Protein
`
`Purification
`
`
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`Second Edition
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`METHODS IN MOLECULAR BIC)LC)G\"FM
`
`__—...._.____-—_—_——_4.———-—u—._._
`
`Protein Purification
`
`Protocols
`
`Second Edition
`
`Edited by
`
`Paul Cutler
`
`Genomic and Pmreomir SL'it’iM'é’S. Medicinex Reward; Center.
`
`Gla.1‘oSmitl1Kiine PhurmnCeuIit'aix, vaenage, UK
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`HUMANA PRESS * TOTOWA, NEW JERSEY
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`Library nl'CL‘Ingrexs Cataloging itl Ptthiieulttin Data
`Ptttteitt Purifieatinn PFI.I[t1L'Ui\.--2llti ed. [edited b} Paul Cutler.
`p.
`: em ~ (Melbnds in molecular biology : \'. 244i
`Include». bibliographical references and index.
`ISBN i‘5882(}-0(17-”Hlik. paper‘idISBN l-S‘tJS‘JLhSSVX
`ISSN Illa-173745
`I. Prnteiits-Purification--Labnratnr_\ manualx.
`[DNLM‘ l. Prnteivatsolatiun ta purification--l.abm-atur)- Manuals. QU
`15 P967 2004]
`l. Cutler. Paul. Nb} lE. Methuda in moieeular binlogy
`(Tulane. NJ.) 1 V. 2-14.
`Ql’SS I .Phg‘tfib jun-t
`547.7‘5U46nd631
`
`2003006803
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`
`
`Contents
`
`Preface ........................................................................................................................... v
`
`Contributors .................................................................................................................. xi
`
`1 General Strategies
`Shawn Doonan and Paul Cutler ............................................................................ 1
`
`2 Preparation of Extracts From Animal Tissues
`I. Mark Skehel ..................................................................................................... 15
`
`3 Protein Extraction From Plant Tissues
`Roger I. Fido, E. N. Clare Mills, Neil M. Rigby, and Peter R. Shewry ................ 21
`4 Extraction of Recombinant Protein From Bacteria
`
`Anne F. McGet‘trick and D. Margaret Worrall ................................................... 29
`
`5 Protein Extraction From Fungi
`Paul D. Bridge, Tet‘suo Kokubun, and Monique 5. I. Simmonds ......................... 37
`6 Subcellular Fractionation of Animal Tissues
`
`Norma M. Ryan ................................................................................................... 47
`7 Suhcellular Fractionation of Plant Tissues: isolation
`of Plastio’s and Mitochondria
`
`Alyson K. Tobin and Caroline G. Bowsher ......................................................... 53
`
`8 The Extraction of Enzymes From Plant Tissues Rich in Phenolic Compounds
`
`William S. Pierpoint ............................................................................................ 65
`
`9 Avoidance of Proteolysis in Extracts
`Robert I. Beynon and Simon Oliver (Revised by Paul Cutler) ............................ 75
`10 Concentration of Extracts
`
`Shawn Doonan .................................................................................................... 35
`
`11 Making and Changing Buffers
`Shawn Doonan .................................................................................................... 91
`
`12 Purification and Concentration by Ultraiiltration
`
`Paul Schratter (Revised by Paul Cutler) ............................................................ 101
`
`13 Bulk Purification by Fractional Precipitation
`.
`Shawn Doonan .................................................................................................. H7
`
`14
`
`Ion-Exchange Chromatography
`Chris Selkirk ...................................................................................................... 125
`
`15 Hydrophobic Interaction Chromatography
`Paul A. O’Farrell ............................................................................................... 133
`
`if) Affinity Chromatography
`Paul Cutler ........................................................................................................ 139
`
`T7 Dye-Ligand‘Aiiinity Chromatography
`Anne F. McGettrick and D. Margaret Worrall ................................................. 151
`
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`Contents
`
`18 Lectin Affinity Chromatography
`iris West and Owen Goldring (Revised by Paul Cutler) ................................... 159
`tmmunoaffinity Chromatrmraphy
`7
`Paul Cutler ........................................................................................................ 167
`
`1‘)
`
`20 immobilized Metalelon Affinity Chromatography
`Tai-Tung Yip and T. William Hutchens (Revised by Paul Cutler) ..................... 179
`21 Chromatography on Hydroxyapatite
`Shawn Doonan
`
`191
`
`22 Thiophiiic Affinity Chromatography and Related Methods
`Paul Matejtschuk ............................................................................................... 195
`
`2,5 Affinity Precipitation Methods
`lane A. Irwin and Keith F. Tipton ..................................................................... 205
`
`24
`
`Isoeiectric Focusing
`Reiner Westermeier .......................................................................................... 225
`
`25 Chromatot'ocusing
`Timothy 1. Mantle and Patricia Noone ............................................................. 233
`
`2b SizevExclusion Chromatography
`Paul Cutler ........................................................................................................ 239
`
`27
`
`Fast Protein Liquid Chrormttography
`David Sheehan and Siobhan O’Sullivan ........................................................... 253
`
`28 Reversed-Phase Chromatography of Proteins
`William A. Neville ............................................................................................. 259
`2‘) Extraction of Membrane Proteins
`
`Kay Ohlendieck ................................................................................................. 283
`
`30 Removal of Detergent From Protein Fractions
`Kay Ohlendieclr ................................................................................................. 295
`Purification of Membrane Proteins
`
`31
`
`Kay Ohlendieck
`32 Lyophiiization of Proteins;
`
`301
`
`Ciarén O’Fa’géin ................................................................................................ 309
`33 Storage of Pure Proteins
`Ciarén O’Féga’in
`34 Eler'troelution of Proteins From Polyacryiamide Gels
`Michael }. Dunn ................................................................................................ 339
`
`323
`
`35 Electroblotting of Proteins From Poiyacrylamide Gels
`Michael I. Dunn
`
`345
`
`36 Two-Dimensional Poiyacrylamide Gel Electrophoresis
`for Proteome Analyses
`Neil A. lanes ...................................................................................................... 353
`
`37 Microscale Solution lsoelectrofocusing: A Sample Preh‘ar'tionation
`Method for Comprehensive Proteome Analysis
`Xun Zuo and David W. Speicher ...................................................................... 361
`
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`Contents
`
`ix
`
`38 Practical Column Chromatography
`Shawn Doanan .................................................................................................. 377
`
`39 Detection Methods
`
`jacek Mozdzanowsiri and Sudhir Barman ........................................................ 389
`
`40 Peptide Proteomics
`
`Dean E. McNuity and ]. Randal! Slemmon ....................................................... 411
`
`41 Multidimensional Liquid Chromatography of Proteins
`Rod Watsou and Tim Nadier ............................................................................. 425
`
`42 Mass Spectrometry
`David I. Be“ ...................................................................................................... 447
`
`43 Purification of Therapeutic Proteins
`
`[Lilian Bonnerjea ................................................................................................ 455
`
`44 Purification Process Scale-Up
`Kari Prince and Martin Smith ........................................................................... 463
`
`Index ......................................................................................................................... 481
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`14
`
`Ion-Exchange Chromatography
`
`Chris Selkirk
`
`1. Introduction
`
`Ion-exchange chromatography is one of the most widely used forms of column chro—
`matography. It is used in research, analysis, and process-scale purification of proteins.
`Ion exchange is ideal for initial capture of proteins because of its high capacity, rela-
`tively low cost, and its ability to survive rigorous cleaning regimes. [on exchange is also
`ideal for “polishing” of partially purified material on account of the high-resolution at—
`tainable and the high capacity giving the ability to achieve a high concentration of prod-
`uct. Ion—exchange chromatography is widely applicable because the buffer conditions
`can be adapted to suit a broad range of proteins rather than being applicable to a single
`functional group of proteins.
`Ion—exchange chromatography matrices are available as dry granular material or as
`preswollen loose beads. but ptepacked columns (Bio—Rad, Amersham Bioticience) are
`now common, particularly for sm all—scale analytical and method development work. Ion
`exchange can now also be carried out on monolithic columns (Bio—Rad), on membranes
`(Pall, Sartorius). and on ion-exchange high-perfonnance liquid chromatography (HPLC)
`columns. The method is essentially the same whichever of these formats is employed.
`The method described in this chapter will assume that the column to be used is packed
`ready for use.
`Ion-exchange chromatography relies on the interaction of charged molecules in the
`mobile phase (butter + sample) with oppositely charged groups coupled to the station—
`ary phase (column packing matrix). The charged molecules in a buffer solution come
`from the buffer components (e.g., salts). The charged groups on a protein are provided
`by the different amino acids in the protein. Lysine, arginine. and histidine have a posi-
`tive charge at physiological pH. whereas aspartic acid and glutamic acid have a nega—
`tive charge at physiological pH.
`
`Charges on amino acids at physiological pH:
`+ve
`lysine, arginine. histidine
`—vc
`aspartic acid. glutamic acid
`Charges on ion-exchange matrices
`+ve DEAE, QAE
`~ve CM. SP. sulfonic acid
`
`From: Methods in Molecular Biolagv, vol. 244: Protein Purification Protocols: Second Edition
`Edited by: I" Cunt-tr © Humana Press Inc. Totowa. NJ
`
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`l2 6
`
`Table 1
`
`Selkirk
`
`Ion-Exchange Groups
`
`Ion-exchanger type
`Strong exchangers
`Weak exchangers
`
`Cation exchangers
`
`Anion exchangers
`
`SP t'sulfopropyl)
`3 {Methyl sulfonale}
`Q (quaternary ammonium}
`QAE (quaternary aminoethyl)
`
`CM {carboxymethylj
`
`DEAE {diethylaminoethyli
`
`The net charge on a protein molecule will depend on the combination of positively
`and negatively charged amino acids in the molecule. The charges of the amino acid
`groups varies depending on the hydrogen ion concentration (acidity) of the solution and,
`thus. the overall charge on a protein varies according to the pH. The more acidic the
`solution, the more grDUps will be positively charged; the more alkaline the solution, the
`more negatively charged the protein will become. The pH at which the negative charges
`on a protein balance the positive charges and, therefore, the overall charge on that pro-
`tein is zero is called the isoelectric point (pl) for that protein. It is useful to know the pl
`of a protein if it is to be purified by ion-exchange chromatography. This information will
`assist in deciding on the best starting conditions for optimization of the purification con-
`ditions (see Note 1}.
`
`Binding and elution of proteins is based on competition between charged groups on
`the protein and charged counterions in the butler for binding to oppositely charged
`groups on the stationary phase. The higher the concentration of charged salt molecules in
`the solution, the greater is the competition for binding to the ligands on the matri it, so the
`greater is the tendency for the protein to dissociate from the ion—exchange matrix.
`The protein sample is applied to the ion—exchange column in a solution of low salt
`concentration. The counterions with which the column has been charged are not per—
`manentl y bound but are held by electrostatic interaction. Therefore, there is a continual
`binding and unbinding of counterions. Under low—salt conditions. charged groups on the
`protein have a greater probability of binding to charged counterions on the ion ex—
`changer and become bound to the ion-exchange column. During elation. the salt con«
`centration is increased, so that when a protein group dissociates from an ionic group an
`the stationary phase, there is an increased probability that ions in the mobile phase will
`bind to the charged group on the protein and the ionic group on the stationary phase.
`Thus. the proteins dissociate from the ion-exchange matrix and are eluted as the salt
`coucentration increases. The more strongly bound the protein, the greater is the salt con-
`centration required to elute it.
`Ion—exchange matrices are divided into two major types according to the charge on
`the ion—exchange ligands (see Table 1'):
`
`Cation Exchange: Cation-exchange resins have negatively charged groups on the surface.
`These are used to bind proteins that have an overall positive charge. Proteins will have an
`overall positive charge at a pH below their isoelectric point Therefore. cation exchange is
`used at a pH below the isoelectric point of the proteinfs} to be bound.
`Anion Etta-hangs: Anion—exchange resins have positively charged groups on the surface.
`These are used to bind proteins that have an overall negative charge. Proteins will have an
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`ion-Excite nge Chromatography
`
`t2?
`
`Table 2
`
`Buffers: Anion Exchange
`
`Buffer
`Anion
`pH Range
`
`N-methyl piperazinc
`Piperazine
`L—Histidine
`Bis-Tris
`
`CI—
`CI
`(31'
`Cl'
`
`4.5-5.0
`5.0—6.0
`5.54.5
`5.8458
`
`6.4—7.3
`CI
`Bis~Tris propane
`Jill-8.2
`Cl—
`Triethanolamine
`7.5—8.0
`Cl
`Tris
`
`Diethanolamine 3.4—9.4 Cl—
`
`
`overall negative charge at a pH above their isoelectrie point. Therefore. anion exchange is
`used at a pH above the isoelectric point of the proreints) to be bound.
`
`[on exchangers are also divided into strong and weak ion exchangers. Strong ion—ex-
`change ligands maintain their charge characteristics. and therefore ion—exchange capacity,
`over a wide pH range. whereas weak ion—exchange ligands show a more pronounced
`change in their exchange capacity with changes in pH (1). DEAE—Sepharose Fast Flow
`{ weak anion) has a working pH range of 2.0—9.0, whereas Q—Sepharosc Fast Flow (strong
`anion) has a working pH range of 2.04.2.0. CM-Sepharose Fast Flow (weak cation) has
`a working pH range of (iii—[0.0 and SP—Sepharose Fast Flow (strong cation) has a work-
`ing pH range of 14.0—13.0 If your purification is to be carried out at pH above 9 for anion
`exchange or below 6 for cation exchange. then it is likely that you will need to use a strong
`ion exchanger. However, if your purification is to be carried out at a less extreme pH. then
`the slight differences in selectivity mean that it may be worth comparing the results ob-
`tained with both weak and strong ion exchangers to optimize your purification.
`
`2. Materials
`
`1. Binding buffer of appropriate pH and composition for binding of protein to matrix (see
`Notes 2—4). Tables 2 and 3 list some suitable buffers and the pH ranges over which they
`are useful is included.
`
`in.)
`
`Elution buffer (often the same as binding buffer but with higher salt concentration) (see
`Note 5}.
`3. Regeneration buffer (eg, 1 M NaCl). Note: Buffers and samples should be filtered (0.45
`pm} before applying to the column to avoid blockage of the column flow path by particu—
`lates. Buffers should be stored sterile or with addition of a bacteriostat (cg. 0.02% lwr‘v]
`sodium azide).
`
`5.
`
`4. Desalting column. These can be purchased ready to use (e.g.. Amersham Bioscience Hi—Trap
`desalting, Bio-Rad Econo—Pac P6 or Perbio D-Salt columns} or can be prepared in the lab.
`lon~cxchange column. Ion—exchange columns can be purchased ready to use (e.g., Amer—
`sham Bioscience or Bio-Rad) or can be prepared in the lab by packing a column with loose-
`ion—exchange beads according to the manufacturer’s instructions.
`Chromatography equipment (see Notes 6 and 7‘).
`Assay methods for analysis of the purified materials will be required to determine the suc—
`cess of the purification.
`
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`Table 3
`
`Selkirk
`
`Buffers: Cation Exchange
`
`Buffer
`Cation
`pH range
`Maleic acid
`Na+
`1.5—2.5
`Forrnic acid
`Na”r
`3.3—4.3
`Citric acid
`Na+
`2.64510
`Lactic acid
`Na+
`3.64.3
`Acetic acid
`Na+
`4.3—5.3
`MESfNaOH
`Na+
`5.54).?
`
`Phosphate
`MOPS
`HEPES
`
`Na+
`Na+
`Na+
`
`653—7.?
`6.5—7.5
`7.5—8.2
`
`3. Methods
`
`3.1. Preparation
`
`I. The starting material must first be equilibrated in the binding buffer before ion exchange can
`be commenced. if the sample is not already prepared in a suitable bu ffer for the desired com-
`ponents to bind to the matrix. then the buffer can be replaced either by dialysis or by using a
`desalting column (see Chapter 26). [f recovery of the protein of interest is to be measured fol+
`lowing ion—exchange chromatography, then it is worthwhile also measuring recovery of the
`protein after the preparatory desalting. Some proteins precipitate in low—ionic—strength
`buffers close to the protein p! and theSe are the conditions employed in ion—exchange buffers.
`Small changes in buffer pH. ionic strength. or buffer composition can make major changes
`in the recovery of protein both on the desalt step and on the ion-exchange purification.
`2. The sample should be liltered (0.45 pm} before applying to the column to reduce the risk
`of column blockage.
`3. Before use, the ion—exchange column should be charged with the counterion. The most
`commonly used counterions are sodium (Na‘i for cation exchange and chloride {Cl') for
`anion exchange. Many ion exchangers are supplied charged with Na‘ or Cl‘; however. if
`this is not the case or if a different counterion is to be used, then the column will need to
`be charged with the appropriate counterion. This is most easily done by pumping l—E col-
`umn volumes of hi gh-ionic-strength elution buffer through the column (see Note 8).
`4. Once charged with the counterion, the column needs to be thereughly washed with the bind+
`ing buffer {S—IU column volumes) to ensure equilibration in the low-salt buffer prior to ap-
`plication of the protein. Measure pH and conductivity of the buffer eluted from the column
`and compare this with the pH and conductivity of the binding buffer being appiied to the
`column. Once the column is cquilibratcd, then the measurements for eluate and binding
`buffer should be the same.
`
`3.2. Chromatography
`
`1. The sample. in the binding buffer. is applied to the column either by gravity flow or prefer-
`ably using a pump. The recommended flow rate for the ion-exchange medium should be
`included in the suppliers instructions. The chromatography steps are often carried out at a
`lchr flow rate than column washing and equilibration because the proteins are larger than
`the buffer ions so it will take longer to diffuse into the pores of the stationary phase.
`The eluate from the column should be collected for analysis to confirm that the protein of
`interest has bound to the column.
`
`to
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`ion-Exchange Chromatography
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`1'29
`
`3. Once the sample has been applied, the column is washed with several column volumes of
`binding buffer (around 5 column volumes, depending on sample and on column packing) to
`ensure that all nonbound proteins are washed out of the column. Monitoring the column elu-
`ate with an ultraviolet (UV) detector at 280 nm gives an immediate visual indication of the
`amount of protein or other UV~ahsorbing material present in the cluate from the column.
`4. Elute bound proteins by washing the column with an increasing salt gradient of 0—500 mM
`NaCl in binding buffer over 10—15 column volumes {see Notes 5 and 9—11).
`Collect the eluted protein in fractions for analysis.
`.U!
`6. Analyse both nonbound material and eluted fractions to determine in which fractions the
`protein of interest has been isolated and whether contaminants have coeluted. Based on this
`analysis. required modifications to the chromatographic: conditions can be planned {see
`Note 10).
`
`3.3. Column Regeneration and Storage
`
`I.
`
`2,
`
`Ion—exuhange columns should be cleaned and regenerated betWeen purifications. otherwise
`the binding can rapidly be reduced and the column can become blocked by contaminants.
`Ion-exchange columns can be washed with a high—salt solution as part of the elution gradi—
`ent or as a separate cleaning step. Use of l M NaCl will elute most covalently bound con-
`taminants not eluted during the purification (see Note 8).
`Ion exchange columns can be stored packed provided they contain a bacteriostatic solution.
`The column can be equilibrated in buffer containing 0.02% (wiv) sodium azide or (de-
`pending on the bead material) in a buffer containing 20% ethanol. Consult the manufac—
`turer‘s instructions for the recommended storage options. Ensure that the column tubing is
`sealed to prevent drying of the column packing during storage. Columns should preferably
`be stored at 4°C.
`
`3. Before subsequent reuse, any cleaning or storage solution must be washed out and the col-
`umn must again be fully equilibrated by pumping 5—10 column volumes of the appropriate
`binding buffer through the column.
`4. During long-term storage. there is a likelihood that the column may start to dry out. There-
`fore for extended storage periods. it is recommended that the column should be unpacked
`and the ion—exchange matrix stored in a buffer containing a bacteriostatic agent (c.g., 20%
`ethanol}.
`
`4. Notes
`
`I. The isoelectric point (pf) can be determined experimentally by isoelectric focusing. [so-
`electric points for many proteins can be found in the literature (2). The p! of novel proteins
`can be predicted from the amino acid sequence of a protein if this is known. Software pack-
`ages are available that will calculate the pl and there are sites accessible on the Internet
`where pl and other properties for a protein can be calculated if the amino acid sequence or
`the base sequence of the DNA coding for the protein is entered (e.g., Swissprot. www.cx-
`pasyeh). Once into the Swissprot site, go to "Proteomics Tools” and then click on “Primary
`structure analysis.“ There are several options within this part of the site that offer predic—
`tions of protein properties. including pl.
`2. When scouting for the best pH for ion-exchange purification. start by trying a pH around
`1—1 .5 pH units from the pl of the protein being purified: one unit above the p] for anion ex—
`change and one unit below the pi for cation exchange. Having analyzed the separation
`achieved at this pH. the buffer can be adjusted slightly in subsequent runs to improve the
`results.
`
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`130
`
`3.
`
`It is often desirable to choose conditions such that the protein of interest elutes early in the
`elution gradient. Under such conditions. little of the binding capacity of the column will be
`occupied by weaker binding species, so the column will have a greater capacity for the pro-
`tein of interest.
`
`Selkirk
`
`4. The ionic strength of the buffer is as important as the pH when carrying out ion-exchange
`purification. In most cases, a starting buffer of 20—50 111114 is suitable. Many proteins tend
`to aggregate in solutions close to the protein’s p1: this aggregation is increased in solutions
`of lower ionic strength. Hydrophobic interactions between proteins and the chromatogra-
`phy matrix will also increase at low ionic strength and these can affect
`the separation
`achieved creating a “multimode” chromatography. It
`is therefore not advisable to use
`buffers of less than around 10—20 mM for most applications.
`5. Proteins are most commonly eluted from the ion—exchange matrix by increasing the ionic
`strength of the solution. Usually, this is achieved by increasing the sodium chloride concen-
`tration, but it can also be achieved by increasing the molarity of the buffer components. Pro-
`teins can also be eluted by a change in buffer pH, raising the pH to elute from cation ex—
`changers and lowering the pH to elute from anion exchangers. However, it is more difficult
`to control pH gradients on standard ion—exchange columns than to control changes in ionic
`strength. so pH gradients are only commonly applied with chromatofocusing columns.
`Ion-exchange chromatography can be carried out in short. wide columns because the bed
`volume is more important than the bed height. Bed heights (ifs-‘10 cm are frequently used.
`Once the capacity of the column has been determined for your protein under your condi—
`tions, then the column volume can be set to fit the amount of protein to be purified in one
`full.
`
`6.
`
`'1'. The equipment used for ion—exchange chromatography can be as basic as a gravity—fed col-
`umn with the eluate fractions collected manually. However. the use of programmable chro-
`matography control equipment is recommended. as it will make it easier to carry out pu-
`rification reproducibly and aid in analysis of the chromatography results.
`In addition to the equipment used for other chromatography methods, a conductivity
`meter is desirable to measure the changes in buffer conductivity during the chromatogra—
`phy process. An in-line conductivity cell and a conductivity meter allow continuous moni—
`toring of the eluate conductivity. This can be recorded alongside the ultraviolet (UV) mon—
`itor trace on a two—channel chart recorder, allowing determination of the conductivity at the
`point each protein peak is eluted.
`A gradient mixer is required for clution of proteins by salt gradient. Gradient mixers
`allow the formation of a controlled and reproducible salt gradient that is essential for run
`to run consistency.
`It is common to charge the ionic groups on the column matrix with the counterion by flush—
`ing the column with the high—salt buffer used for protein elution. However. other high-salt
`solutions can be used. Sodium chloride (1 M) is often used for cleaning ion-exchange
`columns between purification runs. This will charge the column with chloride {anion ex—
`change) or sodium (cation exchange) at the same time as cleaning. Sodium hydroxide
`{0.5—l M) can be used for cleaning and sanitizing of Sepharose ion-exchange columns; on
`cation-exchange columns. it will also serve to charge the matrix with sodium. Washing the
`column with NaOH {0.5 M for 30 min) acts as a bacteriocide and will aim destroy endo—
`
`8-
`
`toxin bound to the column. The instructions provided by the ion—exchange resin manufac-
`turer should be consulted for appropriate methods of sanitizing or sterilizing the chro—
`matography medium you are using.
`
`12 of 79
`
`Fresenius Kabi
`
`Exhibit 1028
`
`12 of 79
`
`Fresenius Kabi
`Exhibit 1028
`
`

`

`ion-Exchange Chromatography
`
`131
`
`9.
`
`[f the bound proteins are to be eluted using a continuous gradient, then a low—salt buffer,
`normally the binding buffer. and a high—salt buffer (binding buffer plus 0.1Ll M sodium
`chloride) are prepared. The gradient mi xer can then be programmed to produce a gradient
`by mixing a low-salt solution with increasing amounts of a high—salt solution. The volume
`over which the gradient is run can be altered according to the required resolution. For ini-
`tial investigations. a gradient of 0—500 mM NaCl over lO—l 5 column volumes is often suit—
`able.
`
`IO. After initial purification runs have been analyzed it may be desired to alter the gradient to im-
`prove separation of eluted proteins. The gradient can be altered either by increasing or de-
`creasing the ionic strength of the high-salt buffer or by altering the volume over which the
`gradient is applied. Applying a more gradual gradient will have the effect of increasing peak
`separation but will also spread peaks, reducing the concentration at which each protein is
`collected. Applying a steeper gradient sharpens the eluted peaks, but may cause closely elut—
`ing peaks to merge, increasing contamination ofthe product. By careful analysis oithe peaks
`eluted, the gradient can be fine—tuned to optimize purification of Lhe desired protein. Al-
`though ien—exchange columns have a high capacity, the resolution achieved can be improved
`by loading the column to well below the maximum binding capacity (IO—20%}.
`l l. Elution by stepwise increases in sodium chloride can be used ifa gradient mixer is not avail—
`able or once elution conditions have been determined well enough to determine the appro—
`priate salt concentrations for each step. In this case. prepare solutions of (binding) buffen
`containing sodium chloride at appropriate concentrations over the desired range. Step
`elution using appropriate. concentrations can achieve the highest elution concentrations for
`a protein. However, if the wrong concentrations are used. peak splitting or coelution of
`peaks can result. The solutions are run through the column in turn from the lowest salt con-
`centration to the highest. With each solution. the volume applied should be sufficient to
`elute all of the proteins that can be eluted at that concentration. This should be monitored
`using an UV monitor.
`
`References
`
`l. Amcrsharn Pham‘tacia Biotech { 1999} [on Exchange Chromatography Principles and Meth-
`ods, Amersham Pharmacia Biotech, Uppsala, Sweden.
`2. Righetti. P. G. and Carravaggio, T. (1976) lsoelectrie points and molecular weights of pro—
`teins: a table. J. Cltmnmmgr. 127, 1—28.
`
`13 of 79
`
`Fresenius Kabi
`
`Exhibit 1028
`
`13 of 79
`
`Fresenius Kabi
`Exhibit 1028
`
`

`

`15
`
`Hydrophobic Interaction Chromatography
`
`Paul A. O'Farrell
`
`1. Introduction
`
`Hydrophobic interaction chromatography (HIC) is a technique for the separation of
`biological macromolecules based on their surface hydrophobicity. Although proteins
`maintain their tertiary structure by burying a hydrophobic core and exposing polar
`residues to the solvent. it is still the case that they have hydrophobic areas on their sur—
`faces [as much as 50% of the surface area (1 )1. Indeed, these areas are often critical to
`a protein’s function, as many of them are involved in protein—protein interactions. It is
`the variety in the extent and character of these hydrophobic patches that is exploited
`for separatiOn in HIC. It can be a privverful technique, especially becaus