PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com

Abstract 1

Charge variants screening: Key assay for the evaluation of therapeutic mAb production processes, at various stages of development

Anticipated applicability in: (1) Clone selection, (2) Cell culture optimization, (3) Purification optimization, (4) BioProcess scale-up, (5) Formulation optimization, (6) Manufacturing optimization

Charge variant profile can be obtained using capillary zone electrophoresis (CZE) – separation based primarily on charge (no sieving matrix):

Charge variants with low pI (Acidic variants) migrate slower than variants with high pI (Basic variants)

Dynamic coating is used to suppress electro-osmotic flow within microchannel, to improve separation of charge variants

Relative areas of basic, main, and acidic variants are consistent across all concentrations

CVs : 1.7% (basic); 0.9% (main); 2.1% (acidic) SOFTWARE FEATURES

Label expected peaks

Exclude peaks from analysis

Determine relative amounts (%)

Manual peak integration

Export to Empower

Instrument: LabChip GXII

Sample sipped from 96-well plate

Washing/conditioning between samples not required

Labeling reaction: 10 min at room temperature

Input concentration: 2 mg/ml is optimal, 0.5-10 mg/mL is allowed

Workflow:

Conclusions

Developed a high-throughput microchip-CZE assay for profiling the charge variants of therapeutic mAbs Key features of the assay:

Speed: < 2 hrs / 96 samples (includes sample preparation)

Resolution: comparable to or better than conventional CZE

Reproducibility: %CV ~1 % for main peak and < 4% for variant peaks

Sensitivity: detection down to 1% of minimum input

Automatability: 96-well plate format

Assay launch: July 23, 2012

Introduction 2

5

Materials & Methods 3

High-Throughput Screening of mAb Charge Variants Using Microchip-CZE Authors: Tobias Wheeler, Lucy Sun, Rajendra Singh, Bahram Fathollahi and Hans Pirard

Caliper - A PerkinElmer Company, Alameda, CA, USA

Conventional vs. Microfluidic Screening

High-throughput screening methods are required to support process development (and, potentially, quality control) for the production of mAbs

Typical analysis times of charge variant screening methods:

Traditional methods are unable to meet the demand for analysis of 100s of samples in a reasonable amount of time (< 8 hrs)

IEC/CEX cIEF CZE Microchip-

CZE

Per Sample 10 – 90 min 15 min 10-30 min ~1 min

Per 96-Well Plate 16-144 hrs 24 hrs 16-24 hrs <2 hrs

Microchip

Mix the pH 5.9 and pH 7.4 Running Buffers to the desired pH for optimal resolution

Add 75 µL of the Running Buffer

mixture to wells 3, 4, 7, 8, & 10 of the Chip

Add 750 µL of the Running Buffer to Buffer Tube

Place chip, plate, and buffer tube in instrument

and run assay

2) Chip Prep:

3) Run Assay:

1) Sample Labeling:

Dye Mixture: 5 µL

Dye + 145 µL

DMF

96-well plate

∆t = 10 min.

T = Troom

60 µL H2O

LabChip GXII

Microchip-CZE achieves similar resolution between charge variants and reduces analysis time to ~1 minute per sample

Results 4

Charge Variant Profiles Comparison of charge profile of mAb samples

obtained with microchip-CZE to those obtained with conventional CZE and iCE280 methods

Intermediate Assay Reproducibility

Independently labeled a mAb five times; nine repeat injections in two chips on two instruments (n = 90)

Relative amount = 100(Peak Area / Total Area)

Total coefficient of variance (CV) for relative amount of each peak is less than 4%

Limit of Detection

Detect reliably down to ~1% of minimum recommended sample concentration (1 mg/mL)

Variation in Relative Amounts

10, 5, 2, and 1 mg/mL of mAb

Shape of profile is maintained across all

concentrations

ACKNOWLEDGEMENTS We thank Dr. Yan He and Dr. Nathan Lacher for providing mAb samples and feedback

Conventional CZE: 15 min

(capillary wash between

samples is not factored into

time)

Cs = 1.6mg/mL

C = 1.26% Cs

C = 0.41% Cs

We have developed an automated, high-throughput

microfluidic platform, the LabChip® GXII, that

performs multiple protein characterization assays.

The current assays include (1) purity assessment

by microchip CE-SDS, and (2) profiling of N-

glycans, both with an analysis time of < 60 s per

sample. More recently, we have developed a third

assay: a high-throughput, microchip-CZE method

for the characterization of mAb charge variants. In

this method, mAbs with 7 < pI < 9.5 are

fluorescently labeled while conserving net charge.

This labeling is performed in a 96-well plate format

that is amenable to automation. Labeled sample is

then drawn into a microchip through a capillary

sipper and applied vacuum. Once in the microchip,

the sample is electrokinetically injected and

separated in a microchannel that is dynamically

coated to suppress electroosmotic flow. Sufficient

resolution of charge variants is achieved in < 68 s

and the labeling and analysis of 96 samples

requires < 2 hours. This poster (1) describes the

separation method and the sample workflow, and

(2) demonstrates the resolution, speed, sensitivity,

reproducibility, and ease-of-use of the method, for

the high-throughput screening of mAb charge

variants.

Microchip-CZE: 60 s

~13X Faster Separation

Free Dye

25 µL

Sample

5 µL

Labeling

Buffer

Enough for 24

samples

5 µL

Dye Mixture

8.5

85

8.7

12

Main

Isofo

rm -

8.8

10

8.9

48

9.0

44

Absorb

ance

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

pI

6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60

mAb 4

6.14 pI

9.50 pI

iCE280

Analysis Time:

~15 min

Basic

Acidic

Chip 1 / Instr. 1 Chip 2 / Instr. 2 Total

Peak CV (%) CV (%) CV (%)

P1 2.1 2.2 2.5

P2 3.2 4.1 3.7

P3 0.8 0.7 1.1

P4 2.9 2.6 3.2 P1

P2

P3

P4

Basic

Acidic

Basic

Acidic