instructions28-4082-02ac plasmidselectxtra

Instructions 28-4082-02 AC

PlasmidSelect XtraPlasmidSelect Xtra Starter KitPlasmidSelect Xtra is the chromatography medium used in the selectivecapture step of a three-step chromatographic process to purify super-coiled, covalently closed circular plasmid DNA (sc pDNA).

This process comprises:

1 RNA removal and buffer exchange by group separation onSepharose™ 6 Fast Flow.

2 Capture and selective desorption of supercoiled plasmid DNA withPlasmidSelect Xtra.

3 Final polishing on SOURCE™ 30Q.

PlasmidSelect Xtra Starter Kit contains these three BioProcess™ mediain prepacked, ready to use columns in quantities sufficient to obtain upto 5 mg of pure supercoiled plasmid DNA.

The columns are:

HiPrep™ 26/10 Sepharose 6 FF, 53 ml

HiTrap™ PlasmidSelect Xtra, 5 ml

HiTrap SOURCE 30Q, 5 ml

Note that PlasmidSelect Xtra Starter Kit does not include buffers.

PlasmidSelect Xtra medium is also supplied in 25 and 200-ml laboratorypacks as well as 1 and 5-l process-sized packs (seeOrdering information).

Table of Contents31 Introduction .................................................................................................32 Prepacked columns and media characteristics .........................83 Purification protocol ................................................................................144 Scaling up .....................................................................................................195 Packing columns for scaling up .........................................................226 Evaluating column packing ..................................................................

257 Adjusting pressure limits in chromatography system soft-

ware ................................................................................................................278 Maintenance ...............................................................................................309 Ordering Information ..............................................................................

Please read these instructions carefully before using theproducts.

Intended use

The products are intended for research use only, and shall notbe used in any clinical or in vitro procedures for diagnosticpurposes.

Safety

For use and handling of the products in a safeway, please referto the Safety Data Sheets.

2 28-4082-02 AC

1 IntroductionPlasmidSelect Xtra medium and PlasmidSelect Xtra Starter Kit havebeen developed to purify supercoiled plasmid DNA.We recommendthe three-step purification process using PlasmidSelect Xtra StarterKit illustrated in Figure 1, Section 3, for obtaining pure supercoiledplasmidDNA. This process does not require the use of RNase enzyme.Figure 1 also shows typical results. The prepacked columns in thestarter kit provide fast and easy separations in a convenient format.In addition, the process can be scaled up for cGMP purification ofsupercoiled plasmid DNA suitable for gene therapy.

PlasmidDNA content and quality can also be determined in a simpleand easy way using PlasmidSelect Xtra Starter Kit (see Instructions28-4082-03).

2 Prepacked columns and mediacharacteristics

HiPrep 26/10 Sepharose 6 FF columns andSepharose 6 FF medium

HiPrep 26/10 Sepharose 6 FF column is prepacked with Sepharose6 Fast Flow. This medium consists of 90-μm diameter highly cross-linked 6% agarose beads. It displays high chemical stability and isuseful for group separation of large biomolecules such as RNA andplasmid DNA. Table 1 describes key column and mediumcharacteristics.

HiPrep columns cannot be opened or refilled.Note:

28-4082-02 AC 3

Table 1. HiPrep 26/10 Sepharose 6 FF and Sepharose 6 Fast Flowcharacteristics

Sepharose 6 Fast FlowMediumHighly cross-linked agarose, 6%Matrix90 μmAverage particle size53 mlBed volume100 mmBed height26 mmInternal diameterPolypropyleneColumn material30 to 300 cm/h (2.7 to 27 ml/min)Recommended flow

velocity 1

30 to 60 cm/h (2.1 to 5.3 ml/min)Recommended flowvelocity for sc pDNA purification

450 cm/h (40 ml/min)Maximum flow velocity 1

0.5 MPa, 5 bar, 73 psiColumn hardware pressure limit

3 to 13pH stability 2

2 to 14Working rangeCleaning-in-place

4°C to 40°COperating temperature20% ethanol at 4°C to 30°CStorage

1 H2O at room temperature. For viscous buffers and samples the flow velocity must beoptimized. Starting with a low flow velocity is recommended.

2 Working range: pH interval where themediumcanbe handledwithout significant changein function.Cleaning-in-place: pH interval where the medium can be subjected to cleaning-in-placewithout significant change in function.

4 28-4082-02 AC

HiTrap column characteristicsThe columns are made of biocompatible polypropylene that doesnot interact with biomolecules.

The columns are delivered with a stopper at the inlet and a snap-offend at the outlet. Table 2 lists the characteristics of HiTrap columns.

HiTrap columns cannot be opened or refilled.Note:

Make sure that the connector is tight to prevent leakage.Note:

Table 2. Characteristics of HiTrap columns5 mlColumn volume (CV)1.6 x 2.5 cmColumn dimensions5 bar (0.5 MPa)Column hardware pressure limit

The pressure over the packed bed varies depending on arange of parameters such as the characteristics of thechromatography medium, sample/liquid viscosity and thecolumn tubing used.

Note:

Supplied Connector kit with HiTrap columnNo. suppliedUsageConnectors supplied

1For connection of syringe to HiTrapcolumn

Union 1/16” male/luer female

2, 5 or 7For sealing bottom of HiTrap columnStop plug female, 1/16”

28-4082-02 AC 5

HiTrap PlasmidSelect Xtra and PlasmidSelect XtraHiTrap PlasmidSelect Xtra columns are prepackedwith the thiophilicaromatic adsorption medium PlasmidSelect Xtra. This mediumconsists of 34-μm diameter highly cross-linked agarose beads withan immobilized 2-mercaptopyridine ligand. Its main application ispurifying the supercoiled form of plasmid DNA. Table 3 describeskey medium characteristics.

Table 3. HiTrap PlasmidSelect Xtra and PlasmidSelect Xtracharacteristics

Highly cross-linked agarose, 6%Matrix34 μmAverage particle size2-mercaptopyridine, 3,5 mg/mlLigand> 2 mg/mlDynamic binding capacity for sc

pDNA (6125 bp) 1

150 cm/h (5 ml/min)Recommended flowvelocity 2

≤ 120 cm/h (4 ml/min)Recommended flowvelocity for sc pDNA purification




0.5 M NaOHCleaning-in-place15°C to 30°COperating temperature20% ethanol at 4°C to 30°CStorage

1 Conditions for determining dynamic binding capacity:Sample: Approx. 0.3 mg plasmid/ml in binding buffer (capacity at 10% breakthrough)Column volume: 1 ml (Tricorn™ 5/50)Flow rate: 0.2 ml/minBinding buffer: 2.1 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris, pH 7.0Elution buffer : 0.3 M NaCl, 1.7 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5



6 28-4082-02 AC

HiTrap SOURCE 30Q and SOURCE 30QHiTrap SOURCE 30Q columns are prepacked with SOURCE 30Qmedium that consists of 30-μm diameter rigid, mono-sized poly-styrene/divinyl benzene beads with quaternary ammonium groups.The ion exchange group is attached to the matrix via hydrophilicspacer arms following hydrophilization of the polymeric basematrix.SOURCE 30Q is used for reproducible, high-productivity separationsat large scale. Table 4 describes key medium characteristics.

Table 4. HiTrap SOURCE 30Q and SOURCE 30Q characteristicsHighly rigid polystyrene/divinyl ben-zene

Matrix

30 μmAverage particle sizeQuarternary ammoniumFunctional group150 cm/h (5 ml/min)Recommended flow

velocity 1

≤ 120 cm/h (4 ml/min)Recommended flowvelocity for sc pDNA purification




≤ 1.0 M NaOHCleaning-in-place4°C to 40°COperating temperature20% ethanol at 4°C to 30°CStorage



28-4082-02 AC 7

3 Purification protocolFlow scheme for PlasmidSelect Xtra Starter Kit

Clarified alkaline lysate

Purified supercoiled pDNA

HiT

rap

™H

iTra

p™

HiP

rep

™ 2

6/1

0

PlasmidSelect Xtra Starter Kit

HiPrep 26/10 Sepharose 6 FF

26 mm x 100 mmVt: 53 ml

RNA Removal

HiTrap PlasmidSelect Xtra

16 mm x 25 mmVt: 5 ml

Supercoiled pDNA capture

HiTrap SOURCE 30Q


Supercoiled pDNA polishing

8 28-4082-02 AC

mS/cm

0

50

100

0.0 4.0 8.0 12.0 16.0 CV

150

0

500

1000

2000

2500

mAU

1500

RNA

mS/cm

0

50

100

200

0.0 4.0 8.0

0.0 1.00.5 1.5 2.0

16.0 20.012.0 CV

150

0

1000

2000

4000

mAU

3000

CV

Step 1. HiPrep 26/10 Sepharose 6 FF

Step 2. HiTrap PlasmidSelect Xtra

Step 3. HiTrap SOURCE 30Q

oc - sc -

oc - sc -

5000

3000

Fig 1. The process for purifyingsupercoiled plasmid DNA.(A) Typical chromatograms of thethree steps run on ÄKTAexplorer10 (see flow scheme for details).(B) Ethidium bromide-stained0.8%agarose electrophoresis gelsof the key fractions eluted in Steps1 and 2.

mS/cm

0

50

100

0.0 4.0 8.0 12.0 16.0 CV

150

0

500

1000

2000

2500

mAU

1500

RNA

mS/cm

0

50

100

200

0.0 4.0 8.0

0.0 1.00.5 1.5 2.0

16.0 20.012.0 CV

150

0

1000

2000

4000

mAU

3000

CV

Step 1. HiPrep 26/10 Sepharose 6 FF

Step 2. HiTrap PlasmidSelect Xtra

Step 3. HiTrap SOURCE 30Q

oc - sc -

oc - sc -

5000

3000

pDNA = plasmid DNA; oc = opencircular; sc= supercoiled, covalent-ly closed circular; M = molecularweight ladder (DRIgest III).

28-4082-02 AC 9

EquipmentFor convenience and efficiency, we recommend using a chromato-graphy systemwith a gradient-forming pump and UV detection, forexample ÄKTA™ systems. If you use ÄKTAexplorer, remove the flowrestrictor to avoid high back-pressure when working with viscoussamples such as clarified bacterial cell lysate. A temperature-com-pensated device tomeasure conductivitymay prove useful. In addi-tion, clarification by centrifugation or filtration may be required toprepare the bacterial alkaline cell lysate. You can assess the qualityof the plasmid preparation after each step by running samples in0.8% to 1% agarose gel electrophoresis and staining the gels withethidium bromide 1 .

BuffersYou need the following buffers:For Step A

2.1 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5Buffer A:For Step B

2.0 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5Buffer B:Buffer C: 0.3 M NaCl, 1.7 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl,

pH 7.5For Step C

0.4 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5Buffer D:1.0 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5Buffer E:

Use high purity water and chemicals. Filter buffers through a0.22-μm or 0.45-μm filter before use.

1 Sambrook, J. and Russel, D. W., Molecular cloning. A laboratorymanual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,NY, (2001).

10 28-4082-02 AC

Sample preparationThe starting material for plasmid DNA (pDNA) is usually clarifiedbacterial cell lysate containing the desired plasmid1. Generally, freshor frozen cell paste is suspended in 50 mM Tris-HCl buffer, pH 7.5,containing 10mMEDTA to reduceDNAse activity andapproximately50 mM glucose. Lyse cells by adding 0.2 M NaOH, 1% SDS at roomtemperature. The suspension normally changes color and becomesviscous.

Perform this step quickly and use only gentle stirring to avoidshear stress breaking chromosomal DNA and damaging theplasmids.

Note:

Flocculate cellular debris and SDS complexes by gently adding cold3 M potassium acetate, pH 5 and incubating on ice. Clarify the plas-mid DNA extract using filtration or centrifugation. This procedurenormally gives an initial concentration of approximately 0.05 to0.1 mg/ml plasmid DNA in an alkaline lysate.

To obtain up to 5 mg supercoiled plasmid DNA (sc pDNA) of highpurity at the end of the three-step purification, the plasmid-containing extractmay have to be concentrated using, for example,ultrafiltration. We suggest using hollow fiber ultrafiltration with aMWCO of 300, for example MidGee™ Cartridge UFP-300-C-MM12AfromGE. Typically, a plasmidDNA concentration from0.5 to 1mg/mlprior to RNA removal and buffer exchange on HiPrep 26/10Sepharose 6 FF should minimize the number of cycles run on thiscolumn before capture of the supercoiled plasmid DNA on HiTrapPlasmidSelect Xtra.

Filter the sample through a filter (1 or 5 μm, for example) beforeapplying it to the column.

You can readily scale up this process. For other column sizes anddetails of column packing, see Sections 4 and 5. Custom-packedcolumns are available on request (see Ordering information).

28-4082-02 AC 11

Protocol

Step A: RNA removal and buffer exchange

ActionStep

Equilibrate HiPrep 26/10 Sepharose 6 FF with at least 110ml (2 CV) of Buffer A at 2.7 to 5.3 ml/min (30 to 60 cm/h).

1

Load up to 16 ml (0.3 CV) of concentrated, clarified alkalinelysate at a flow rate of 2.7 ml/min (30 cm/h).

2

Collect the void volume containing the partially purifiedplasmid DNA preparation. Discard the RNA-containingsecond (large) fraction.Note:RNA tends to elute with the yellowish colored band charac-teristic of the clarified cell lysate. Make sure that all materialhas eluted from the previous run before applying a newsample.

3

Dependingon the initial concentrationof plasmidDNA, you can loadseveral portions of the same sample on the columnwithout extensiveequilibration between consecutive loadings (see Fig 2). However, werecommend cleaning the column with water and re-equilibrate atleast every fifth cycle orwhen the chromatogrambecomesdistorted.

For recommendations onmedia regeneration and cleaning-in-place(CIP), see Maintenance.

Fig 2. Three consecutivesample loadings onHiPrep 26/10 Sepharose6 FF.

mS/cm

0

50

100

200

0.0 1.0 2.0 3.0 4.0 5.0 6.0 CV

150

0

1000

2000

4000

mAU

3000

5000

12 28-4082-02 AC

Step B: Capture and selective desorption of supercoiledplasmid DNA

ActionStep

Equilibrate HiTrap PlasmidSelect Xtra with at least 10 ml (2CV) of Buffer B at 4 ml/min (120 cm/h).

1

Load the partially purified plasmid DNA sample from Step Aat 4 ml/min (120 cm/h).

2

Wash with 20 ml (4 CV) of buffer B.3

Open circular forms of plasmid DNA are not retained andelute with the wash.

Elute supercoiled plasmid DNA with 25 ml (5 CV) of Buffer C(up to 3.3 ml/min [100 cm/h]).

4

Collect the peak of highly pure supercoiled plasmidDNA (Fig1, Step 2).

5


Step C: Final polishing

ActionStep

Equilibrate HiTrap SOURCE 30Q with at least 10 ml (2 CV) ofBuffer D at 4 ml/min (120 cm/h).

1

Measure the volume of the supercoiled plasmid DNA eluatefrom Step B and dilute with two volumes of pure water or100mMTris-HCl buffer, pH 7.5 to decrease the ionic strengthof the sample.

2

Load the diluted sample at 4 ml/min (120 cm/h).Note:The resulting conductivity may still be higher than in BufferD.

3

Wash with at least 10 ml (2 CV) of Buffer D and elute with atleast 25 ml (5 CV) of Buffer E (up to 4 ml/min [120 cm/h]).

4

28-4082-02 AC 13

ActionStep

Collect the elution peak containing the purified supercoiledplasmid preparation (Fig 1, Step 3).Note:The purified sample now contains more than 0.5 M NaCl.

5


4 Scaling upIntroduction

Optimize the purification using the small prepacked columns in thePlasmidSelect Xtra Starter Kit. After screening, optimizing, and veri-fying the method with PlasmidSelect Xtra Starter Kit, transfer theprocess to the bed heights required for further scale-up.

30-fold scale-upFigure 3 shows a 30-fold scale-up from PlasmidSelect Xtra StarterKit with increased bed heights but constant flow velocity and sampleloading. Even though the change in bed height might be significant,onlyminormodifications to the conditionswould beneeded to furtheroptimize the dynamic binding capacity/recovery for each specificplasmid. Scale-up details are stated opposite each image.

14 28-4082-02 AC



HiPrep 26/10 Sepharose™ 6 FF

26 mm x 100 mmVt: 53 ml



HiTrap SOURCE 30Q


HiT

rap

™H

iTra

p™

HiP

rep

™ 2

6/1

0

PlasmidSelect Xtra Starter Kit



Sepharose 6 Fast FlowBPG 100

100 mm x 250 mmVt: 1960 ml

PlasmidSelect XtraFineLINE Pilot 35

35 mm x 150 mmVt: 145 ml

SOURCE 30QFineLINE PIlot 35

35 mm x 150 mmVt: 145 ml

30 fold scale-up

RNA Removal



Fig 3. An approximately 30-fold scale-up from PlasmidSelect XtraStarter Kit.

28-4082-02 AC 15

Sepharose 6 Fast Flow packed in BPG 100 columnToFromIncreaseMaintain250100Column bed height

(mm)10026Column diameter

(mm)196053Column volume (ml)39-782.7-5.3Volumetric flow rate

(ml/min)30-60Flow velocity (cm/h)

>2Equilibration (CV)≤ 590 ml≤ 16 mlSample volume0.3Sample loading (CV)

(proportionally)

PlasmidSelect Xtra and SOURCE 30Q packed in FineLINE35 column

ToFromIncreaseMaintain15025Column bed height


(mm)1455Column volume (ml)14-193-4Volumetric flow rate


29010(ml)>2Equilibration (CV)Sample volumeUp to

2 mg/mlSample loading (CV)

(proportionally)

Further scaling upIt is possible to further scale-up of the purification process as outlinedin the following image. Here both bed height and flow velocity areessentially kept constant, while bed diameter and volumetric flowrate are increased. Scale-up details are stated opposite each image.

16 28-4082-02 AC




300 mm x 280 mmVt: 19 620 ml

PlasmidSelect XtraFineLine 100

100 mm x 180 mmVt: 1440 ml

SOURCE 30QFineLINE 100

100 mm x 180 mmVt: 1440 ml

Further 10-fold scale-up




100 mm x 250 mmVt: 1960 ml

PlasmidSelect XtraFineLINE Pilot 35

35 mm x 150 mmVt: 145 ml

SOURCE 30QFineLINE Pilot 35

35 mm x 150 mmVt: 145 ml

30 fold scale-up

RNA Removal



Fig 4.A further 10-fold scale-up from thepurificationprocess outlinedin Fig 3.

28-4082-02 AC 17

Sepharose 6 Fast Flow packed in BPG 100 columnToFromIncreaseMaintain250250Column bed height




400003900(ml)>2Equilibration (CV)≤ 5900 ml≤ 590 mlSample volume0.3Sample loading (CV)

(proportionally)

PlasmidSelect Xtra and SOURCE 30Q packed in FineLINE35 column

ToFromIncreaseMaintain180150Column bed height




3000290>2Equilibration (CV)Sample volumeUp to

2 mg/mlSample loading (CV)

(proportionally)

18 28-4082-02 AC

5 Packing columns for scaling upRecommended pilot-scale columns

Empty column 1MediumXK 50/30 2 , BPG 100Sepharose 6 Fast FlowXK 16/20, XK 26/20, FineLINE Pilot 35PlasmidSelect XtraXK 16/20 2, XK 26/20 3 , FineLINE Pilot 35SOURCE 30Q

1 For inner diameter andmaximumbed volumes andbedheights, seeOrdering information.2 Custom-packed columns are available on request, see Ordering information.3 A special filter kit is needed, see Ordering information.

The three media are supplied in 20% (v/v) ethanol. Prepare a slurryby decanting the 20%ethanol solution and replacing it with packingsolution in a ratio of 50% to 70% settled medium to 50% to 30%packing solution. The packing solution should not contain agentsthat significantly increase viscosity. Distilled water or a low ionicstrength buffer are suitable packing solutions for the media.

Packing XK columns

ActionStep

Equilibrate all materials to the temperature at which thechromatography will be performed.

1

De-gas the slurry to minimize the risk of air bubbles in thepacked bed.

2

Eliminate air from the column dead spaces by flushing theend pieces with packing solution (or 20% ethanol). Makesure no air has been trapped under the column net.

3

For SOURCE 30Q and an XK 16 column, place Filter HR 16on top of the bottom plunger.For SOURCE 30Q and XK 26, place Filter 26 on the top of thebottom plunger.

Close the columnoutlet. Leave a few centimeters of packingremaining in the column.

4

28-4082-02 AC 19

ActionStep

To pack an XK 16 column: use Packing Connector XK 16 toconnect an extra XK 16 column as a packing reservoirTo pack an XK 26 column: use Packing Connector XK 26.To pack an XK 50 column: use RK 50 packing reservoir.

5

Pour the slurry into the column in one continuous motion.Pouring the slurry down a glass rod held against the wall ofthe column will minimize the formation of air bubbles.

Immediately fill the remainder of the column with packingsolution, mount the column top piece onto the column andconnect the column to a pump. Avoid trapping air bubblesunder the adapter or in the inlet tubing.

6

Open the bottom outlet of the column and set the pump torun at the desired flow rate.

7

Packing Sepharose 6 Fast Flow in an XK 50/30 columnLet the gel (50% slurry in pure water) settle at a flow rate of10 ml/min (30 cm/h) for 70 min, and then compress it at 30ml/min (90 cm/h) for 10min. Remove the PackingConnectorand lower the adapter until it reaches the surface of thepacked bed. Continue compressing at 60ml/min (180 cm/h)for 10 min.

Packing PlasmidSelect Xtra in XK 16/20 or XK 26/20columnsPack at a constant flow rate of 5ml/min (150 cm/h, XK 16/20)or 11.5 ml/min (130 cm/h, XK 26/20) in the first step, and 9ml/min (270 cm/h, XK 16/20) or 21.2 ml/min (240 cm/h, XK26/20) in the second step. Do not exceed 1.0 bar (0.1 MPa)in the first step and 3.5 bar (0.35 MPa) in the second step.

Packing SOURCE 30Q in XK 16/20 or XK 26/20 columnsPack at 10 ml/min (300 cm/h) for the first 5 min (approx. 3bar, 0.3 MPa). Then increase the flow rate until a maximumpressure of 5 bar (0.5 MPa) is achieved (20 to 30ml/min [600to 900 cm/h]).Note:Do not exceed 70% of the packing flow rate in subsequentchromatographic procedures.

20 28-4082-02 AC

ActionStep

Maintain the packing flow rate for 3 CV after a constant bedheight is reached.

8

After packing is completed,mark the level of the packed bedon the column tube before stopping the pump. Then stopthe pump and close the outlet. Disconnect the inlet tubingfrom the pump and remove the Packing Connector.

9

For SOURCE 30Q, place Filter HR 16 on top of the packedbed. Then lower the adapter until it reaches the surface ofthe packed bed. Tighten the O-ring so that the adapter willslide when pushed. Finally, lower the adapter until it isapproximately 3 mm below the mark on the column tube.

Packing FineLINE Pilot 35 columnFor packing PlasmidSelect Xtra or SOURCE 30Q in a FineLINE Pilot35 column, follow the packing procedures in the instructions suppliedwith the column.

Recommended process-scale columnsEmpty columnMediumBPG, BioProcess LPLC and Chromaflow™Sepharose 6 Fast FlowFineLINE, BPG, BioProcess MPLC andChromaflow

PlasmidSelect Xtra

FineLINE and BioProcess MPLCSOURCE 30Q

For process-scale columns, follow the packing procedures in theinstructions supplied with the column.

28-4082-02 AC 21

6 Evaluating column packingIntroduction

To adhere to good laboratory practice (GLP) andgoodmanufacturingpractice (GMP), you should check the quality of the packing andmonitor it during the working life of the column. Test columnefficiency directly after packing, at regular intervals afterwards, andwhen separation performance is seen to deteriorate. The methodswe recommend for expressing the efficiency of a packed columnare height equivalent to a theoretical plate, HETP, and asymmetryfactor, As. These values are easily determined by applying a samplesuch as 1.0 M NaCl in water with 0.5 NaCl in water as eluent.

The calculated plate number varies according to testconditions and you should therefore use it only as a referencevalue. It is also important that you keep conditions andequipment constant so that results are comparable. Changesin solute, solvent, eluent, sample volume, flow rate, liquidpathway, temperature, etc., will influence the results.

Note:

For optimal results, make sure that the sample volume does notexceed 2.5% of the CV, and keep the flow velocity between 15 and30 cm/h.

If an acceptance limit is defined in relation to column performance,you can use the column plate number as part of the acceptancecriteria for column use.

Method for measuring HETP and asymmetry factor (As)To avoid diluting the sample, apply it as close to the column inlet aspossible.Conditions

1% of CVSample volume1 M NaClSample concentration0.5 M NaCl in water, or dilute bufferEluent30 cm/hFlow velocityConductivityDetection NaCl, buffer

22 28-4082-02 AC

Calculating HETP and As

VR

Wh

50%

10%

Volume

a b

Absorbance

Fig 5. A typical test chromatogram showing HETP and As valuecalculations.

Calculating HETPHETP = L/N

andN = 5.54 (Ve/Wh)2

whereL = Bed height (cm)N = Number of theoretical platesVe = Peak elution distanceWh = Peak width at half peak heightVe and Wh are in the same units.

28-4082-02 AC 23

To facilitate comparing columnperformance, the concept of reducedplate height is often used.

Reduced plate height (h) is calculated as:h = HETP/d

whered is the mean diameter of the beads. As a guide, a value of hbetween 2 and 4 indicates good performance.

The peak should be symmetrical, and the asymmetry factor as closeto one as possible. A change in the shape of the peak is usually thefirst indication of bed deterioration due to use.

Calculating AsAs = b/a

wherea = 1st half peak width at 10% of peak heightb = 2nd half peak width at 10% of peak heightAs = 0.8 to 1.5 (guide)

24 28-4082-02 AC

7 Adjusting pressure limits inchromatography system softwarePressure generated by the flow through a columnaffects the packedbed and the columnhardware, see Figure below. Increased pressureis generated when running/using one or a combination of thefollowing conditions:

• High flow rates

• Buffers or sample with high viscosity

• Low temperature

• A flow restrictor

Exceeding the flow limit (see Tables 1, 3, and 4) may damagethe column.

Note:

pre-column pressure

post-column pressure

pressure over thepacked bed

Fig 6. Pre-column and post-column measurements.

28-4082-02 AC 25

ÄKTA avant and ÄKTA pureThe system will automatically monitor the pressures (pre-columnpressure and pressure over the packed bed, Δp). The pre-columnpressure limit is the column hardware pressure limit (see Tables 1and 2).

The maximum pressure the packed bed can withstand depends onmedium characteristics and sample/liquid viscosity. The measuredvalue also depends on the tubing used to connect the column to theinstrument.

ÄKTAexplorer, ÄKTApurifier, ÄKTAFPLC and othersystems with pressure sensor in the pump

To obtain optimal functionality, the pressure limit in the softwaremay be adjusted according to the following procedure:

ActionStep

Replace the column with a piece of tubing. Run the pumpat the maximum intended flow rate. Note the pressure astotal system pressure, P1.

1

Disconnect the tubing and run the pump at the same flowrate used in step 1. Note that there will be a drip from thecolumn valve. Note this pressure as P2.

2

Calculate the new pressure limit as a sum of P2 and thecolumnhardware pressure limit (see Tables 1 and 2). Replacethe pressure limit in the software with the calculated value.

3

The actual pressure over the packed bed (Δp) will during runbe equal to actualmeasured pressure - total systempressure(P1).

Repeat the procedure each time the parameters are changed.Note:

26 28-4082-02 AC

8 MaintenanceRegeneration

For best performance, wash bound substances from the columnafter each chromatographic cycle (see Table 5).

Table 5. Recommended conditions for column regenerationRe-equilibrationwith starting buffer(ml)

Volume (ml)WashColumn

110-160 (2-3 CV)110 (2 CV)WaterHiPrep 26/10 Sepharose 6FF

10-15 (2-3 CV)10 (2 CV)WaterHiTrap PlasmidSelect Xtra10-15 (2-3 CV)10 (2 CV)2 M NaClHiTrap Source 30Q

To prevent the slow build up of contaminants on the column overtime, more rigorous cleaning protocols may be needed on a regularbasis.

Cleaning-in-Place (CIP)CIP is the removal of very tightly bound, precipitated or denaturedsubstances generated in previous purification cycles. If suchcontaminants accumulate on the column, they may affect itschromatographic properties.

Use the following CIP protocols as a guide when formulating acleaning protocol specific for the raw material used. The frequencyof CIP will depend on the amount of raw material applied to thecolumn. Nevertheless, we recommend CIP after every cycle whenpurifying plasmids from clarified alkaline cell lysate.

28-4082-02 AC 27

HiPrep 26/10 Sepharose 6 FF

ActionStep

Wash with at least 110 to 160 ml (2 to 3 CV) of water at aflow rate of 2.7 to 5.3 ml/min (30 to 60 cm/h).

1

Wash with 110 to 160 ml (2 to 3 CV) of 0.5 M NaOH at a lowflow rate of 0.4 to 1.3 ml/min (5 to 15 cm/h).

2

Incubate the column for at least 15 min.3

Wash with at least 110 to 160 ml (2 to 3 CV) of water at aflow rate of 2.7 to 5.3 ml/min (30 to 60 cm/h).

4

Re-equilibrate the column with 110 to 160 ml (2 to 3 CV) ofBuffer A.

5


ActionStep

Wash with at least 10 to 15 ml (2 to 3 CV) of water at a flowrate of 4 ml/min (120 cm/h).

1

Wash with 10 to 15 ml (2 to 3 CV) of 0.5 M NaOH at a lowflow rate of 1.3 ml/min (40 cm/h).

2



4

Re-equilibrate the column with 10 to 15 ml (2 to 3 CV) ofBuffer B.

5

28 28-4082-02 AC

HiTrap SOURCE 30Q

ActionStep


1

Wash with 10 to 15 ml (2 to 3 CV) of 0.5 M NaOH at a lowflow rate of 1.3 ml/min (40 cm/h).

2

Wash with 10 ml (2 CV) of 2.0 M NaCl at a low flow rate of1.3 ml/min (40 cm/h).

3



5

Re-equilibrate the column with 10 to 15 ml (2 to 3 CV) ofBuffer D.

6

SanitizationSanitization is the reduction ofmicrobial contamination in the columnand related equipment to an acceptable minimum. You need todesign a specific sanitization protocol for each process accordingto the type of contaminants present and local regulatory demands.We recommendwashing the columnwith 0.5MNaOHwith a contacttime of 30 to 60 min.

StorageStore media and columns at 4°C to 30°C in 20% ethanol.

28-4082-02 AC 29

9 Ordering InformationCode No.QuantityProduct

28-4052-681 pack 1PlasmidSelect Xtra Starter Kit28-4024-0125 mlPlasmidSelect Xtra28-4024-02200 ml28-4024-031 l28-4024-045 l

1 Contains oneHiPrep 26/10 Sepharose 6 FF column, oneHiTrapPlasmidSelect Xtra column,one HiTrap Source 30Q column and accessories.

Code No.QuantityAccessories18-1112-5121/16" male/luer female

(For connection of syringe to top of HiTrap col-umn)

18-1003-682Tubing connector flangeless/M6 female(For connection of tubing to bottom of HiTrapcolumn)

18-1017-982Tubing connector flangeless/M6 male(For connection of tubing to top of HiTrap col-umn)

18-1112-576Union 1/16" female/M6 male(For connection to original FPLC System throughbottom of HiTrap column)

18-3858-015Union M6 female /1/16" male(For connection to original FPLC System throughtop of HiTrap column)

18-1027-122Union luerlock female/M6 female28-4010-818HiTrap/HiPrep, 1/16" male connector for ÄKTA11-0004-645Stop plug female, 1/16"

(For sealing bottom of HiTrap column)11-0003-555Fingertight stop plug, 1/16"

Code No.QuantityRelated media products17-0159-011 lSepharose 6 Fast Flow17-0159-0510 l17-1275-0150 mlSOURCE 30Q17-1275-02200 ml17-1275-031 l17-1275-045 l28-4052-691 pack 1PlasmidSelect Xtra Screening Kit

1 Contains five 5-ml HiTrap Sepharose HP columns, five 1-ml HiTrap PlasmidSelect Xtracolumns, and accessories.

Code No.Custom-packed columns90-1003-68Sepharose 6 Fast Flow XK 50/30

30 28-4082-02 AC

Code No.Custom-packed columns90-1003-80SOURCE 30Q XK 16/20

Code No.Empty pilot-scale columns and accessories18-8773-01XK 16/20 (16 mm i.d.)

Max. 30 ml bed volume or 15 cm bed height18-1000-72XK 26/20 (26 mm i.d.)

Max. 65 ml bed volume or 12.5 cm bed height18-8751-01XK 50/30 (50 mm i.d.)

Max. 550 ml bed volume or 28.5 cm bed height18-1102-02FineLINE Pilot 35

Max. 144 ml bed volume or 15 cm bed height18-1103-01BPG 100/500 (100 mm i.d.)

Max. 2 l bed volume or 26 cm bed height18-1153-44Packing Connector XK1618-1153-45Packing Connector XK 2618-8790-01XK 50 Reservoir18-1000-70XK 50 Fast Flow Kit18-3585-01Filter Kit HR 1618-1101-28Filter Kit 2618-1112-44Online Filter (50 and 100 ml/min system)18-1027-11Online Filter Kit (20, 50, and 100 ml/min systems)

Code No.DNA molecular weight markers27-4060-01DRIgest III (restriction enzyme digest of DNA, 25 μg)

Code No.Related literature18-1052-52Data File, Sepharose 6 Fast Flow18-1107-12Data File, SOURCE 30Q18-1115-23Data File, BPG 100, 140, 200, 30018-1060-59Data File, BPG 45018-1138-92Data File, Chromaflow18-1104-95Data File, FineLINE Pilot 3518-1130-00Data File, FineLINE 100/100L, 200/200L18-1167-76Data File, BioProcess LPLC columns18-1167-75Data File, BioProcess MPLC columns18-1168-09Data File, Hollow fiber Start AXM and Start AXH cross flow

cartridges for the ÄKTAcrossflow automated system18-1165-29Selection Handbook, Hollow fiber cartridges and systems for

membrane separations.

28-4082-02 AC 31

For local office contact information, visitwww.gelifesciences.com/contact

GE Healthcare UK LimitedAmersham PlaceLittle ChalfontBuckinghamshire, HP7 9NAUnited Kingdom

www.gelifesciences.com/protein-purification

GE and GE monogram are trademarks of General Electric Company.

ÄKTA, BioProcess, Chromaflow, FineLINE, HiPrep, HiTrap, MidGee, Sepharose, SOURCE, and Tricornare trademarks of are trademarks of General Electric Company or one of its subsidiaries.

© 2006-2014 General Electric Company – All rights reserved.First published May 2006

All goods and services are sold subject to the terms and conditions of sale of the company withinGE Healthcare which supplies them. A copy of these terms and conditions is available on request.Contact your local GE Healthcare representative for the most current information.

GE Healthcare Bio-Sciences ABBjörkgatan 30, 751 84 Uppsala, Sweden

GE Healthcare Europe GmbHMunzinger Strasse 5, D-79111 Freiburg, Germany

GE Healthcare Bio-Sciences Corp.800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA

GE Healthcare Japan CorporationSanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan

28-4082-02 AC 04/2014 a1762

instructions28-4082-02ac plasmidselectxtra

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