PhyNexus tip concentrating effect
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CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip® Column Concentration Effect
PhyNexus, Inc.
CAPTURE PURIFY ENRICH™ PhyNexus
Why are high concentrations of purified
proteins needed?
• Low concentration protein samples are not stable
– Loss of protein mass (plate out and precipitate)
– Loss of protein activity (denature, unfold)
• Centrifuge concentrator membrane filters is the most common way
to increase protein concentration but time consuming and
substantial mass and activity cannot be recovered
• Assays require high protein concentration
• Accurate quantification (protein mass and concentration) requires
higher concentration
CAPTURE PURIFY ENRICH™ PhyNexus
High concentration recovery is needed
from small sample volumes
• High throughput expression: 96 well format
• Plates for 1-96 and 1-12 at-a-time robots: 96 well format
• Sample volumes 0.2 – 2.2 mL
• Applications:
– Process monitoring
– Assay screening
– Resin and condition screening (optimum recovery, purity and activity)
– Etc. (any established process that needs miniaturization)
CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip Columns
4x
Capture
Pro Pro
Pro Pro
Pro Pro
Pro
Pro
Pro
Target Protein
Contaminants
Buffer
Pro
CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip Columns
4x
Purify
Pro
Pro Pro Pro
Pro
Pro Pro
Pro
Pro
Target Protein
Contaminants
Wash Buffer
Pro
CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip Columns
4x
Enrich
Pro
Pro Pro
Pro
Pro Pro Pro
Pro
Pro
Target Protein
Elution Buffer
Pro
CAPTURE PURIFY ENRICH™ PhyNexus
Why back and forth flow?
• The affinity interaction equilibrium reaction is slow.
RESIN ProA + Antibody RESINProAAntibody
• Single pass columns (spin columns) don’t have enough time to
interact.
• Diffusion (magnetic beads, bulk process) takes time
• Back and forth flow type active transport solves both issues:
– Allows enough contact time until the equilibrium reaction is
completed
– Therefore conditions used or developed are scaleable to any
size column
– Enables the PhyTip® Column Concentration Effect
CAPTURE PURIFY ENRICH™ PhyNexus
Antibody purification
20 µL ProA PhyTip 200+ column vs. spin column
Pierce Spin Column 20 mL PhyTIp Column
A LY S FT E FT E FT E FT E FT E FT E
L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Vol.
(uL) mAU2 [ ]
(mg/mL) Mass
(ug) %
rec.
Pierce-1 434 190.3 0.08 36 71
Pierce-2 440 170.5 0.07 30 59
Pierce-3 441 163.6 0.06 27 55
200+ 20uL-1 74 690.4 0.46 35 69
200+ 20uL-2 69 704.2 0.47 33 66
200+ 20uL-3 72 762.2 0.52 38 75
HPLC data
= ladder
= hIgG
= e. coli lysate
= A + LY
= flow through
= elution
L
A
LY
S
FT
E
CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip® Column Concentration Effect
• What happens when you decrease the column size to 5 µL from 20
µL?
• The PhyTip Column Concentration Effect becomes stronger
• The Concentration Effect is counter intuitive:
– 5 µL bed very small column bed but will give very high sample
concentrations
– To increase the concentration of recovered protein, use a
smaller column
CAPTURE PURIFY ENRICH™ PhyNexus
Antibody recovery from ProA Spin column and 20 µL PhyTip® column
vs. 5 µL ProA PhyTip ® column
HPLC data
Vol. (uL) mAU2 [ ] (mg/mL) Mass (ug) % rec.
Pierce-1 434 190.3 0.08 36 71
Pierce-2 440 170.5 0.07 30 59
Pierce-3 441 163.6 0.06 27 55
200+ 20uL-1 74 690.4 0.46 35 69
200+ 20uL-2 69 704.2 0.47 33 66
200+ 20uL-3 72 762.2 0.52 38 75
200+ 5uL-1 15 272.2 1.45 22 45
200+ 5uL-2 16 259.2 1.35 22 45
200+ 5uL-3 17 273.2 1.45 25 50
CAPTURE PURIFY ENRICH™ PhyNexus
Pierce Spin Column 20 mL PhyTIp Column 5 mL PhyTIp Column
A LY S FT E FT E FT E FT E FT E FT E FT E FT E FT E
L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 L
Antibody recovery from ProA spin column and 20 µL PhyTip column
vs. 5 µL ProA PhyTip column
gel data
= ladder
= hIgG
= e. coli lysate
= A + LY
= flow through
= elution
L
A
LY
S
FT
E
CAPTURE PURIFY ENRICH™ PhyNexus
Why does this happen? How does it work?
• Compared to spin columns, PhyTip® pipette tip columns have
– Smaller elution volumes
– Higher bed loading
• The bed loading increases as the column bed size is decreased
• For the same ratio of elution volume as the bed size is decreased,
the concentration of recovered protein increases
CAPTURE PURIFY ENRICH™ PhyNexus
The PhyTip® Column Concentration Effect
• As the bed size is decreased, the concentration of recovered protein
increases
• Works with any “on/off type” high selectivity resin
– ProA/ProG
– IMAC
– Streptavidin
– Ion exchange
– Ion pairing / reverse phase
– Any column/analyte chemistry where the isotherms are sharp i.e. they change
rapidly and completely with changes in solvent composition
• Match bed size to sample concentration and volume to load up the
bed