Protein PROProtein PRO Parallel Capillary Electrophoresis Parallel Capillary Electrophoresis System OverviewSystem Overview

Advanced Analytical Technologies, Inc.

Ames, IA USA

OutlineOutline

• Overview of Protein PRO System

• Protein Sizing and IgG Purity Analysis

• Parallel Capillary Zone Electrophoresis (CZE) on the Protein PRO

• Summary

Protein PROProtein PRO System System

• 24-Capillary protein electrophoresis separation and quantification system

• Superior resolution, quantification and automation vs. traditional slab gel methods

• Direct on-line UV absorbance detection of protein with no labeling required

• Proprietary capillary conditioning solution paired with gel sieving matrix

• Pressure of up to 300 psi can be applied to capillary array

• System is open and flexible – can vary capillary i.d., effective length, # of capillaries or separation matrices/methods

• Key protein applications: Sizing and Purity by CGE, Charge Heterogeneity by CZE

The Protein PRO blends the high resolution and automation of CE with the parallel sampling capabilities of SDS-PAGE

• 24 capillaries are arranged in a linear array at detection window

• UV light is passed through capillary array and imaged onto photodiode array detector

• Capillary inlets are arranged 2 x 12 for direct sample injection from 96-well plates or 12-well PCR tube strips

• Capillary outlets are bundled to a common reservoir connected to pumping system

• Sample injection by vacuum or voltage

• 24 individual CE separations are performed in parallel

Multiplexed (Parallel) CE-UV TechnologyMultiplexed (Parallel) CE-UV Technology

Protein PROProtein PRO:: 24-Capillary Array24-Capillary Array

• Capillaries are spread apart throughout entire effective length

• Outlet reservoir, ground electrode permanently housed in system

Protein PROProtein PRO:: 24-Capillary Array Optical Alignment24-Capillary Array Optical Alignment

• Continuous measurement of UV light intensity simultaneously in 24 individual capillaries; software automatically assigns and tracks each capillary of array

• Absolute light intensity does not have to be equal as the relative absorbance is measured in each capillary

24-Capillary Array System Configuration24-Capillary Array System Configuration

High Pressure Pump

HV Power Supply

Capillary Array Outlet Reservoir (8 mL)

Optics and PDA Detector

UV Lamp

Protein PROProtein PRO: AATI Protein Gel Matrix: AATI Protein Gel Matrix

• Proprietary gel matrix for performing high resolution protein sizing by CE

• Uses bare fused silica capillaries and pre-rinse with capillary conditioning solution

• Relatively low gel viscosity allows for efficient capillary filling and vacuum sample injection

• Low UV background absorbance provides sensitive on-line detection at 214 nm

• Relatively low current reduces bubble generation

• Gel and conditioning solution can be utilized in single channel CE instruments as well as in Protein PRO parallel CE system

• Initial testing with customer demonstrated improved peak shape and reduced tailing for challenging mAb samples as compared to existing single capillary gel and method

Capillary Array:

• 24 capillaries; 75 µm i.d./200 µm o.d.; 33 cm eff/48 cm total

New Array Conditioning:

• 1M NaOH (-2 psi 10 min, hold 20 min), water (300 psi, 5 min), conditioning solution (300 psi, 10 min), protein gel buffer (300 psi, 10 min)

Between Run Conditioning:

• Empty/fill with protein gel @ 300 psi, 10 min

CGE Experimental conditions:

• Pre-run : -4.5 kV 4 min

• Injection : -0.3 psi 60-120s and hold for 60s (vacuum) or -5 kV 30-60s (ek)

• Separation : -10 kV to -12 kV

Protein PROProtein PRO: Separation Conditions: Separation Conditions

Protein PROProtein PRO: Sample Preparation Conditions: Sample Preparation Conditions

Non-Reduced CE-SDS:• Prepare 250 mM Iodoacetamide (IAM) alkylating reagent stock solution

• Add 10X sample buffer to protein/IgG sample along with IAM and protein markers (optional); load 40 uL solution into wells of 96-well sample plate

• Incubate at 70ºC for 10 min; cool to RT for 3 min

• Place into Protein PRO instrument for analysis

Reduced CE-SDS:• Add 10X sample buffer to protein/IgG sample along with protein markers (optional)

and 2-Mercaptoethanol (BME) or similar reducing agent

• Load 40 uL solution into wells of 96-well sample plate

• Incubate at 70ºC for 10 min; cool to RT for 3 min

• Place into Protein PRO instrument for analysis

Protein PROProtein PRO: Protein Sizing Standard Reduced Separation: Protein Sizing Standard Reduced Separation

200.0Myosin

116.0β-Galactosidase

97.0Phosphorylase b

66.0Albumin

45.0Ovalbumin

29.0Carbonic Anhydrase

20.0Trypsin inhibitor

14.2α-Lactalbumin

8.6Ubiquitin

Molecular weight (kD)Protein

200.0Myosin

116.0β-Galactosidase

97.0Phosphorylase b

66.0Albumin

45.0Ovalbumin

29.0Carbonic Anhydrase

20.0Trypsin inhibitor

14.2α-Lactalbumin

8.6Ubiquitin

Molecular weight (kD)Protein

• Run time for reduced 200 kDa protein < 40 min

• Raw Data

• After normalization to 8.6 kDa and 200 kDa markers

Protein PROProtein PRO: Protein Sizing Standard Reduced Separation: Protein Sizing Standard Reduced Separation

Maximum Sample Throughput: Up to 24 different samples in a single CE run

Sizing Range: 9 kDa – 200 kDa (other markers may be substituted to adjust sizing range)

Dynamic Range: 2-2000 ng/l Carbonic Anhydrase

Sensitivity (S/N > 3): 2 ng/l Carbonic Anhydrase

Sizing Accuracy: Carbonic Anhydrase +/- 3%

Sizing Reproducibility: Carbonic Anhydrase < 2% CV

Quantitation Reproducibility: CA 14%CV; whole standard series 20%CV (relative concentration to upper marker)

Detection Wavelength: 214 nm

Run Time: Typical run time 40 min to separate 200 kDa protein

Sample Volume Required: Typical 50 L/sample well (minimum 20 L/well)

Injection Modes: Vacuum or electrokinetic

Protein PROProtein PRO: Protein Sizing Specifications: Protein Sizing Specifications

y = 0.0169x + 0.0201

R2 = 0.9999

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

0 200 400 600 800 1000

Concentration of Lysozyme (ng/ul)

Pe

ak

Are

a R

ati

o (

10

0 n

g/u

l UB

IS)

Protein PROProtein PRO: Lysozyme Calibration Curve: Lysozyme Calibration Curve

100 ng/ul UB IS

500 ng/ul

200 ng/ul

100 ng/ul50 ng/ul

20 ng/ul

5 ng/ul2 ng/ul

1 ng/ul• Relative to 8.6 kDa ubiquitin

internal standard (100 ng/l)

Protein PROProtein PRO: α-Lactalbumin and CAII Calibration Curves: α-Lactalbumin and CAII Calibration Curves-Lactalbumin 2.0 – 2000 ng/L

CAII 2.0 – 2000 ng/L

• Phosphorylase B (97 kDa) used as internal marker• Dynamic range corresponds to 0.1% level detection

PB IS

UB IS

LCAII

1000

500

200

100

50

20105210.5

y = 0.0068x

R2 = 0.9932

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.0 500.0 1000.0 1500.0 2000.0

Carbonic Anhydrase Concentration (ng/ul)

Rel

ativ

e C

orr

ecte

d P

eak

Are

a

y = 0.0097x

R2 = 0.9917

0.02.04.06.08.0

10.012.014.016.018.020.0

0.0 500.0 1000.0 1500.0 2000.0

-Lactalbumin Concentration (ng/ul)

Rel

ativ

e C

orr

ecte

d P

eak

Are

a

Protein PROProtein PRO: IgG Purity/Heterogeneity: IgG Purity/Heterogeneity

• Sample: Beckman IgG Control Standard, Reduced Conditions

LC

NGHC

HCIS

• Sample: 1 mg/mL Beckman IgG Control Standard, Reduced Conditions

• Non-glycosylated heavy chain can be resolved from heavy IgG chain, allowing for evaluation of % glycosylation

• Direct UV detection of protein – exact same method as “gold standard” single capillary method – with > 20X increase in throughput

Protein PROProtein PRO: Reduced IgG Separation: Reduced IgG Separation

NGHC

HC

LC

Reduced IgG Separation using Protigel MatrixReduced IgG Separation using Protigel Matrix

• Sample: 1 mg/mL Beckman IgG Control Standard, Reduced Conditions

• Six consecutive run profile on Beckman MDQ instrument using AATI Protigel CE-SDS separation matrix and method

• Kit is available for single capillary CE systems

Protein PROProtein PRO: Non-Reduced IgG with 10 kDa Marker: Non-Reduced IgG with 10 kDa Marker

10 kD

Intact IgG (~150 kDa)

HCLC

• Some light chain/heavy chain as well as other lower MW impurities are observed

• Both reduced and non-reduced analysis can be performed in parallel in different capillaries during a single experiment

Customer Reduced IgG Sample with 10 kDa MarkerCustomer Reduced IgG Sample with 10 kDa Marker

10 kD

LC

HC

NGHC

• Result was comparable to customer’s existing single capillary CGE method

Customer Non-Reduced IgG Sample with 10 kDa MarkerCustomer Non-Reduced IgG Sample with 10 kDa Marker

10 kD

Intact IgG

HCLC

• Result showed slightly improved resolution compared to customer’s existing single capillary CGE method

Standard Protein Mixture: 50 um i.d. CapillariesStandard Protein Mixture: 50 um i.d. Capillaries

200.0Myosin

116.0β-Galactosidase

97.0Phosphorylase b

66.0Albumin

45.0Ovalbumin

29.0Carbonic Anhydrase

20.0Trypsin inhibitor

14.2α-Lactalbumin

8.6Ubiquitin

Molecular weight (kD)Protein

200.0Myosin

116.0β-Galactosidase

97.0Phosphorylase b

66.0Albumin

45.0Ovalbumin

29.0Carbonic Anhydrase

20.0Trypsin inhibitor

14.2α-Lactalbumin

8.6Ubiquitin

Molecular weight (kD)Protein

•System can be easily switched from 75 um to 50 um i.d. capillary array to optimize or adjust methods

•50 um i.d. capillaries may further increase separation resolution, improve peak shape

Parallel CZE-UV: Small Molecule MixtureParallel CZE-UV: Small Molecule Mixture

Sample:

2 Pyridinium Cations, DMSO, 2 Benzene Sulfonic Acid Anions

Capillary Array:

50 um i.d.; 55 cm EFF, 80 cm TOT; 24-capillary

BGE:

10 mM Sodium Tetraborate pH 9.2

CZE Conditions:

-0.5 psi, 20 sec inj+12 kV (150 V/cm)214 nm UV detection

Parallel CZE: Migration Time and Peak Area Parallel CZE: Migration Time and Peak Area ReproducibilityReproducibility

•Reproducible, high throughput CZE methods can be developed on same platform used for CGE methods

Normalized Corrected Peak Area (Peak 5 vs. Peak 4)

Normalized Migration Time (Peak 5 vs. Peak 4)

• Parallel CE-UV technology utilized with the Protein PRO system can be applied to a broad range of high throughput bioanalytical applications

• Parallel CE-UV provides many benefits:

Significantly increased sample throughput Improved laboratory efficiency Lower turnaround times Decreased reagent and sample consumption Reduction in labor and operational/maintenance costs ONLY technology providing high throughput CGE and CZE capabilities

• Many methods previously developed for single capillary CE instruments can be successfully transferred to a parallel format

• The parallel CE configuration format provides an open, flexible format to vary capillary length, i.d., # capillaries, internal markers and/or separation conditions to adjust resolution or accommodate different applications as needed

Key Benefits and SummaryKey Benefits and Summary

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