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
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