5. SOURCE................................................................................................34
Properties ......................................................................................37
Chemical stability ....................................................................37
Flow rate..................................................................................38
Capacity ..................................................................................40
Recovery..................................................................................40
Reproducibility ........................................................................41
Availability ....................................................................................41
6. Sepharose based ion exchangers.......................................................42
Chemical stability ..........................................................................42
Physical stability ............................................................................43
Sepharose High Performance ion exchangers.........................43
Properties ......................................................................................44
Physical stability ......................................................................44
Capacity ..................................................................................44
Flow rate..................................................................................46
Availability ....................................................................................46
Sepharose Fast Flow ion exchangers ........................................46
Properties ......................................................................................47
Physical stability ......................................................................47
Capacity ..................................................................................47
Flow rate..................................................................................49
Availability ....................................................................................50
Sepharose Big Beads ion exchangers.........................................50
Properties ......................................................................................51
Physical properties ...................................................................51
Capacity ..................................................................................51
Flow rate..................................................................................51
Availability ....................................................................................51
STREAMLINE SP and STREAMLINE DEAE ......................52
Properties ......................................................................................53
Physical stability ......................................................................53
Capacity ..................................................................................53
Availability ....................................................................................54
DEAE Sepharose CL-6B and CM Sepharose CL-6B.............54
Properties ......................................................................................54
Physical stability ......................................................................54
Capacity ..................................................................................55
Flow rate..................................................................................56
Availability ....................................................................................57
7. DEAE Sephacel.....................................................................................58
Properties ......................................................................................58
Chemical stability ....................................................................58
Physical stability ......................................................................59
Capacity ..................................................................................59
Flow rate..................................................................................60
Availability ....................................................................................60
8. Sephadex ion exchangers ...................................................................61
Properties ......................................................................................61
Chemical stability ....................................................................61
Physical stability ............................................................................62
Swelling ...................................................................................62
Ionic strength dependence........................................................62
pH dependence ........................................................................62
Capacity ..................................................................................62
Availability ....................................................................................64
9. Experimental design ............................................................................65
Choice of ion exchanger ..............................................................65
Specific requirements of the application .........................................65
Column separation, batch separation or...................................65
expanded bed adsorption
The scale of the separation .......................................................65
The required resolution............................................................65
The required throughput..........................................................66
Scaleability...............................................................................66
Reproducibility ........................................................................67
Economy .................................................................................67
The molecular size of the sample components ................................67
Choice of exchanger group ............................................................68
Determination of starting conditions .......................................69
The isoelectric point .................................................................69
Test-tube method for selecting starting pH ...............................69
Electrophoretic titration curves (ETC) .....................................70
Chromatographic titration curves (retention maps) ..................74
Choice between strong and weak ion exchangers ...........................76
Choice of buffer.............................................................................76
Choice of buffer pH and ionic strength.....................................76
Choice of buffer substance .......................................................77
Test-tube method for selecting starting ionic strengths..............79
10. Experimental Technique.....................................................................80
Column chromatography............................................................80
Choice of column...........................................................................80
Column design.........................................................................80
Column dimensions .................................................................81
Quantity of ion exchanger .............................................................81
Preparation of the ion exchanger ...................................................81
Pre-swollen ion exchangers ......................................................81
Pre-packed ion exchange media ...............................................81
Sephadex ion exchangers .........................................................82
Alternative counter-ions ...........................................................82
Decantation of fines.................................................................82
Packing the column........................................................................82
Column Packing Video Film.....................................................82
Checking the packing...............................................................83
Equilibrating the bed ...............................................................84
Sample preparation........................................................................85
Sample concentration...............................................................85
Sample composition.................................................................85
Sample volume.........................................................................85
Sample viscosity .......................................................................85
Sample preparation ..................................................................86
Sample application ........................................................................87
Sample application with an adaptor .........................................87
Other methods of sample application.......................................89
Sample application onto a drained bed .....................................89
Sample application under the eluent .........................................89
Elution ..........................................................................................90
Change of pH ..........................................................................90
Change of ionic strength ..........................................................91
Gradient direction....................................................................91
Choice of gradient type ............................................................91
Resolution using a continuous gradient ....................................93
Choice of gradient shape ..........................................................94
Sample displacement ................................................................95
Gradient generation.......................................................................96
Gradient formation with two pumps or a single pump .............96
in combination with a switch valve
Gradient Mixer........................................................................96
Batch separation............................................................................97
Expanded bed adsorption ...........................................................98
Expanded bed technology ..............................................................99
Basic principle of operation............................................................99
STREAMLINE adsorbents ..........................................................100
STREAMLINE columns ..............................................................100
Auxiliary equipment ....................................................................100
Regeneration ................................................................................101
Cleaning, sanitization and sterilization procedures ............101
Cleaning ......................................................................................101
Sanitization .................................................................................101
Sterilization .................................................................................101
Protocols for cleaning-in-place (CIP),...........................................102
sanitization and sterilization
SOURCE and Sepharose Based ion exchangers......................102
MonoBeads and MiniBeads columns......................................102
DEAE Sephacel and Sephadex based ion exchangers..............103
Storage of gels and columns......................................................103
Prevention of microbial growth..............................................103
Storage of unused media ........................................................104
Storage of used media ............................................................104
Storage of packed columns.....................................................104
Determination of the available and dynamic capacities.....104
Calculation ............................................................................106
11. Process considerations .....................................................................107
Defining the purpose ..................................................................108
The strategic focus ......................................................................109
Capture .......................................................................................109
Intermediate purification .............................................................111
Polishing......................................................................................111
Selection of chromatography media .......................................112
Base matrix properies and derivitization chemistry.................113
Bead size ................................................................................113
Documentation and technical support....................................113
Regulatory support ................................................................113
Vendor certification ...............................................................114
Delivery capacity ...................................................................114
Method design and optimization ............................................114
Binding conditions.......................................................................114
Elution ........................................................................................115
Sample load .................................................................................116
Flow rate .....................................................................................117
Selecting a column ......................................................................118
Aspects of column design .............................................................119
Flow distribution system ........................................................119
Material resistance and durability ..........................................119
Sanitary design.......................................................................119
Pressure vessel safety..............................................................120
Regulatory support ................................................................120
Ergonomics............................................................................120
Packing large scale columns......................................................120
Column configuration .................................................................120
Packing the column......................................................................121
Scale-up .........................................................................................121
12. Applications ........................................................................................124
The design of a biochemical separation ........................................124
Application examples ..................................................................127
Enzymes ................................................................................127
Isoenzymes ............................................................................128
Immunoglobulins...................................................................129
Nucleic acid separation ..........................................................129
Polypeptides and polynucleotides...........................................130
Antisense phosphorothioate oligonucleotides.........................132
Areas of application.....................................................................133
Purification of a recombinant Pseudomonas ...............................136
aeruginosa exotoxin A, PE553D
Strategy..................................................................................141
13. Fault-finding chart .............................................................................143
14. Ordering information .......................................................................151
15. References............................................................................................155
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