A High Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation

Scott Fulton BioSystem Development, LLC Madison, WI USA www.biosystemdevelopment.com

Protein Sample Processing Bottleneck

Manufacturing process

Biological system

Clinical patient

Samples

Mass spectrometer

HPLC

Plate reader

Instruments

AssayMAP® Cartridges

Two Operating Modes

• Fully quantitative binding & elution

• Compatible with microplate systems

• Uses standard liquid handling

• Limited flow control & resin options

• No special equipment (lab centrifuge)

• Fully quantitative binding & elution

• Compatible with microplate systems

• Precise, bidirectional flow control

• All resins & applications

• Special equipment required

Spin Format Probe Syringe

Probe Syringe Liquid Handling

AssayMAP Bravo 96-Channel Workstation

96 probe syringe head

AssayMAP vs. Pipet Tip Columns

AssayMAP Cartridge Pipet Tip Column

• Single entry/exit port

• Multi-pass contact (equilibrium adsorption)

• Highly variable flow rate

• Air entrainment likely

• Non-quantitative extraction

• Separate entry & exit ports

• Single-pass contact (“true chromatography”)

• Controlled flow rate

• No air entrainment

• Fully quantitative binding & elution

Ab Purification on Protein A (PA-W)

PA-W: Effect of Flow Rate

Binding (Dynamic Capacity)

Elution

PA-W: Reproducibility & Precision

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 50 100 150 200 250

A2

80

µg hIgG Loaded

µg hIgG loaded 11 15 19 25 33 44 58 76 100 132 173 228

%CV 1.6 1.3 1.4 1.0 1.4 1.2 1.4 1.4 1.9 1.9 1.5 1.0

N = 8

100 µg load N = 96

%CV = 1.26

Streptavidin (SA-W) Cartridge

Ligand Immobilization

Antigen Purification

Specific Antibody Purification

Anti-FLAG Ab immobilized on SA-W Cartridges Washed 1X in PBS

Human IgG Purified from Rat Serum

• Camelid VHH domain ligands (14 kD, manufactured in yeast)

• Available ligands for antibodies

– Human Fc, all-species Fc

– Kappa, lambda light chain

– IgM, IgA, IgG4

• Ligands for plasma proteins, fusion proteins & biosimilars

20 – 0 µg hIgG spiked into PBS or rat serum, purified using the ligands shown

PBS PBS Rat Serum Rat Serum

Protein A CaptureSelect® hFc Ligand

Fluorescent Ligand Binding Assay

Neutralize

CaptureSelect hFc

Protein A

20 10 5 2.5 1.3 0.63 0.31 0.16 µg hIgG

AssayMAP ELISA vs. Microplate ELISA

Coat Block Sample Conjugate Substrate Read

Micron scale diffusion path

15 – 30 minutes turnaround

Millimeter scale diffusion path

4 – 24 hours turnaround

Microplate

AssayMAP IA

Leached Protein A ELISA

R² = 0.992

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10

A45

0

ng/mL Protein A

Run 1

Run 2

Microplate Format

ng/mL

Protein A Run 1 Run 2 Mean St Dev %CV

0 0.083 0.077

0 0.090 0.083 0.083 0.005 6.4

0.1 0.103 0.112

0.1 0.098 0.099 0.103 0.006 6.2

0.6 0.183 0.211

0.6 0.199 0.194 0.197 0.012 5.9

1.5 0.468 0.478

1.5 0.473 0.508 0.482 0.018 3.7

4 1.211 1.340

4 1.198 1.419 1.292 0.106 8.2

10 2.692 2.731

10 2.798 2.919 2.785 0.099 3.6

Reagents from commercial microplate format kit (Cygnus Technologies, Southport, NC)

Non-Porous Bead Technology

Non-Porous (N) Wide Pore (W)

Loading capacity ~1 µg ~100 µg

Elution volume 3 µL 20 µL

1000 fmol load

Peptide Mapping Analysis Workflow

• Integrated automation of purification, digestion & cleanup

• Rapid digestion with immobilized trypsin cartridge

• High throughput (up to 96 samples/run)

• Excellent reproducibility

Enzyme Digest Purify

Denature Reduce Alkylate

Peptide Cleanup LC/MS

Automated Tryptic Digestion

2 separate digestions run side-by-side

Protein: bSA Mass load: 125 µg Digestion volume: 50 µL Flow rate: 2 µL/min Digestion time: 25 min

Reversed-Phase Peptide Cleanup

Elution Profiles Dynamic Capacity

N-Glycan Profile Analysis

• Current method – Manual only

– 3 days to complete

– 10 - 30 samples/run

• AssayMAP method – Manual or automated

– No drying steps

– 3 hours to complete

– Up to 192 samples/run

GlykoPrep System Results

Peak

Average

% Area

% CV

(n = 96)

1 9.8% 3.2%

2 20.0% 1.2%

3 20.3% 2.5%

4 11.1% 3.4%

5 13.1% 1.6%

6 9.5% 2.3%

0

5

10

15

20

25

0 20 40 60 80 100

% o

f To

tal P

eak

Are

a

Sample

Reproducibility

12 consecutive runs α-1 Acid Glycoprotein

Comparison to Standard Methods

HILIC HPLC chromatograms with fluorescence detection

Parallel Micro-Chromatography

Cytochrome C/ribonuclease A on cation exchange resin

Columns 1 – 12 = 0 – 100 mM NaCl wash

Cation Exchange Gradient Scouting

0%

10%

20%

30%

40%

50%

0 100 200 300 400 500

% o

f Fe

ed

mM NaCl

pH 4.5

RNase A

Cytochrome C

0%

10%

20%

30%

40%

50%

0 100 200 300 400 500%

of

Fee

dmM NaCl

pH 5.0

RNase A

Cytochrome C

0%

10%

20%

30%

40%

50%

0 100 200 300 400 500

% o

f Fe

ed

mM NaCl

pH 5.5

RNase A

Cytochrome C

0%

10%

20%

30%

40%

50%

0 100 200 300 400 500

% o

f Fe

ed

mM NaCl

pH 6.0

RNase A

Cytochrome C

0%

10%

20%

30%

40%

50%

0 100 200 300 400 500

% o

f Fe

ed

mM NaCl

pH 6.5

RNase A

Cytochrome C

0%

10%

20%

30%

40%

50%

0 200 400 600

% o

f Fe

ed

mM NaCl

pH 7.0

RNase A

Cytochrome C

Equilibratecartridge

Loadsample Wash Elute

4.5

5.0

6.0

6.5

5.5

8.0

pH

(mM) NaCl

7.0

7.5

0 100 200 300 400 500

Purification Method Development

Conventional

AssayMAP

System Method Development Multi-Dimensional Separation

Second Column First Column

AssayMAP Cartridge Pipeline

• Affinity purification – Protein A

– Streptavidin (porous)

– Streptavidin (non-porous)

– Protein G, L, etc.

– BAC Vhh ligands

– IMAC

– Lectins

• Physical Purification – Cation exchange

– Anion exchange

– Reversed-phase

– Hydrophobic interaction

• Cleanup/Extraction – Reversed-phase

– Cation exchange

– Anion exchange

– Hydrophilic interaction (HILIC)

– Porous graphitic carbon

– Specialty phases (e.g. boronate, MIP)

• Enzymes – Trypsin

– Lys C, etc.

– Papain/FabRICATOR®

Summary of AssayMAP Bravo

• High throughput microchromatography platform

– 5 µL packed bed cartridge with any 15 – 100 µm particle resin

– 96-channel ANSI/SBS microplate format

– Single pass, fully quantitative binding & elution

– Positive displacement, bidirectional flow control

– Integrated workflows involving purification, quantitation & reaction

• Multiple applications in biopharmaceutical development

– Affinity purification or bind/elute assays (e.g. cell culture titer, PK studies)

– ELISA (e.g. process impurity or biomarker assays)

– Sample preparation for peptide mapping

– Sample preparation for glycan profiling

– Chromatographic method development