Manialt
`Purification
`
`Exhibit 1028
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`Second-Edition
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`METHODS IN MOLECULAR BIOLOGY™
`
`Protein Purification
`Protocols
`
`Second Edition
`
`Edited by
`Paul Cutler
`
`Genomic and Proteomic Sciences, Medicines Research Center,
`GlaxoSmithKline Pharmaceuticals, Stevenage, UK
`
`HUMANAPRESS * Totowa, NEW JERSEY
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`© 2004 HumanaPressInc.
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`All rights reserved. Nopart of this book may be reproduced, stored in aretrieval system, or transmitted in any form or by any
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`Printedin the UnitedStates of America. 10 9 8 765432 1
`e-ISBN 1-59259-655-X
`Library of Congress Cataloging in Publication Data
`Protein Purification Protocols.--2nded. / edited by Paul Cutler.
`p. cm -- (Methods in molecular biology; v. 244)
`Includes bibliographical references and index.
`ISBN 1-58829-067-0 (alk. paper) e-ISBN 1-59259-655-X
`ISSN 1064-3745
`|. Proteins--Purification--Laboratory manuals.
`[DNLM: 1. Proteins--isolation & purification--Laboratory Manuals. QU
`25 P967 2004]
`I. Cutler, Paul, 1962- 11. Methods in molecular biology
`(Totowa, N.J.) ; v. 244.
`QP551.P69756 2004
`547.7'5046--de21
`
`2003006803
`
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`
`
`Contents
`
`PROPACE:
`
`sete ssracesascceescass as cuceos sexe ccawe tava ctecscrerd ener sas sess dicct ie eer auyenensqceuee st ben ci Ans ote etenneeeemecner marca Vv
`
`Contributors sssecssnceeereeseeerseeeien te sera ease ererrierenereanvices stems car rnneemestuesseeess xI
`
`1 General Strategies
`Shawn Doonan and Paul Cutler ........ cc cccsesssssscscessssnnesssssesnsesssscecsscsssecessesssnnsseeeess 1
`
`2 Preparation of Extracts From Animal Tissues
`Fe Ma fk Shehel,...cc.cescrsecerssennssannnancsnnzsessceninaasacniecsssddsdassadeaeiiieatadidssiissveonavGiSisFCeOs 15
`3 Protein Extraction From Plant Tissues
`Roger J. Fido, E. N. Clare Mills, Neil M, Rigby, and Peter R. Shewry..........2++ 21
`4 Extraction of Recombinant Protein From Bacteria
`AnneF. McGettrick and D. Margaret Worrall......:.ssssseessesssessssessessensssassssensens 29
`5 Protein Extraction From Fungi
`Paul D. Bridge, Tetsuo Kokubun, and MoniqueS.J. SimMOnds........1.ssseeeeseees i ef
`6 Subcellular Fractionation of Animal Tissues
`Norma M. RYal..sccssssessscecesseecssssnsnsssenssnsassssnsessnncccsssscesescessaseanecnsaaeesanonsenssssaoooass 47
`7 Subcellular Fractionation of Plant Tissues: /solation
`of Plastids and Mitochondria
`Alyson K. Tobin and Caroline G. Bowshe’r.........scsccssssseressscsercersensseensenrnesseesesses 53
`8 The Extraction of Enzymes FromPlant Tissues Rich in Phenolic Compounds
`Willan: Piecing! siiscmniananiianmnmannneAn 65
`9 Avoidance of Proteolysis in Extracts
`Robert J. Beynon and Simon Oliver (Revised by Paul Cutler) ......s.sssssesesseersees 75
`10 Concentration of Extracts
`Shawi' DOONAN sisscrsssessssccesesesssesasssssarcaassseescsverensanvevssssuneecccvernstescssessseosacsetsssssueass 85
`11. Making and Changing Buffers
`SHAWN DOONAN .usssesssseccessesccssssacsssssesnscessccsensssnsssenseseensnsnenscseasesessseesssensaseanseseenreas 91
`
`12 Purification and Concentration by Ultrafiltration
`Paul Schratter (Revised by Paul Cutler) ....cccscsessscescccssccsressssescceeeseseesnesseeensaeens 101
`13 Bulk Purification by Fractional Precipitation
`.
`SIVA WIN PIOOMIAND occcaccisaaucezecnccvcseescosvacssduveresavecsservsceesencnsccesssuatessscetsee aasecesaawesevse 117
`14
`lon-Exchange Chromatography
`CRPIS SOUP. siscssrccsecszsezserarnerracarsnicicsscsvsasesvevsiveasccseenivseesvassusi sevavesrasceseucssuseeas 125
`
`15 Hydrophobic Interaction Chromatography
`Pat As OFaN cscccccsvececececsesseseraesesaesicsssssssesvsannsacssiessas tenses eotwosovsevnnvassseeonnatenee 133
`
`16 Affinity Chromatography
`Paul Cutler .ecccccccsssscssessscecsecsscccsscccsssseessceesscscecsesneeesnssseessusessscevasssasscaeceeatecesnees 139
`
`17 Dye-Ligand: Affinity Chromatography
`Anne F, McGettrick and D. Margaret Worrall ......:cccccsccsessseseesesensrensecenceneneaee 151
`
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`Contents
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`19
`
`20
`
`18 Lectin Affinity Chromatography
`Iris West and Owen Goldring (Revised by Paul Cutler) .........:.scsssssssssssseseesseses 159
`Immunoaffinity Chromatography
`Part Cather ssvaserceserweunsevavssisinsscenesigiciiaistsccdbt ncn aranenavavmermocenemoanusrncesacocccsusversiia 167
`Immobilized Metal-lon Affinity Chromatography
`Tai-Tung Yip and T. William Hutchens (Revised by Paul Cutler) .....2......0.000++. 179
`21 Chromatography on Hydroxyapatite
`SHAWN DOONAN woeecsesssssesesesseseresessesssssseseseseseseecsesesssesacsrsrersssssasssstsssesasasseseeseseee, 191
`22. Thiophilic Affinity Chromatography and Related Methods
`Paul Matejtschuk .....ccccccsssssesssssssssssesssessesssssssssscscsssssssacsssssscssssssearatesussssetasseseee 195
`23 Affinity Precipitation Methods
`Jane A, Irwin and Keith F. Tipton .....sscsecesesssessesscesssssesesscsseststsssnssscssscscsseccascens 205
`Isoelectric Focusing
`RGNWESTEHICIED serssesseuszscnsscsvatnssseanenenerenrnennceanxesasenecosusnsseveeveesxasiepeavasvessias 225
`25 Chromatofocusing
`Timothy J. Mantle and Patricia NOONE ....cccccsssssssssssssssssssesesesessssssessisssssececscesees 233
`26 Size-Exclusion Chromatography
`Pat SREP semesscmennssecvsssnsauebsvareaueteniodideenneeaneiaTiacsse0 oon, 239
`Fast Protein Liquid Chromatography
`David Sheehan and Siobhan O/Sullivan ..cccccccscsccsssssssssssesssssssscssessssssscesssceesseee 253
`28 Reversed-Phase Chromatography ofProteins
`William A. Neville ....cccscscsssssssessssssssssssssessssssssssesscssasssacsssssssssssessscessststassesseasenes 259
`29 Extraction of MembraneProteins
`Bea DOREOI sess usaoxinenigus pasa cotses sss ttsnonceetetosaneararermxereeesevnrsnsvexnooosruxvanevsessens 283
`30 Removal of Detergent From Protein Fractions
`Kay Ovtlendieehe nescssasccossscssssssucsassnenesnsssiubiieieasiisitasivaiistcemanmanrennnenremesacacsoce 295
`Purification of Membrane Proteins
`IAS ETCGN anrxcoemnmsn cosa secretive anesaissiesilficial H SRNR GRIEG Siavincccnnamen TOT
`32 Lyophilization of Proteins
`EO 309
`33 Storage of Pure Proteins
`CTPA OFAcco eseszacsasuszrstihasscnsesereoeonearsonsosmsararaarssneceonsosvewsvosvsieseaneenesne BOD
`34 Electroelution of Proteins From Polyacrylamide Gels
`Mibectiael ) PRA ses sxcceuscosiivsi ins cxueesisueasaisecssGis Usicilisdesidsiéienammanenenaverevnsoureuemaunerres 339
`35 Electroblotting of Proteins From Polyacrylamide Gels
`Michael J. Dunn coccccsssssssssssssssesessesssssessersceecscesrscssterssassssssssasssasessasatsssssassasseseceee 345
`36 Two-Dimensional Polyacrylamide Gel Electrophoresis
`for Proteome Analyses
`Nei] A. JOOS ...sssscsesececessssessesssssssscsnsassnssssssesesesssescesacssazstsnssucussesesasatsesessatsesssane 353
`37 Microscale Solution Isoelectrofocusing: A Sample Prefractionation
`Method for Comprehensive Proteome Analysis
`Xun Zuo and David W. Speicher ....cccccccscessseesssessreresesssssessersescsssrasstsssssssssseses 361
`
`24
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`27
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`Contents
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`IX
`
`38 Practical Column Chromatography
`Shawn DOGAN scsscessessscscsesvossasesssvsssscssasnsstosseseeevscsssvevssssvavessssasvusvensevenssvcees 377
`
`39 Detection Methods
`Jacek Mozdzanowski and Sudhir Burman .......::cccsssssscscessscscesssscseesssssesesesssesees 389
`40 Peptide Proteomics
`Dean E. McNulty and J. Randall SlOMMoOn ...ccccsscccseenecceesseeeeteneseetetseeneeessntaes 411
`41 Multidimensional Liquid Chromatography of Proteins
`Rod Watson: and. Tint Nagler vs ccss cccevsczsszececesscsucesesceececesseuansctuuep vinsvedststesiecousncees 425
`
`42 Mass Spectrometry
`David } BOW csscsccccccvecasessseacessscsevscscssanssasatesecsvisetecvevesceseeeiescierssevstessecncctvietevetes 447
`43 Purification of Therapeutic Proteins
`Jaliaty BONMET{Odiecciccccvcrsdessscssevsassusnrsdinsdsvttivesccsyeces¥dedesteViderenetsndebecssnendersenrades=5 455
`44 Purification Process Scale-Up
`Karl Prince and Martin Smith .....cccccccccseesccsssessecsesssccssssescsssaesesesnssscessassseneasaase 463
`
`WCU hace reece ene veo vcr rranicot rvs vetceatc cen eevee evn Pace oem ear ee eee stead een Neer MRED REO 481
`
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`14
`
`lon-Exchange Chromatography
`
`Chris Selkirk
`
`1. Introduction
`
`lon-exchange chromatographyis one of the most widely used forms of column chro-
`matography.It is used in research, analysis, and process-scale purification of proteins.
`lon exchangeis ideal for initial capture of proteins because of its high capacity, rela-
`tively low cost, andits ability to survive rigorous cleaning regimes. Ion exchangeis 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, lon-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 prepacked columns (Bio-Rad, Amersham Bioscience) are
`now common,particularly for small-scale analytical and method development work. lon
`exchange can now also be carried out on monolithic columns (Bio-Rad), on membranes
`(Pall, Sartorius), and on ion-exchange high-performanceliquid chromatography (HPLC)
`columns. The methodis 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.
`lon-exchange chromatographyrelies on the interaction of charged molecules in the
`mobile phase (buffer + sample) with oppositely charged groups coupledto 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 aminoacids 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 aminoacids at physiological pH,
`+ve
`lysine, arginine, histidine
`—ve
`aspartic acid, glutamic acid
`Charges on ion-exchange matrices
`+ye DEAE, QAE
`—ve CM,SP, sulfonic acid
`
`From: Methods in Molecular Biology, vol. 244: Protein Purification Protocols: Second Edition
`Edited by: P, Cutler © Humana Press Inc. Tatowa, NJ
`
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`126
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`Selkirk
`
`Table 1
`lon-Exchange Groups
`
`Ion-exchanger type
`Strong exchangers
`Weak exchangers
`
`Cation exchangers
`
`Anion exchangers
`
`SP (sulfopropyl)
`S (Methyl sulfonate)
`Q (quaternary ammonium)
`QAE(quaternary aminoethy!)
`
`‘CM (carboxymethyl)
`
`DEAE(dicthylaminoethyl)
`
`The net charge on a protein molecule will] depend on the combination ofpositively
`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 groups 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 (p/) for that protein.It is useful to know the p/
`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 Nate 1).
`Binding and elution of proteins is based on competition between charged groups on
`the protein and charged counterions in the buffer for binding to oppositely charged
`groupsonthe stationary phase. The higher the concentration of charged salt molecules in
`the solution, the greater is the competition for bindingto the ligands on the matrix, 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-
`manently 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 boundto the ion-exchange column. During elution, the salt con-
`centration is increased, so that when a protein group dissociates from an ionic group on
`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
`concentration 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 ts
`used at a pH below the isoelectric point of the protein(s) to be bound.
`Anion Exchange: 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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`lon-Exchange Chromatography
`
`127
`
`Table 2
`Buffers: Anion Exchange
`
`Buffer
`Anion
`pH Range
`
`N-methyl piperazine
`Piperazine
`L-Histidine
`Bis-Tris
`Bis-Tris propane
`Triethanolamine
`Tris
`Diethanolamine
`
`Ccl-
`cl
`a
`clr
`Cl
`cl-
`Cl
`cl-
`
`4.5-5.0
`5.0-6,0
`5.5-6.5
`5.8-6.8
`6.4-7.3
`7.3-8.2
`7.5-8.0
`8.49.4
`
`overall negative charge at a pH abovetheir isoelectric point. Therefore, anion exchange 1s
`used at a pH abovethe isoelectric point of the protein(s) to be bound.
`
`lon exchangers are also divided into strong and weak ion exchangers. Sirong ion-ex-
`changeligands 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—-Sepharose Fast Flow(strong
`anion) has a working pH range of 2.0-12.0. CM—Sepharose Fast Flow (weak cation) has
`a working pH range of 6.0-10.0 and SP—Sepharose Fast Flow (strong cation) has a work-
`ing pH range of 4.0-13.0. If your purification is to be carried out at pH above9 for anion
`exchange orbelow 6 for cation exchange. thenit 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 yourpurification.
`
`2. Materials
`
`Mm
`
`|. Binding buffer of appropriate pH and composition for binding of protein to matrix (see
`Notes 24). Tables 2 and 3 list some suitable buffers and the pH ranges over which they
`are useful is included.
`Elution buffer (often the same as binding buffer but with higher salt concentration) (see
`Note 5).
`| M NaCl). Note: Buffers and samples should befiltered (0.45
`3. Regeneration. buffer (e.g.,
`um) 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 (e.g., 0.02%[w/v]
`sodium azide),
`4. Desalting column, These can be purchased readyto 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-exchange 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.
`6. Chromatography equipment (see Notes 6 and 7).
`7, Assay methodsfor analysis of the purified materials will be required to determine the suc-
`cess of the purification.
`
`5.
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`128
`
`Selkirk
`
`Table 3
`Buffers: Cation Exchange
`
`Buffer
`Cation
`pH range
`Maleic acid
`Na*
`1.5-2.5
`Formic acid
`Nat
`3.3—4.3
`Citric acid
`Nat
`2.6-6.0
`Lactic acid
`Na+
`3.64.3
`Acetic acid
`Nat
`4.3-5.3
`MES/NaOH
`Nat
`5.5-6.7
`Phosphate
`Na*
`6.7-7.7
`MOPS
`Nat
`6.5-7.5
`HEPES
`Nat
`7.58.2
`
`3. Methods
`
`3.1. Preparation
`|, The starting material mustfirst be equilibrated in the binding buffer before ion exchange can
`be commenced.If the sample is not already prepared in a suitable buffer 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). If recovery of the protein ofinterest 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 theseare 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-exchangepurification.
`2. The sample should be filtered (0.45 sm) 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 mast
`commonly used counterions are sodium (Na+) for cation exchange and chloride (C]—) 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 1-2 col-
`umn volumes of high-ionic-strength elution buffer through the column (see Note 8).
`4, Once charged with the counterion, the column needs to be thoroughly washed with the bind-
`ing buffer (S—10 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 applied to the
`column. Once the column is equilibrated, 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
`lowerflow 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.
`
`re)
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`lon-Exchange Chromatography
`
`129
`
`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 thatall nonbound proteins are washed out of the column. Monitoring the columnelu-
`ate with an ultraviolet (UV) detector at 280 nm gives an immediate visual indication of the
`amountof protein or other UV-absorbing material present in the eluate from the column.
`4, Elute bound proteins by washing the column with an increasing salt gradient of 0-500 mM
`NaC]in binding buffer over 10-15 column volumes (see Notes 5 and 9-11).
`,ouA
`Collect the eluted protein in fractions for analysis.
`6. Analyse both nonbound material and eluted fractions to determine in which fractions the
`protein ofinterest has been isolated and whether contaminants have coeluted. Based on this
`analysis, required modifications to the chromatographic conditions can be planned (see
`Note 10).
`
`2,
`
`3.3. Column Regeneration and Storage
`|, Ton-exchange columns should be cleaned and regenerated between purifications, otherwise
`the binding can rapidly be reduced and the column can become blocked by contaminants.
`lon-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 1 M NaCl will elute most covalently bound con-
`taminants not eluted during the purification (see Note 8).
`lon-exchange columnscan be stored packed provided they contain a bacteriostatic solution.
`The column can be equilibrated in buffer containing 0.02% (w/v) 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 columntubing 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 ofthe appropriate
`binding buffer through the column.
`4. During long-term storage, there is a likelihood that the column maystart to dry out. There-
`fore for extended storage periods, it is recommendedthat the column should be unpacked
`and the ion-exchange matrix stored in a buffer containing a bacteriostatic agent (e.g., 20%
`ethanol),
`
`4. Notes
`
`|, The isoelectric point (p/) can be determined experimentally by isoelectric focusing. Iso-
`electric points for many proteins can be foundin theliterature (2). The p/ of novel proteins
`can be predicted from the amino acid sequence ofa protein if this is known. Software pack-
`ages are available that will calculate the p/ and there are sites accessible on the Internet
`where p/ and other properties for a protein can be calculatedif the amino acid sequence or
`the base sequence of the DNA coding for the protein is entered (¢.g., Swissprot, www.ex-
`pasy.ch). Once into the Swissprotsite, go to "Proteomics Tools” and then click on “Primary
`structure analysis.’ There are several opuons within this part of the site that offer predic-
`tions of protein properties, including p/.
`2. When scouting for the best pH for ion-exchange purification, start by trying a pH around
`1-1.5 pH units from the p/ ofthe protein being purified: one unit above the p/ for anion ex-
`change and one unit below the p/ for cation exchange, Having analyzed the separation
`achieved at this pH, the buffer can be adjusted slightly in subsequent runs to improve the
`results.
`
`11 of 79
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`11 of 79
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`Fresenius Kabi
`Exhibit 1028
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`130
`
`Selkirk
`
`3.
`
`Itis 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.
`4. The ionic strength of the buffer is as important as the pH when carrying oul ion-exchange
`purification. In most cases, a starting buffer of 20-50 mM is suitable. Many proteins tend
`to aggregate in solutions close to the protein’s p/; 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 pHto elute from anion exchangers. However,it is more difficult
`to control pH gradients on standard ion-exchange columnsthan to control changes in ionic
`strength, so pH gradients are only commonly applied with chromatofocusing columns,
`6. Ton-exchange chromatography can be carried out in short, wide columns because the bed
`volume is more important than the bed height. Bed heights of 5-10 cm are frequently used.
`Once the capacity of the column has been determined for your protein under your condi-
`tions, then the column volumecan be setto fit the amount of protein to be purified in one
`run.
`
`7, The equipmentused 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 chromatographyresults.
`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 elution of proteins by salt gradient. Gradient mixers
`allow the formation of a controlled and reproducible salt gradientthat is essential for run
`to run consistency.
`It is commonto charge the ionic groups on the column matnx 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—1 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 also destroy endo-
`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.
`
`8.
`
`12 of 79
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`Fresenius Kabi
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`12 of 79
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`Fresenius Kabi
`Exhibit 1028
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`

`

`lon-Exchange Chromatography
`
`131
`
`9,
`
`If 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 0.1-1 M sodium
`chloride) are prepared. The gradient mixer 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 10-15 column volumesis often suit-
`able,
`10, After initial purification runs have been analyzed it may be desired to alter the gradientto im-
`prove separation of eluted proteins. The gradient can be altered either by increasing or de-
`creasing the ionic strength ofthe 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 of the product. By careful analysis of the peaks
`eluted, the gradient can be fine-tuned to optimize purification of the desired protein. Al-
`though ion-exchange columns have a high capacity, the resolution achieved can be improved
`by loading the columnto well below the maximum binding capacity (10-20%).
`11. Elution by stepwise increases in sodium chloride can be usedif a gradient mixeris 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) buffer-
`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
`
`1. Amersham Pharmacia Biotech (1999) lon Exchange Chromatography Principles and Meth-
`ods, Amersham Pharmacia Biotech, Uppsala, Sweden.
`2. Righetti, P. G. and Carravaggio, T. (1976) Isoelectric points and molecular weights of pro-
`teins: a table. J. Chromatogr. 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% ofthe surface area (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 powerful technique, especially because it operates on
`a principle different from either of the two most commonly used general chromato-
`graphic methods, ion exchange and size exclusion, and can thus be used to separate
`components that these techniques cannot. Careful manipulation of the conditions can
`enable it to be very sensitive. For example,it is capable of separating proteins that dif-
`fer by as little as one amino acid