Chromatography

Question 1

DEAE

Answer 1

-diethyl-amino monolith
-anion exchange, uses buffers of increasing salt concentrations
-remove proteins, RNA, non-pDNA

Question 2

HLD

Answer 2

C4-HLD monolith. Contains butyl ligands to make hydrophobic surface. Affinity (hydrophobic interaction) chromatography, use buffers of decreasing salt concentration

Question 3

fluid control modes (5)

Answer 3

-pump mode - select pump-heads A/B
-pump out mode - control if fluid flows through system drain or rest of flowpath
-filter mode - controls if fluid flows through filter / system vent
-column mode - forward to flow through column, bypass to skip column
- outlet mode - select outlet for flowthrough

Question 4

4 things to monitor DEAE Loading

Answer 4

1. Pressure - Precolumn should reach ~3.5 bar or # cycles/loading values may need to be changed
2. no particulates
3. UV should be constant after initial increase
4. conductivity around 35-40 mS/cm

Question 5

3 things to monitor HIC loading

Answer 5

1. no particulates
2. Pre-column pressure should reach ##, otherwise may need to change # cycles/loading values
3. UV constant after initial increase

Question 6

2 things to monitor DEAE wash 2

Answer 6

1. Pressure - increases before RNA elutes and may spike and cause system to interlock
2. pre-column conductivity - should be around 58-62 mS/cm, higher than 63 mS/cm means plasmid could elute early

Question 7

DEAE buffers/phases

Answer 7

1. Equilibration - 40% SL017, 60% SL018; 7.5-8.5 pH, 58-62 mS/cm
2. Loading - 4.5-5.5 pH, 35-40 mS/cm
3. Wash 1 - SL018; 7.5-8.5 pH, 4-8 mS/cm
4. Wash 2 - 40% SL017, 60% SL018; 7.5-8.5 pH, 58-62 mS/cm
5. Elution - SL017; 7.5-8.5 pH, 85-95 mS/cm
6. Resin Strip - SL019/40
7. NaOH - 1M NaOH
8. WFI
9. Storage - SL020

Question 8

HLD

Answer 8

1. Equilibration - SL018/SL021
2. Loading
3. Wash 1 - SL018/SL021
4. Wash 2 - SL018/SL021
5. Elution - SL018/SL021
6. Resin Strip - SL018
7. NaOH - 1 M NaOH
8. WFI
9. Storage - SL020