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CAPTURE PURIFY ENRICH™ PhyNexus

Lee Hoang, Ph.D.

R&D Manager

PhyNexus, Inc. San Jose, CA

Automated, high throughput protein purification and sample prep using PhyTip® columns for therapeutics

leads screening and process development


PhyNexus, Inc.

• PhyNexus applies analytical principles to biological problems. •The PhyNexus team offers validated, accurate, and rugged protein sample preparation technology. The PhyTip® column technology can be scaled as needed.

Greek for nature, natural, life or living

Latin for linking or connecting


PhyTip® column design

Top screen

Bottom screen

Resin maintained in discrete area


PhyTip® column design

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

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


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


Antibody Purification


Small volume, high throughput, automated mAb purification and analysis


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 mg/mL is equivalent to manufacturer’s specifications • Resin performance is not compromised by miniaturization


PhyTip columns for small volume purification


PhyTip columns for process development

24 conditions (capture, wash, elution) tested for each of 4 clones Yield measured by ELISA, activity by SPR


Antibody purifications: robust, high performance, reproducible

Sample Conc. mg/mL %Recovery %CV Sample

Conc. mg/mL %Recovery %CV Sample

Conc. mg/mL %Recovery %CV Sample

Conc. mg/mL %Recovery %CV

1 0.653 84 1 0.373 82 2 0.368 79 2 0.349 80 1 0.667 86 1 0.385 84 2 0.399 86 2 0.360 83 1 0.641 83 1 0.389 85 2 0.407 88 2 0.347 80 1 0.651 84 3% 1 0.387 85 4% 2 0.394 85 4% 2 0.367 84 7% 1 0.618 80 1 0.399 87 2 0.380 82 2 0.344 79 1 0.643 83 1 0.407 89 2 0.405 87 2 0.335 77 1 0.694 89 1 0.400 88 2 0.401 86 2 0.352 81 1 0.692 89 1 0.360 79 2 0.357 77 2 0.341 78 1 0.647 83 1 0.373 82 2 0.379 82 2 0.353 81 1 0.643 83 1 0.399 87 2 0.403 87 2 0.354 81 1 0.643 83 1 0.388 85 2 0.410 88 2 0.272 62 1 0.650 84 1 0.394 86 2 0.390 84 2 0.320 73 3 0.405 88 3 0.320 69 4 0.413 86 4 0.347 80 3 0.422 92 3 0.323 70 4 0.349 73 4 0.411 94 3 0.454 99 3 0.337 73 4 0.399 83 4 0.369 85 3 0.439 96 10% 3 0.325 70 4% 4 0.402 84 6% 4 0.390 90 5% 3 0.442 96 3 0.326 70 4 0.389 81 4 0.393 90 3 0.429 94 3 0.336 73 4 0.389 81 4 0.409 94 3 0.431 94 3 0.353 76 4 0.390 81 4 0.380 87 3 0.418 91 3 0.344 74 4 0.389 81 4 0.374 86 3 0.442 96 3 0.338 73 4 0.383 80 4 0.401 92 3 0.379 83 3 0.354 76 4 0.351 73 4 0.393 90 3 0.386 84 3 0.325 70 4 0.339 71 4 0.409 94 3 0.304 66 3 0.321 69 4 0.380 79 4 0.422 97


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 Screening with PhyTip Columns

Target protein

• Screening commercially available resins to find high capacity and low background


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


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


IEX Chromatography


Ion Exchange Method Development

Cation exchange resin capture and release of lysozyme

Anion exchange release of BSA


Ion Exchange Scale-up

• 1mL PhyTip column containing 10 mL anion exchange media • 50 mM to 750 mM NaCl step gradient elution

• 5 mL FPLC anion exchange column • Shallow gradient combined with steps


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.


Glycan Mapping


Challenge of Glycan Mapping

Problem • Small differences in size • Similar • No UV/Vis absorbance

Solution • Label with fluorescent dye • HPLC or CE separation


Glycan Labeling Strategy


Prater et al., “Automated sample preparation facilitated by the PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins,” Analytical Biochemistry, (2007) 369(2), 202-9

Normal Phase Columns for enrichment of oligosaccharides from glycoproteins


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

# of

GFP

pos

itive

cel

ls


Target Discovery & Validation

Drug Screening

& Lead Optimization

Development Clinical Trials Manufacturing

Selected Current Customers: Raven Biotechnologies, Cell Signaling Inc, Pfizer, Astra Zeneca, Domantis, Target Discovery, Sareum

• Protein Interaction IP, Co-IP • Pathway Activation ChIP Phosphoprotein enrichment • Target Expression and Purification Screening • Biomarker Discovery • Biomarker Validation



Drug Screening

& Lead Optimization


• Library Synthesis • Library Screening and Selection • Assay Development Affinity tagged library screening • Lead Characterization Ab specificity, selectivity, affinity • Lead Optimization Affinity maturation • Structural Analysis Crystallization screening Selected Current Customers:

GSK, Merck, Amgen, Dyax, EMD, Wyeth, Genentech, CAT, Pfizer, Astra Zeneca



Drug Screening

& Lead Optimization


• Process Development • Protein Analytics • Pre-Clinical Assays

Selected Current Customers: Merck, Genentech, Inhibitex, FoldRX, Lonza, EMD, Talecris



Drug Screening

& Lead Optimization


• Immunogenicity

Selected Current Customers: Talecris



Drug Screening

& Lead Optimization


Selected Current Customers: Merck

• Manufacturing monitor and Q/C


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 as little as 30 minutes

• 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

Automated, high throughput protein purification and sample ... › wp-content › uploads › 2017...

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