The Tip Concentrating Effect is a process that allows the recovery of high concentrations of proteins in a single purification procedure. PhyTip pipette tip columns from PhyNexus have been designed to produce very high concentrations of recovered proteins compared to competitive techniques such as spin columns, gravity columns, magnetic beads and FPLC. The Tip Concentrating Effect relies on the ability of PhyTip columns to capture protein on very small column bed volumes. Then proteins are recovered in a highly efficient and effective process. All conditions being equal, with any two different resin bed sizes of PhyTip columns, the smaller resin bed size will produce the highest concentration of recovered protein.

1. CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip® Column Concentration Effect
PhyNexus, Inc.

2. 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

3. 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)

4. CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip Columns
4x
Capture
Pro
Pro
ProPro
Pro
Pro
Pro
Pro
Pro
Target Protein
Contaminants
Buffer
Pro

5. CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip Columns
4x
Purify
Pro
Pro ProPro
Pro
ProPro
Pro
Pro
Target Protein
Contaminants
Wash Buffer
Pro

6. CAPTURE PURIFY ENRICH™ PhyNexus
PhyTip Columns
4x
Enrich
Pro
Pro Pro
Pro
ProProPro
Pro
Pro
Target Protein
Elution Buffer
Pro

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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