are kept low. Large volumes of organic solvents may behandled during cleaning which calls for explosion proof equipment. The columndesign has to meet local regulations to be approved.Regulatory supportRegulatory support for process scale columns should be available from thecolumn supplier to provide information on materials necessary for registration ofthe process including chemical stability, toxicological tests, physical data andcolumn construction.ErgonomicsFor easy handling of process columns it is important that they are constructed in astable way and are easy to pack and clean. Large columns should preferably havelockable wheels.In laboratory columns, the bed height can easily be adjusted using moveable adaptors.In large columns, adaptors may be impractical and too heavy to handle.Therefore, columns with fixed end pieces are selected in many applications.Valves should be easy to reach and remove when the column is taken apart.Packing large scale columnsColumn configurationProcess columns with a moveable adaptor are essentially packed in the same wayas laboratory columns with adaptors. In essence, this means that the gel slurry iscompressed by a flowing liquid until the bed height has stabilized, at which pointthe flow is stopped and the adaptor is lowered onto the gel surface and secured inplace.Large scale columns are, however, frequently supplied with fixed end pieces. Thiscalls for a different packing technique. An extension tube is fitted on top of thecolumn as a reservoir for the gel slurry. When the bed has been packed and settledat the join between the extension tube and the column, the extension tube is removedand the top column lid is secured in place. With this method it is important to120

calculate the exact amount of media that is required to get the appropriate bedheight.Packing the columnDetailed packing instructions for ion exchange media in process columns will notbe given here. Please refer to the instructions supplied with respective media andrespective column.Scale-upWhen the IEX step has been optimized at laboratory scale, the method can be scaled-up.Provided that scaleability has been ”designed-in” during the developmentphase, scale-up to final production scale should be straightforward.”Design-in” of scaleability has to do with how the chromatographic step has beendesigned and optimized (robustness, simplicity, costs, capacity etc.) and the choiceof appropriate chromatography media (chemical stability, physical stability, beadsize, cost etc.).Some general guidelines for scaling up are outlined in Table 23.Table 23. Scale-up guidelines.Maintain Increase Check system factorsBed height Column diameter Distribution systemLinear flow rate Volumetric flow rate Wall effectsSample concentration Sample load Extra column zoneGradient volume/bed volumespreadingIncreasing the bed volume by increasing the column diameter and increasing volumetricflow and sample load accordingly, will ensure the same cycle time as in thelaboratory scale method development. The column bed height, linear flow rate,sample concentration and ratio of sample to gel, all optimized at laboratory scale,will be kept the same. If a gradient is used for elution, the ratio of gradient volumeto bed volume will remain constant and, therefore, the time required for the gradientto develop and the effect on resolution, will remain the same on the largercolumn. The same principle is applied for the volume of each step in a step elutionprocedure.Different system factors may affect performance after scale-up. If the large scalecolumn has a less efficient flow distribution system, or the large scale system introduceslarge dead volumes, peak broadening may occur. This will cause extra dilutionof the product fraction or even loss of resolution if the application is sensitiveto variations in efficiency (plate number) in the system used.121

scaling up to a larger diameter column means that most of the bed support generatedby the friction against the column wall is lost. This can give increased bedcompression and poorer flow/pressure characteristics.If all the above aspects are taken into consideration, chromatographic variabilityis normally not a major issue when scaling up.Non-chromatographic factors may have a more significant effect on performanceduring scaling up. These factors include: changes in sample composition and concentrationthat often occur as the fermentation scale increases, precipitation in thefeed stock due to longer holding times when large volumes are handled, nonreproducibilityof the buffer quality due to inadequate equipment for consistentlypreparing large quantities of buffer solutions, and microbial growth in feed-stockor buffers due to increased handling and longer holding times.Figure 69 shows a 700-fold scale up of a model protein separation on SOURCE30S going from a 2.2 ml column to a 1.57 liter column in one step.122

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12. ApplicationsIon exchange has proven to be one of the major methods of fractionation of labilebiological substances. From the introduction of the technique in the 1960s´ to thedevelopment of modern high performance media, ion exchange chromatographyhas played a major role in the separation and purification of biomolecules andcontributed significantly to our understanding of biological processes. The examplesgiven in the following section have been drawn from the published literatureas well as from work in our own laboratories.For detailed information on specific subjects the reader is referred to the originalwork.The design of a biochemical separationIon exchange chromatography, in common with other separation techniques inthe life sciences, is rarely sufficient as the sole purification stage in the separationor analysis of complex biological samples. Ion exchange is frequently combinedwith other techniques which separate according to other parameters such as size(gel filtration), hydrophobicity (hydrophobic interaction chromatography orRPC) or biological activity (affinity chromatography).908070Popularity of fractionation techniquesHomogenization605040PrecipitationIEXGF30AC20101 2 3 4 5 6 7Stage in purification schemeFig. 70. Frequency of useof fractionation techniques(1). (Reproduced bykind permission of theauthors and publisher.)Not only is the choice of techniques important. The order in which they areemployed will also play a role in determining the speed, the convenience and theoverall yield for the purification.124

@?@??@eO@K?e?@@@f?O@??@?@?@?@heW&eW-KO.??@?@?W-XeW.e?@@@eW.h@K?@?@?@?@eW-X?)X?W&@e.R'@U??@?@?7R1e7Ye?@?@e7YeW&?26X@@@??@?26T-KO@?W-Xe@?@??@W2@@@@e?@?@W)X?@@@W-X?@@@@@@@?@?2@6T.?W-X ?@?@?@?@heW&eW-KO.??@?@?W-XeW.e?W26K?e?)X??W26K?e?)X? W.MS@@6K?@)T2@@@6T2@e@?he?@@@?@?@e7R)T@1?7Y@fN@)X?@?@?@?@e@@@??@@@e@@@?7@@@V@@?@?J@@@V@R@@@W&@)e@@@??@@Y@@?@e?@@@@@)?@?@@R1?@?@?@?@W@@@?@@H?7@) @?@?@6KO@?26T&@?W-X?@@e.R'@R@@R1??@6X?@6T&?26X??W2@@@?)X?W&@W)Xhg??@?@?@?@eW-X?)X?W&@e.R'@U??@?@?7R1e7YeW.MS@@6K?@)T2@@@6T2@e@?he?@@@?@?@e7R)T@1?7Y@fN@)X?@?@?@?@e@@@? @?@?@V@@@@@V@Y@?7@)?@@fN@?@@?@??@V1?@V@@@@V1??7Y@@HJ@1?7Y@@@)hg??@?@?@?@?C@T@@U@?3X@e@KC@>,?@?@?3T5e3T5??@f3T5?3X?@W@@?@?*U@@?3T(Y@@@X?e@?@??@@X@@?@e?@?I'Xe@?@@T5?@?@?@?@@U@@?@@L?3X? *UO&@XS@@@>@@?@?@@Y@*UO&@XS@@@>@@?@?@@Y@ V40MI40R+R4@@?@?@@?@e@?he?@?@?@?@@0R+MI4@?V4@e@@0R+Y?@?@?V+YeV+Y??@fV+Y?V/?@(R'?@?V4@@?V+Y?@MI/?e@?@??(R@@5?@e?@eV/e@?(R+Y?@?@?@?(R4@@?(R/?V/? @?@?@W@@X?@?3X@?3Xe@@e@KC@T@@T5??@W5?@?3X?@W5??3X@@?*U@?3X@@X?hg??@?@?@?@?C@T@@U@?3X@e@KC@>,?@?@?3T5e3T5?V40MI40R+R4@@?@?@@?@e@?he?@?@?@?@@0R+MI4@?V4@e@@0R+Y?@?@?V+YeV+Y? @@@?@(MI/?@?V4@?V/e@@e@@0R+MI+Y??@(Y?@?V/?@(Y??N@@5?V4@?V40R/?hg??@0Y ?(Y??@ ?@?@ ?W-Xe@@@?f?O@?he?@f?W&?f?)X?W&hf?@he@?e@?eO2@?g?W-Kg@?e?@@6T-X?W-X?f?O@??@1?7@W-T2@@?W-KO.?@W-X?@@@?@X@?e@W2@@8h?7R@@?@?@?@?e?@X;@R1?7R1??@@@@@@??W&?W&?2@@?@@@@@@??@?@6T&KO)X??W.?W&X@?@ ?W26K?he?@ ?*?,e@Xe?@@@@@@??@6KO-X?W-X@W)X?W&@??@@@@@?@?@?W&?2@@?)X?@e@@6T-X?@@@@@?W.??)X?7@?W-T2@@??W-X?@6T.?@@@??@?2@6T2@?W-X???W26K?he?@W&@>@@6T2@@6T26KC@T&?@hf?S@Ue@)X??@?@?@@??@V@@R1?7R@@@@)?7Y@??@?@ N@W@@@?7@@@?@W@1?@e@@V@R1?@?@?@?7H?J@1?@@?7R@@?@??7@)?@@@H?@?@?J@@@?@@Y@?7@)??W&@>@@6T2@@6T26KC@T&?@hf?@@?@@@R@@?@?7R@@H?@@R1?@?@W@@@?e@@Y@@?@@g?@?@HJ@@@?@?e?@)X@?@?@?@??@?@?@@??*@W&@@@?@?@@?@?@??@?@V@@@@@)??7H?7@@@?@*@S@@@@@Y@?@@YS@@R@5 ?*?)X??S,??@?@?@@??@W@@T5?3T@@@Xe3X@??@?@?@@U@@?3X?@?@@U@?@e@@?3T5?@?@?@?3L?*U@?@@?3T@@?@??3X??@@@L?@?@?*U@@?@@X@?3Xe??@@T@@@T@@?@?3T@@L?@@T5?@?@@U@@?e@@X@@9V@g?@?@?*U@@?@?fS@@T5?3T5??@?@?@@??N@@@X?@?@?@@?@?@??@?@?3XI'Xe?3L?3X?@?@ *@S@@@@@Y@?@@YS@@R@5V40MI4@@?@?3@@0R'?(Y?@hf?(R+R+R+R'?@?V+MI/?(R+Y?@?(R4@@?e(R@@V4@@e?@e?3T5?V4@@?@?@?e.MI+Y?V+Y??@?@?@@?e@MI/?@?@@@@?@?@??@?@?V/?V/e?V/?V/?@?@@@g?@e?V+Yg@?@? ?N@? ?(Y??N@??@@5 @?@?W2@@heW&f?V@HW&K?e@@@?W-KO-XgO@e@?@?eW26XW&@K @Ke?W.?@?7@?@@?@?@6T-X??W&@@@?@?@@?W&KO&@e@@@@@?7@@@e@Xe7R@@R1e@@@@@@e@?@?e7

matography solutes bind to the gel at low ionic strength and are eluted from thecolumn at a higher ionic strength. The converse situation occurs in hydrophobicinteraction chromatography. Thus if these two techniques are to be used in a separationscheme it is logical to have them adjacent to each other. This principle isillustrated in Figure 72 which shows the purification of human a 2 -macroglobulinfrom Cohn Fraction III.After initial purification by affinity chromatography on Blue Sepharose CL-6B toremove albumin, the sample was applied to a Q Sepharose High Performancecolumn and eluted with an increasing salt concentration gradient. Relevant fractionswere then pooled and a 2 -macroglobulin was purified to homogeneity byhydrophobic interaction chromatography on a Phenyl Sepharose HighPerformance column.Fig. 72. Purification of human a 2 -macroglobulin. (Work by Pharmacia Biotech,Uppsala, Sweden.)126J(?'L?.Y?V/?@?@??@eO@K?e?@@@f?O@? @@?@@@@@@?@?@?@?h?W&??W-KO.e@?@?W-X??W26X?eW.h@K@?@?@?@??W-X?)X?W&@??.R'@Ue@?@?7R1??7@@?@?@@Y@he@?@?@?@?C@T@@U@?3X@??@KC@>,?@?@?3T5??3T&@?e3T5?3X?@W@@?@?*U@@?3T(Y@@@X?e@?@??@@X@@?@e?@?I'Xe@?@@T5?@?@?@?@@U@@?@@L?3X?V40MI40R+R4@@?@?@@?@e@?g@?@?@?@@0R+MI4@?V4@??@@0R+Y?@?@?V+Y??V4@@?eV+Y?V/?@(R'?@?V4@@?V+Y?@MI/?e@?@??(R@@5?@e?@eV/e@?(R+Y?@?@?@?(R4@@?(R/?V/??@0Y @?e.R/??(Y?@??@K?e@? @?e@Kf@@@??@?@@@?W-XgO@f?@f@Kg@?@?he?@K?he?O@Ke?@he?@hW.?@e@?W-X??W26K?he?@hf?@eI@?7R1e@@@@@@eW-X@e@?@@6?)Xe@?@W-X?@@@@@?@?@@@@6T-T&?@?W2@@@@??@?@@?@KO.?@?@W-T2@@e7H?@e@?7R1??@@@@6T2@??@6X?@6T-T&?W&X@@@@?@?W&@>@@6T2@@6T26KC@T&?@h?@f?@?@e@?@?@@e7R@@e@?@?@@@)e@?@@R1?@@@@@?@?@?@@V@R@@?@?7Y@@?@??@?@HJ@@@H?@?@@R@@?@e@??@e@?@?@??@?@?@@Y@??@V1?@V@R@@?7@@@@?@?@?*@S@@@@@Y@?@@YS@@R@5he?@e@??3T5e@?@?@@e3T@@e@?@?@@X?e@?@@T5?@@@@@?@?@?@@?3T@@?@?3X@@?@??@?@?*U@@L?@?@@T@@?@e@??@e@?3T5??@?@?@@X@??@W5?@?3T@@?3X?@@?@?@?V40MI4@@?@?3@@0R'?(Y?@h?@e@??V+Ye@?@?@@eV+R'e@?@?(R/?e@?(R+Y?@0?4@?@?@?@@?V+R4@@?N@@5?@??@?@?V40R/?@?(R+R'?@e3L?@e@?V+Y??@?@?(R@@??@(Y?@?V+R'?V/?@@?@?@??@K? W&eW26Khe@? W2@?hfW&?@K?O@K?fW&K?h?W2@hfW&K?hg?*@@?W26X?@?@?@e*@?@@@?@@?W26T&?@??W2@@@@@f*@@@e'6KO26T&@@?W26X?@@@?e*@@@?W26X?@@@@@?e?@W26X?@@@?eW26Xe@?)T26X@@@e'6X?@@@?W2@@?W26X??W26X?*@e*@@@@??W26X?e@@

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A 280nmb 2mRBP+TSFAGP+a 1mALBSample:Column:Flow rate:Buffer A:Buffer B:Gradient:Detection:0.5 ml desalted urineMono Q HR 5/52 ml/min6.25 mM Bis-Tris propane, pH 7.5BufferA + 0.35 M NaCl, pH 9.50-100% in 25 ml280 nm, 0.05AUFS0 10 20 30 Vol (ml)Urine protein chromatogram from a renal-transplant patient, illustrating thedistinct peaks of the low MW proteins. The retinal-binding proteins (RBP) iscontaminated with a small amount of transferrin (TSF).b 2m = b 2-microglobulin,AGP =a 1-acid glycoprotein,a 1m = a 1-microglobulin,ALB = albumin. Diagonal line illustrates gradient from ferA buf to bufer B.Fig. 87. Anionexchange of urine inrenal proteinuria(39).Purification of a recombinantPseudomonas aeruginosa exotoxin A, PE553DThis application shows a purification process for a genetically modified recombinantPseudomonas aeruginosa exotoxin A (MW 55 000) expressed in the periplasmof E. coli. The process was developed for large scale production of modifiedtoxin, conjugation to a polysaccharide and use as a vaccine. The purificationstrategy used chromatography media differentiated to tackle the problems of capture,intermediate purification and polishing (for further information please referto Chapter 11.)The result was a highly purified exotoxin A from crude cell homogenate usingonly four chromatography steps, and taking less than half the time of a more conventionalapproach.Exotoxin A was captured directly from unclarified E. coli homogenate by expandedbed adsorption using STREAMLINE DEAE adsorbent in a STREAMLINE200 column (Fig. 88). The following intermediate purification step was hydrophobicinteraction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow (highsub) packed in a BPG 200 column (Fig. 89). This step removed a substantial partof the UV absorbing material (including nucleic acids) that could interfere withthe following steps. The second intermediate purification step on SOURCE 30Q,packed in FineLINE 100 column, removed the majority of the remaining contaminants(Fig. 90). The polishing step was HIC on SOURCE 15PHE (Fig 91). Theprocess resulted in a pure protein, according to PAGE and RPC analysis (Fig 92and 93), and the overall recovery was 51 % (Table 24).136

?@@@?@?@?@@@J(?'L?.Y?V/T.?e?@@@@?@@@@@?N@U?@@?@@@@?@?@?@??@)?fI4@?@?@?@??W2@@??7Y??@@@@? ?'@@@@f?W-Xe?W-X?@@X ?@ ?V'XW5f?.R/e?7R1S@@? O26X ?@ N@@Hf?@?@?@@U ?@(MI/X? ?@ J@@Lf?@K?e?3T5e?@@@?O2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@6K S@@?hf?@H??N1? ?@ 7Y@1f?@@@?@?V+Yf?@I4@6X? ?'@U ?@f@? ?@ 3X@5f?@heW2@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@0M??B1? ?S@)X?hf?@f@? ?@ S@@Uf?@h?W.M3L ?7

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aLane 1 LMW markersLane 2 pool fromSOURCE 15 PHELane 3 pool fromSOURCE 30QLane 4 pool fromPhenyl Sepharose 6Fast Flow (high sub)Lane 5 LMW markers1 2 3 4 5b1 2 3Lane 1 pool from Phenyl Sepharose 6Fast Flow (high sub)Lane 2 pool from SOURCE 30QLane 3 pool from SOURCE 15PHEFig. 92. a) SDS-PAGE on PhastGel Gradient 8-25b) Native PAGE on PhastGel Gradient 8-25Table 24Purification step Volume, litre Total protein, gram Exotoxin A, gram* Step recovery*Bacterial extract 180 351 10.8STREAMLINEDEAE 13.5 140 8.54 79Phenyl Sepharose 6Fast Flow (high sub) 11.4 41 6.60 77SOURCE 30Q 30.2 12.6 6.04 91SOURCE 15PHE 12.2 n.d. 5.5 91*Activity was determined with a radial immunodiffusuin assay using Goat anti-exotoxin A antibodies(List, USA).139

?W-X??7R1??@?@?W26Xe?W2@6T26X?7

StrategyThe starting point for developing the purification strategy was experience from asuccessful downstream process for purification of another modified Pseudomonasaeruginosa exotoxin, LysPE38 (Fig. 94). Refinements included reducing the numberof steps by the introduction of STREAMLINE DEAE for capture, and achievinghigh flow rates through the use of high performance SOURCE media for lateintermediate purification and polishing.Fig. 94. Comparison of the two purification schemes.141

This approach of using media designed for different stages in a downstream processspeeded up process development and shortened the production schedule.Comparison with the earlier process for purification of a similar exotoxin demonstratesthe dramatic savings in time achieved.Interestingly, the use of ion exchange at more than one stage is a common characteristicof large-scale processes. Note that the role and mode of use of the ionexchanger is very different in the different stages. In the example shown, theanion exchanger, STREAMLINE DEAE, is used for expanded bed adsorption ofthe product from the crude, unfiltered E. coli lysate. STREAMLINE is optimisedto handle such crude feedstocks. Particles, the bulk of the impurities and much ofthe water are removed in a group separation. Sample application conditions areoptimised to achieve maximum selectivity during capture of the product and thestepwise elution conditions achieve maximum concentration during elution.Later in the process, anion exchange is again used, but this time to remove proteinswhich have very similar characteristics to the product. This time SOURCE30Q is used with gradient elution. The uniform, small diameter particles help toachieve good resolution with excellent flow rates and low back-pressures. Thelinear salt gradient further improves resolution. Note also that a different pH hasbeen chosen to increases the binding strength of the product and further improvethe resolution during elution.In other examples ion exchange can be used repeatedly in the same process undereven more divergent conditions, in one step to bind only impurities and allow theproduct to pass through and later to bind the product. Furthermore, anion andcation exchange can be combined in the same scheme or pH and salt gradient elutioncan be alternated to achieve removal of different impurities. All of this is anindication of why ion exchange is such a useful technique in industrial purification.142

13. Fault finding chartProblem Cause RemedyColumn is clogged. Presence of lipoproteins Prior to chromatography,or protein aggregates. precipitate with 10%Dextran Sulphate or 3%polyvinylpyrrolidone.Precipitation of proteinsin the column caused byremoval of stabilizingagents duringfractionation.Filter is clogged.Microbial growthhas occurred in thecolumn.Modify the eluent tomaintain stability.Replace the filter. Alwaysfilter samples and bufferbefore use.Microbial growth rarelyoccurs in columns duringuse, but steps shouldalways be taken to preventinfection of packedcolumns, buffers and gelsuspensions. Store gel inthe presence of 20%ethanol or an antimicrobialagent, see page 103.No flow through Outlet closed. Open outlet.the columnNo flow from pump.Clogged end-piece oradaptor or tubing.With peristaltic pumpscheck the condition of thetubings. Check for leaks atall connections.Remove and clean,if possible.Reduced or poor Bed surface blocked Clean using recommendedflow through the by precipitated methods.column.proteins.143

Problem Cause RemedyReduced or poor Bed compressed. Repacking the column mayflow through thebe necessary.*column.Microbial growth.Fines.Microbial growth rarelyoccurs in columns duringuse, but steps shouldalways be taken to preventinfection of packedcolumns, buffers and gelsuspensions. Store gel inthe presence of 20%ethanol or an antimicrobialagent, see page 103.Do not use a magnetic stirrer; itcan break the beads.Back pressure Turbid sample has been Improve sample solubilityincreases during a applied to the column. by the addition ofrun or duringmonoethylene glycol,successive runs.detergents or organicsolvents.Clogged column filter.Precipitation of proteinin the column filter and/or at the top of the gel bed.Prefilter buffers andsamples. Change filter.Clean the column andexchange or clean thefilter.Change pH and/or add urea.Develop a procedure with detergents.Additives which were usedfor initial sample solubilizationshould be included in the solutionsused for chromatography.* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilotcolumns.144

Problem Cause RemedyBack-pressure Precipitation of Lipoproteins may beincreases during a lipoproteins at increased precipitated prior torun or during ionic strength. chromatography by thesuccessive runs.addition of 10% dextran sulphateand1 M calcium chlorideto final concentrations of0.2% and 0.5 M respectively.The protein does Incorrect buffer pH. Use a buffer pH closernot elute in theto the pI of the protein.salt gradient.Ionic strength too low.Use a more concentratedlimit buffer.The protein does Solutions have wrong pH. Calibrate your pH meter,not elute.prepare new solutions andtry again.Protein elutes in Ionic strength of start Decrease ionic strengththe wash phase. buffer is too high. of start buffer.The ionic strength of thesample is too high or thepH is wrong.The column is notproperly equilibrated.Ionic detergents or otheradditives are adsorbed tothe column.Buffer exchange on sample.Repeat or prolong theequilibration step.Clean the column.The resolution The gradient slope Use a shallower gradientobtained is less is too steep. or a plateau in the gradient.than expected.Microbial growth hasoccurred in the column.Flow rate is too high.See above.Run the separation at alower flow rate.145

Problem Cause RemedyThe resolution Proteins or lipids have Clean and regenerate theobtained is less precipitated on the column.than expected. column.Improper filtration ofthe sample beforeapplication to thecolumn.Regenerate the column,filter the sample and repeatthe chromatography step.Aggregate formation of Use urea or zwitterions,proteins in sample and betaine up to 10% orstrong binding to gel. taurine up to 4%.Column is poorly packed.Too much sample masshas been loaded ontothe column.The column is dirty.Detector cell volume istoo big.Large mixing spaces inor after column.Check the packing by running acoloured compound and observingthe band. Repack thecolumn if necessary.*Decrease the sample load.Clean and regenerate thecolumn.Change the flow cell.Reduce all post columnvolumes.Leading or very Overloaded column. Decrease the sample loadrounded peaksand repeat the run.observed in thechromatogram.* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilotcolumns.146

Problem Cause RemedyLeading or very Column is poorly packed. Check the packing byrounded peaksrunning a colouredobserved in thecompound and observingchromatogram.the band. Repack thecolumn if necessary.*Column needsregeneration.Tailing of the peak Sample too viscous.is observed in thechromatogram.Precipitation of proteinin the column filter and/or at the top of the gel bed.Clean and regenerate thecolumn. If this does not helpreplace with a new one.Reduce the amountof protein.Remove nucleic acids.Clean the column andexchange or clean thefilter.Previous elution Incorrect buffer pH Prepare new solutions.profile cannot be and ionic strength.reproduced.The sample has alteredduring storage.Proteins or lipids haveprecipitated on thecolumn.Sample has not beenfiltered properly.Incomplete equilibration.Prepare fresh sample.Clean and regenerate thecolumn.Regenerate the column,filter the sample carefullyand repeat this step.Equlibrate untilconductivity is constant.Previous elution Aggregate formation of Use urea or zwitterions,profile cannot be proteins in sample and betaine up to 10% orreproduced. strong binding to gel. taurine up to 4%.* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilotcolumns.147

Problem Cause RemedyLow recovery of Sample substance may Determine the pH andactivity while not be stable in the salt stability of the protein.normal recovery elution buffers and isof protein. therefore inactivated.Enzyme separated fromco-factor or similar.Microbial growth.Test by pooling fractionsand repeating the assay.Microbial growth rarelyoccurs in columns during use,but steps should always be takento prevent infection of packedcolumns, buffers and gelsuspensions. Store gel in the presenceof 20% ethanol or an antimicrobialagent, see page 103.Protein amount in The protein may have Add protease inhibitorsthe eluted fractions been degraded byto the buffers to preventis much less than proteases. proteolytic digestion.expected.Adsorbtion to filterduring samplepreparation.Microbial growth hasoccurred in the column.Use another type offilter or use detergents.Microbial growth rarely occursin columns during use, but stepsshould always be taken to preventinfection of packedcolumns, buffers and gel suspensions.Store gel in the presenceof 20% ethanol or an antimicrobialagent, see page 103.Protein amount in Non-specific adsorption. Try adding ethylene glycolthe eluted fractions(e.g. 10%) to the buffers tois much less thanprevent any hydrophobicexpected.interactions.Sample precipitates.Hydrophobic proteins.May be caused by removalof salts or sample dilution.Chaotropic salts may beused for elution.148

Problem Cause RemedyMore activity is Different assay Use the same assayrecovered than conditions have been conditions for all the assayswas applied to used before and after the in your purificationthe column chromatographic step. scheme.Removal of inhibitorsduring separation.Replace if necessary.Peaks too small. Wrong sensitivity range Adjust.on detector.Sample absorbs poorlyat the chosen wavelength.Recorder rangeincorrectly set.Excessive zonebroadeningBubbles in the bed. Column packed or storedat cool temperature andthen warmed up.Eluent not properlyde-gassed.Use a different wavelength.Adjust.Check the column packingand re-pack if necessary.Small bubbles can often beremoved by passing wellde-gassed buffer upwardsthrough the column. Columnmay need to be re-packed. Takespecial care if buffers are usedafter storage in a fridge or coldroom.Do not allow columnto warm up due to sunshineor heating system. A waterjacketis a good safeguard.Use de-gassed buffers.De-gas the eluentthoroughly.Cracks in the bed. Large air leak in column. Check all connections forleaks. Repack the column*.Distorted bands as Air bubble at the top Re-install the adaptorsample runs into of the column or taking care to avoidthe bed. in the inlet adaptor. air bubbles.* Does not apply for pre-packed MonoBeads, MiniBeads, RESOURCE, HiTrap, HiLoad and BioPilotcolumns.149

Problem Cause RemedyDistorted bands Particles in eluent or Filter or centrifuge theas sample runs sample. sample. Protect eluentsinto the bed.from dust.Clogged or damaged netin upper adaptor.Distorted bands as Column poorly packed.sample passesdown the bed.Negative peaks at Refractive index effects.solvent front.Dismantle the adaptor, cleanor replace the net. Keep particlesout of samples and eluents.Gel suspension too thick ortoo thin. Bed packed at atemperature different fromrun. Bed insufficiently packed(too low packing pressure, tooshort equilibration). Columnpacked at too high pressure.Buffer exchange the sampleto start buffer.Strange peaks in Buffer impurities. Clean the buffer by running itchromatogram.through precolumn. Use highquality reagents.Peaks on blank Incomplete elution. Wash the column accordinggradients.to recommended method.Spikes in Air bubble trapped Use de-gassed solutions.chromatogram. in UV cell.UV baseline rises Salt concentration, Work well below or abovewith gradient. micelle formation. the CMC or change thegradient so that the increase inUV absorption does not occurwhile the samples are eluting.Impurities in buffers.Use high quality reagents.150

14. Ordering informationProduct Quantity/Pack Size Code No.MonoBeadsMono Q, PC 1.6/5 1 17-0671-01Mono Q HR 5/5 1 17-0546-01Mono Q HR 10/10 1 17-0556-01Mono Q HR 16/10 1 17-0506-01Mono Q 35/100 1 17-1001-01Mono Q 60/100 1 17-1002-01Mono S, PC 1.6/5 1 17-0672-01Mono S HR 5/5 1 17-0547-01Mono S HR 10/10 1 17-0557-01Mono S HR 16/10 1 17-0507-01Mono S 35/100 17-1021-01Mono S 60/100 1 17-1022-01MiniBeadsMini Q, PC 1.6/5 1 17-0671-01Mini S, PC 1.6/5 1 17-0671-01Precision Column Holder 1 17-1455-01SOURCE QRESOURCE Q 1 ml 1 17-1177-01RESOURCE Q 6 ml 1 17-1179-01SOURCE 15Q 10 ml 17-0947-20SOURCE 15Q 50 ml 17-0947-01SOURCE 15Q 200 ml 17-0947-05SOURCE 15Q 500 ml 17-0947-02SOURCE 15Q 1 l 17-0947-03SOURCE 30Q 10 ml 17-1275-10SOURCE 30Q 50 ml 17-1275-01SOURCE 30Q 200 ml 17-1275-02SOURCE 30Q 500 ml 17-1275-03SOURCE 30Q 1 l 17-1275-04SOURCE SRESOURCE S 1 ml 1 17-1178-01RESOURCE S 6 ml 1 17-1180-01SOURCE 15S 10 ml 17-0944-10SOURCE 15S 50 ml 17-0944-01SOURCE 15S 200 ml 17-0944-05SOURCE 15S 500 ml 17-0944-02SOURCE 15S 1 l 17-0944-03SOURCE 30S 10 ml 17-1273-20SOURCE 30S 50 ml 17-1273-01SOURCE 30S 200 ml 17-1273-02SOURCE 30S 500 ml 17-1273-03SOURCE 30S 1 l 17-1273-04151

Product Quantity/Pack Size Code No.Q Sepharose High PerformanceHiTrap Q 5 x 1 ml 17-1153-01HiTrap Q 5 x 5 ml 17-1154-01HiLoad 16/10 Q Sepharose HP 1 17-1064-01HiLoad 26/10 Q Sepharose HP 1 17-1066-01BioPilot Column Q Sepharose HP 35/100 1 17-1011-21BioPilot Column Q Sepharose HP 60/100 1 17-1012-21Q Sepharose High Performance 75 ml 17-1014-01Q Sepharose High Performance 1 l 17-1014-03Q Sepharose High Performance 5 l 17-1014-05SP Sepharose High PerformanceHiTrap SP 5 x 1 ml 17-1151-01HiTrap SP 5 x 5 ml 17-1152-01HiLoad 16/10 SP Sepharose HP 1 17-1137-01HiLoad 26/10 SP Sepharose HP 1 17-1138-01BioPilot Column SP Sepharose HP 35/100 1 17-1031-21BioPilot Column SP Sepharose HP 60/100 1 17-1032-21SP Sepharose High Performance 75 ml 17-1087-01SP Sepharose High Performance 1 l 17-1087-03SP Sepharose High Performance 5 l 17-1087-04Q Sepharose Fast FlowHiLoad 16/10 Q Sepharose Fast Flow 1 17-1060-01HiLoad 26/10 Q Sepharose Fast Flow 1 17-1062-01Q Sepharose Fast Flow 25 ml 17-0510-10Q Sepharose Fast Flow 300 ml 17-0510-01Q Sepharose Fast Flow 5 l 17-0510-04SP Sepharose Fast FlowHiLoad 16/10 SP Sepharose Fast Flow 1 17-1135-01HiLoad 26/10 SP Sepharose Fast Flow 1 17-1136-01SP Sepharose Fast Flow 25 ml 17-0729-10SP Sepharose Fast Flow 300 ml 17-0729-01SP Sepharose Fast Flow 5 l 17-0729-04DEAE Sepharose Fast FlowDEAE Sepharose Fast Flow 25 ml 17-0709-10DEAE Sepharose Fast Flow 500 ml 17-0709-01DEAE Sepharose Fast Flow 10 l 17-0709-05DEAE Sepharose Fast Flow 60 l 17-0709-60CM Sepharose Fast FlowCM Sepharose Fast Flow 25 ml 17-0719-10CM Sepharose Fast Flow 500 ml 17-0719-01CM Sepharose Fast Flow 10 l 17-0719-05CM Sepharose Fast Flow 60 l 17-0719-60152

Product Quantity/Pack Size Code No.Sepharose Big BeadsQ Sepharose Big Beads 1 l 17-0989-03Q Sepharose Big Beads 10 l 17-0989-05SP Sepharose Big Beads 1 l 17-0657-03SP Sepharose Big Beads 10 l 17-0657-05STREAMLINESTREAMLINE DEAE 300 ml 17-0994-01STREAMLINE DEAE 7.5 l 17-0994-02STREAMLINE SP 300 ml 17-0993-01STREAMLINE SP 7.5 l 17-0993-02Sepharose CL-6BDEAE Sepharose CL-6B 500 ml 17-0710-01DEAE Sepharose CL-6B 10 l 17-0710-05CM Sepharose CL-6B 500 ml 17-0720-01CM Sepharose CL-6B 10 l 17-0720-05SephacelDEAE Sephacel 500 ml 17-0500-01DEAE Sephacel 10 l 17-0500-05SephadexDEAE Sephadex A-25 100 g 17-0170-01DEAE Sephadex A-25 500 g 17-0170-02DEAE Sephadex A-25 5 kg 17-0170-03DEAE Sephadex A-25 40 kg 17-0170-07DEAE Sephadex A-50 100 g 17-0180-01DEAE Sephadex A-50 500 g 17-0180-02DEAE Sephadex A-50 5 kg 17-0180-03DEAE Sephadex A-50 40 kg 17-0180-07QAE Sephadex A-25 100 g 17-0190-01QAE Sephadex A-25 500 g 17-0190-02QAE Sephadex A-25 5 kg 17-0190-03QAE Sephadex A-50 100 g 17-0200-01QAE Sephadex A-50 500 g 17-0200-02QAE Sephadex A-50 5 kg 17-0200-03CM Sephadex C-25 100 g 17-0210-01CM Sephadex C-25 500 g 17-0210-02CM Sephadex C-25 5 kg 17-0210-03CM Sephadex C-25 40 kg on request153

Product Quantity/Pack Size Code No.CM Sephadex C-50 100 g 17-0220-01CM Sephadex C-50 500 g 17-0220-02CM Sephadex C-50 5 kg 17-0220-03SP Sephadex C-25 100 g 17-0230-01SP Sephadex C-25 500 g 17-0230-02SP Sephadex C-25 5 kg 17-0230-03SP Sephadex C-25 40 kg on requestSP Sephadex C-50 100 g 17-0240-01SP Sephadex C-50 500 g 17-0240-02SP Sephadex C-50 5 kg 17-0240-03154

15. References1. The right step at the right time. Bio/Technology, 4, 954-958 (1986), Bonnerjera, J., Oh, S., Hoare,M., Dunhill, P.2. Chromatography of Proteins. I. Cellulose ion exchange adsorbents. J. Amer. Chem. Soc. 78(1956) 751 755, Peterson, E.A., Sober, H.A.3. Chromatography of proteins on ion-exchange adsorbents. Meth. Enzymol. 22 (1971) 273—286,Himmelhoch, S.R.4. Chromatography: a laboratory handbook of chromatographic and electrophoretic techniques.Heftman, E. (Ed.), Van Noostrand Rheinhold Co., New York (1975).5. Dynamics of chromatography, Part 1, Principles and theory. Giddings, J.C., Keller, R.A. (Eds.),Marcel Dekker Inc., New York (1965).6. Ion exchange chromatographic characterization of stinging insect vespid venoms. Toxicon(Pergamon Press), 22,1 (1984) 154-160, Einarson, R., Renck, B.7. Physicochemical considerations in the use of MonoBeads for the separation of BiologicalMolecules. Protides of the Biological Fluids, 30 (1982) 629-634, Söderberg, L. et al.8. Gel Filtration in Theory and Practice, Pharmacia Biotech, S-75182 Uppsala, Sweden.9. The separation of human globin chains by ion-exchange chromatography onCM Sepharose CL-6B. Hemoglobin 3 (1979)13—20, Sparham, S.J., Huehns, E.R.10. Agar derivatives for chromatography, electrophoresis and gel-bound enzymes. I. Desulphated andreduced cross-linked agar and agarose in spherical bead form. J Chromatogr. 60(1971)161—177,Porath, J., Janson, J.-C., Laas, T.11. Ion exchanger from pearl-shaped cellulose gel. Nature 223 (1969) 499—500, Determann, H.,Meyer, N., Wieland, T.12. Chromatography of mixed oligonucleotides on DEAE-Sephadex. Biochemistry 3 (1964)626—629, Rushizky, G.W., Bartos, E.M., Sober, H.A.13. DEAE-Sephadex chromatography of guanylate oligomers using guanidinium chloride. Biochim.Biophys. Acta 277 (1972) 290-300, Olson, A.C., Volkin, E.14. The synthesis of triaminoacyl-insulins and the use of the t-butyloxy-carbonyl group for thereversible blocking of the amino groups of insulin. Biochemistry 6 (1967) 3559—3568, Levy, D.,Carpenter, F.H.15. A simple method for estimating isoelectric points. Anal. Biochem. 11(1965) 374—377, Lampson,G.P., Tytell, A.A.16. Isoelectric points and molecular weights of proteins: a table. J. Chromatogr.127 (1976)1—28,Righetti, P.G., Caravaggio, T.17. Isoelectric points of proteins: a table. AnaL Biochem. 86 (1978) 620—647, Malamud, D.,Drysdale, J.W.18. Basic principles used in the selection of MonoBeads ion exchangers for the separation ofbiopolymers. Protides of the Biological Fluids, 30 (1982) 621-628, Fägerstam, L.G. et al.19. Use of electrophoretic titration curves for predicting optimal conditions for fast ion exchangechromatography of proteins. J. Chromatogr. 266 (1983) 409-425, Haff, L.A., Fägerstam, L.G.,Barry, A.R.155

20. ”Isoelectric Focusing: Principles and Methodes”, Technical Booklet Series (1982), PharmaciaBiotech, Uppsala, Sweden.21. Interrelationships of human-interferon gamma with lymphotoxin and monocyte cytotoxin.J. Exp. Med. 159 (1984) 824-843, Stone-Wolff, D.S., Yip, Y.K., Kelker, H.C. et al.22. Glass wool as a potential source of artifacts in chromatography. J. Chromatogr. 152 (1978)514—516, Schwartz, D.P.23. Ion Exchange Chromatography. Protein Purification, Principles, High resolution methodsand Applications, Janson, J.C., Ryden, L. (Eds) VCH, Publishers Inc. New York. (1989) 107-148,Karlsson, E., Ryden, L., Brewer, J.24. Gel Filtration Chromatography. L. Fischer. Elsevier, Amsterdam (1980)25. Arthropod hemocyanin structure: isolation of eight subunits in the scorpion. Arch. Biochem.Biophys. 193 (1979)140—149, Lamy, J., Lamy, J., Weill, J.26. Rapid isolation of Escherichia Coli b-galactosidase by fast protein liquid chromatography.J. Chromatogr. 393 (1987) 462-465, Motorin, Y.A. et al.27. Chromatography of proteins and peptides on Sephadex ion-exchangers: dependence of theresolution on the elution schedule. FEBS Lett. 14 (1971) 7—10, Novotny, J.28. High Performance ion-exchange separation of oxidised and reduced nicotinamide adeninedinucleotides. Anal. Biochem. 142 (1984) 232-234, Orr, G.A., Blanchard, J.S.29. FPLC of leukaemia cell N-Acetyl ß-D-Hexosaminidases. Leukaemia Res.11 (1987) 437-444,Scott, C.S., Patel, M., Stark, A.N., Roberts, B.E.30. Presented at Sixth International Congress on Methods in Protein Sequence Analysis, Seattle,Washington, USA. (1986) Bhikhabhai, R., Lindblom, H., Källman, I., Fägarstam, L.31. Fractionation of DNA restriction fragments with ion exchangers for high performance liquidchromatography. European Journal of Biochemistry 155 (1986) 203-212, Müller, W.32. Inositol triphosphates in carbochal-stimulated rat parotid glands. Biochem. J., 223 (1984)237-243, Irvine, R.F., Letcher, A.J., Lander, D.J., Downes, C.P.;33. Inositol bis-, tris-, and tetrakis- phosphate(s): Analysis in tissue by HPLC. Proc. Natl. Acad. Sci.USA., 83 (1986) 4162-4166, Meek, J.L.34. Release of intra-cellular Ca 2+ and elevation of inositol triphosphates by secretagogues in parietaland chief cells isolated from rabbit gastric mucosa. Biochim. Biophys. Acta., 88 (1986) 116-125,Chew, C.S., Brown, M.R.35. Albumin from human plasma: preparation and in vitro properties. in Separation of Plasmaproteins. J.M. Curling, ed., Pharmacia Fine Chemicals AB, Uppsala, Sweden. (1983) 51-58.Berglöf, J.H., Eriksson, S., Suomela, H., Curling, J.M.36. FPLC for monitoring microbial and mammalian cell cultures. Bio/Tecchnology 2 (1984) 777-781,Frej, A.K. et al.37. Varietal identification by rapid chromatography (FPLC) of wheat gliadins. 3rd Conference, RoyalAustralian Institute, Brisbane, Australia. (1983). Batey, I.38. Rapid extraction and separation of plasma b-endorphin by cation exchange chromatograpy.J. Chromatogr., 297 (1984) 399-403, Stenman, U-H., et al.39. Applications of Fast Protein Liquid Chromatography in the separation of plasma proteins in urineand cerebrospinal fluid. Clin. Chem., 29 (1983) 1635-1640, Cooper, E.H. et al.156

Before any part of this handbook is reproduced, please request permission ofPharmacia Biotech. The following designations are trademarks owned byPharmacia AB: Sephadex, Sephacel, Sepharose, STREAMLINE, HiLoad, HiTrap,MonoBeads, MiniBeads, SOURCE, RESOURCE, FPLC, FPLCdirector,UNICORN, SMART, OligoPilot II, FineLine, BPG, BioPilot, BioProcess,PhastSystem, PhastGel.157

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