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