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CAPTURE PURIFY ENRICH™ PhyNexus

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Anne Burke

Lee Hoang

Jo Walter


Lee Hoang, Ph.D.

R&D Manager

PhyNexus, Inc.

San Jose, CA

Innovative Solutions for Automated

Protein and Nucleic Acid Purification


Current small-scale sample preparation

have drawbacks


PhyTip® column design

Top screen

Bottom screen

Resin maintained in discrete area


PhyTip® column design

5 20 10 20 40 80 160 320 L 200 L 600 L

Traditional PhyTip Columns Gel Filtration PhyTip Columns


PhyTip Columns

4x

Capture

Pro Pro

Pro Pro

Pro Pro

Pro

Pro

Pro

Target Protein

Contaminants

Buffer

Pro


PhyTip Columns

4x

Purify

Pro

Pro Pro Pro

Pro

Pro Pro

Pro

Pro

Target Protein

Contaminants

Wash Buffer

Pro


PhyTip Columns

4x

Enrich

Pro

Pro Pro

Pro

Pro Pro Pro

Pro

Pro

Target Protein

Elution Buffer

Pro


Reciprocal flow, small resin bed size, and low dead volume

allow for efficient processing of small samples


Beckman FX

Caliper Sciclone & Zephyr

Perkin Elmer Minitrak

Tecan Freedom Evo Dynamic Devices Oasis

Hamilton STAR

PhyTip Columns – Compatible with Popular Liquid Handling Robots ®

Velocity 11 Bravo


MEA for fully automated, medium throughput sample processing

“Makes optimisation at small

scale feasible for multiple

conditions. Data is

reproducible at scale up

process. No limitations.”

Isabelle Green, AstraZeneca

"Easy liquid handling platform

that anyone can operate."

Jessika Feliciano, Merck &

Co.

http://www.selectscience.net/products/micro-extractor-automated-instrument-(mea)/?prodID=93919&u=AEDF1BCD-A529-4740-9812-01A5BC229369&techBID=


MEA Deck Positions

Chilled Positions R

eserv

oir

Plate Positions

Tip/PhyTip Column

Positions


MEA: Large volume, parallel processing

• Purify 1-12 samples at a time

• Each sample > 80 mL

• Recover 120 µL pure protein

• Yields in the milligram

range depending upon expression


MEA: Process Development


MEA: Process Development

• 12 types of resins in a box of 96

• 4 Capture conditions (optimize surfactant).

• 2 Wash conditions – vary pH of wash buffers

• 3 Elution conditions – step elution at 3 different salt concentrations

• 288 conditions to be tested

Row 1

elutions Row 2

elutions

Row 8

elutions Rows 1-8


Gel Filtration Chromatography


1 2 3 4 5 6 7

2 3 4 5 6 7

1

Gel Filtration Columns

Protein recovery and salt removal

Tips from left:

1. 600 µL column

2. 200 µL sample load

3-6. 4x100 µL chaser

7. 400 µL chaser

Tubes from left:

1. Starting sample

2. 200 µL fraction

3-6. 100 µL fractions

7. 400 µL fraction

• Routinely obtain 80% protein recovery &

remove >95% salt

• Separation of myoglobin (brown MW

16.7kD) from DNP-glutamate (yellow, MW

313D)

• Elution fractions collected in separate

wells


10K Gel Filtration Columns

Epitope Mapping

Digest Ab-Antigen

complex

Scanning peptide

library

Gel Filtration Separation –

• 500-10,000MW fractionation

Range

• Inject as little as 20 µL

MS analysis


Plasmid DNA Purification


Plasmid purification is difficult to automate


Solution: Process crude lysate

A B


Performance Data

Sample Yield Conc A260/A280 A260/A230

1 18.5 201 1.97 2.12

2 19.4 208 1.98 2.02

3 18.5 204 1.95 1.97

4 16.5 188 1.97 2.12

5 17.7 195 1.95 2.00

6 19.1 206 1.94 1.96

7 19.5 206 1.96 1.96

8 19.5 209 1.95 1.90

0

100

200

300

400

500

Fugene 6 Fugene HD TransIT LT1

Transfection Efficiency

96 hours – Reverse Transfection

# o

f G

FP

po

sitiv

e c

ells


Antibody Purification


PhyTip columns are scale-able

• Linear relationship between resin bed

volume and recovery

• Back-and-forth flow drives binding and

elution to equilibrium

• Measured dynamic binding capacity of

29 g/ L is equivalent to manufacturer’s

specifications

• Resin performance is not compromised

by miniaturization


PhyTip columns for small volume purification


His-tagged Protein Purification


Courtesy Vertex Pharmaceuticals

• Magnetic Beads, Spin Columns and PhyTip Columns

• PhyTip columns outperform competition through yield and concentration

Ni-IMAC purification platform comparisons


Ni-NTA IMAC Resin Wash Optimization

• E.coli culture lysed and cleared

• Spike with His-Rubredoxin, MW 7.5

kDa and purified on 20 µL IMAC

PhyTip columns

• All 8 samples first wash in 5 mM

imidazole

• Second wash in varying

concentrations of imidazole

• Note from 30 mM imidazole the high

MW contaminating band begins to be

removed

• Stringent washing did not affect the

concentration of eluted Rubredoxin

Contaminant

His-rubredoxin


IEX Chromatography


Marc D. Wenger et al., “An automated microscale chromatographic purification of virus-like particles as a strategy for

process development,” Biotechnol. Appl. Biochem, 2007.

Provide feedback on upstream process changes

Examples of Manufacturing and Q/C Monitoring: Merck


Merck Case Study: Comparison of lab-scale and PhyNexus-scale

purification systems

Marc D. Wenger et al., “An automated microscale chromatographic purification of virus-like particles as a strategy for

process development,” Biotechnol. Appl. Biochem, 2007.


Conclusions

PhyTip columns

• Miniaturization: Obtain information early in the process

• Maintain protein activity: Compatible with cell-based assays and screening

• Automation: Process 1 to 96 samples in a complete walk-away manner

• Flexible: Choose optimal column size and resin


Select Sub-set of Current Customers

http://www.gsk.com/index.htm

http://www.inhibitex.com/default.asp

http://www.dyax.com/about/index.html

http://www.dnaxresearch.com/index.html

http://www.lilly.com/index.html

http://www.criver.com/

http://www.wyeth.com/

http://www.organon-usa.com/

http://www.talecris.com/

http://www.astrazeneca.com/ncm.aspx


Thank you for joining the PhyNexus Webinar

Please contact us with any questions

Email:

JoanneWalter - Phynexus Europe

[email protected]

Anne Burke

Lee Hoang

Jo Walter

mailto:[email protected]

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