This poster describes the development of a high-throughput microchip-CZE assay for profiling the charge variants of therapeutic mAbs. Authors: Tobias Wheeler, Lucy Sun, Rajendra Singh, Bahram Fathollahi and Hans Pirard Affiliations: PerkinElmer (Caliper), Alameda, CA, USA For further information on the Microfluidics Technology (PerkinElmer) presented in this poster, please visit http://bit.ly/12j68ol

1. 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
Intermediate Assay Reproducibility  Relative areas of basic, main, and acidic variants
1 Abstract Conventional vs. Microfluidic Screening 4 Results  Independently labeled a mAb five times; nine repeat
are consistent across all concentrations
injections in two chips on two instruments (n = 90)  CVs : 1.7% (basic); 0.9% (main); 2.1% (acidic)
We have developed an automated, high-throughput  High-throughput screening methods are required to Charge Variant Profiles
support process development (and, potentially,  Relative amount = 100(Peak Area / Total Area)
microfluidic platform, the LabChip® GXII, that
quality control) for the production of mAbs  Comparison of charge profile of mAb samples SOFTWARE FEATURES
performs multiple protein characterization assays.
obtained with microchip-CZE to those obtained with Chip 1 / Instr. 1 Chip 2 / Instr. 2 Total
The current assays include (1) purity assessment  Typical analysis times of charge variant screening conventional CZE and iCE280 methods P3  Label expected peaks
Peak CV (%) CV (%) CV (%)
by microchip CE-SDS, and (2) profiling of N- methods: P1 2.1 2.2 2.5  Exclude peaks from analysis
glycans, both with an analysis time of < 60 s per Microchip-
IEC/CEX cIEF CZE P2 3.2 4.1 3.7  Determine relative amounts (%)
sample. More recently, we have developed a third CZE Microchip-CZE: 60 s P3 0.8 0.7 1.1 P4
10 – 90 min  Manual peak integration
assay: a high-throughput, microchip-CZE method Per Sample 15 min 10-30 min ~1 min P2
~13X Faster Separation P4 2.9 2.6 3.2 P1
for the characterization of mAb charge variants. In Per 96-Well Plate 16-144 hrs 24 hrs 16-24 hrs <2 hrs  Export to Empower
 Total coefficient of variance (CV) for relative amount
this method, mAbs with 7 < pI < 9.5 are of each peak is less than 4%
fluorescently labeled while conserving net charge.  Traditional methods are unable to meet the demand
This labeling is performed in a 96-well plate format for analysis of 100s of samples in a reasonable Limit of Detection
amount of time (< 8 hrs)
that is amenable to automation. Labeled sample is Acidic  Detect reliably down to ~1% of minimum
then drawn into a microchip through a capillary recommended sample concentration (1 mg/mL)
Basic
sipper and applied vacuum. Once in the microchip, Free Dye
the sample is electrokinetically injected and
3 Materials & Methods
separated in a microchannel that is dynamically
coated to suppress electroosmotic flow. Sufficient  Instrument: LabChip GXII
resolution of charge variants is achieved in < 68 s
0.45
Main Isoform - 8.810
0.40 iCE280
mAb 4
and the labeling and analysis of 96 samples
0.35
requires < 2 hours. This poster (1) describes the
0.30
separation method and the sample workflow, and Cs = 1.6mg/mL
0.25
(2) demonstrates the resolution, speed, sensitivity,
Absorbance
Analysis Time:
C = 1.26% Cs
0.20 ~15 min
reproducibility, and ease-of-use of the method, for Acidic
9.50 pI
C = 0.41% Cs
0.15
the high-throughput screening of mAb charge
8.712
LabChip GXII Microchip Basic
5
6.14 pI
variants.
0.10
Conclusions
8.948
8.585
9.044
 Sample sipped from 96-well plate
0.05
Developed a high-throughput microchip-CZE
0.00
 Washing/conditioning between samples not required
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
Variation in Relative Amounts
2 Introduction  Labeling reaction: 10 min at room temperature
pI
 10, 5, 2, and 1 mg/mL of mAb assay for profiling the charge variants of
 Input concentration: 2 mg/ml is optimal, 0.5-10 mg/mL is therapeutic mAbs
allowed Conventional CZE: 15 min
 Charge variants screening: Key assay for the
evaluation of therapeutic mAb production (capillary wash between Key features of the assay:
samples is not factored into
processes, at various stages of development
 Workflow: time)
 Anticipated applicability in: (1) Clone selection, (2)  Speed: < 2 hrs / 96 samples (includes
Cell culture optimization, (3) Purification 1) Sample Labeling: Acidic sample preparation)
optimization, (4) BioProcess scale-up, (5)
Basic
Formulation optimization, (6) Manufacturing 5 µL
Dye Mixture: 5 µL  Resolution: comparable to or better than
5 µL
optimization 25 µL
Labeling
Dye + 145 µL
Dye Mixture conventional CZE
Sample DMF
Buffer 60 µL H2O
 Charge variant profile can be obtained using
capillary zone electrophoresis (CZE) – separation  Reproducibility: %CV ~1 % for main peak
based primarily on charge (no sieving matrix):
and < 4% for variant peaks
96-well plate ∆t = 10 min.
Enough for 24
samples
T = Troom
 Shape of profile is maintained across all  Sensitivity: detection down to 1% of
concentrations minimum input
2) Chip Prep:
 Mix the pH 5.9 and pH 7.4 Running  Automatability: 96-well plate format
Buffers to the desired pH for optimal
resolution
 Add 75 µL of the Running Buffer
 Assay launch: July 23, 2012
mixture to wells 3, 4, 7, 8, & 10 of the
Chip
 Charge variants with low pI (Acidic variants)
 Add 750 µL of the Running Buffer to
migrate slower than variants with high pI (Basic
Buffer Tube
variants)
 Dynamic coating is used to suppress electro- 3) Run Assay:
osmotic flow within microchannel, to improve ACKNOWLEDGEMENTS
 Microchip-CZE achieves similar resolution between We thank Dr. Yan He and Dr. Nathan Lacher for
separation of charge variants Place chip, plate, and buffer tube in instrument
and run assay charge variants and reduces analysis time to ~1 providing mAb samples and feedback
minute per sample
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com