1

EVALUATION OF NEW TECHNOLOGY

FOR THE QUALITY CONTROL

LABORATORIES

JEFF SCHNEIDERHEINZE, PH.D.

• Background on Microchip CE (MCE)

• Extended Qualification and Robustness of MCE

• Troubleshooting Case Studies

OUTLINE

2

3

Technology Resolution Maximum

Throughput

Run Time Schematic Instrument Example Result

PAGE Low ~ 20

(Depends

on gel size)

3.5 hours

CE High 24 35 mins

MCE High 384 40 s per sample

(16 mins for 24

samples)

EVOLUTION OF ELECTROPHORESIS

4

MCE USING PERKINELMER LABCHIP® INSTRUMENTS

GXII GXII Touch

WHT_LabChip_GXII_Touch_Antibody_Analysis.pdf

Protein PICO Assay Reagent Kit

PRINCIPLE OF THE PICO MCE ASSAY

5

A fluorescent dye is chemically attached to free amines contained in the protein of interest. The labeled sample

is subsequently analyzed by MCE using fluorescence detection

Heat

LDS

Alkylating agent Dye

6

COMPARISON OF CE-SDS RESULTS TO MCE USING PICO

7

ADVANTAGES OF MCE GXII/GXII TOUCH INSTRUMENTS

• High Throughput

– Bottleneck becomes data processing rather than sample analysis time

– Fewer instruments are needed to match the same throughput of CE

• Ease of use/interface

– No manipulation of capillaries, cutting capillaries, etc.

– Minimal method parameters

• Compatibility with high salt matrices without sample pretreatment

EXTENDED QUALIFICATION AND ROBUSTNESS

9

WORKFLOW OF PICO MCE ASSAY

9

Image credit: https://pixabay.com/en/tube-pellet-empty-clear-open-lab-308643/

https://www.thermofisher.com/order/catalog/product/AB2396

http://www.perkinelmer.com/product/protein-express-labchip-760499

http://www.perkinelmer.com/product/ht-labchip-gx-ii-touch-cls138160

Samples Add Buffers Denature

Incubate to

Attach Dye

Electropherograms

• Temperature

• Volume

• Pooled vs. unpooled

• Cooling time

• Heated vs. unheated lid

• Cooling time

• Sitting time

• Sitting location• Humidity

• Temperature

• Integration parameters

• Analyst integration

• Identity

• pH

• Ionic strength

• Concentration of alkylating agent

• Detergent (SDS vs. LDS)

Add Dye

Add Stop

Solution

Load Plate

and ChipRun Assay

GXII AND GXII TOUCH COMPARABILITY STRATEGY

10

Current Qualified Molecules (n=10

at the time of study, 15 to date)New Molecules

Bridging study between with

GXII and GXII Touch to allow

analysis by either model

Qualify using the GXII Touch

instrument

11

COMPARABILITY BETWEEN INSTRUMENT MODELS

Instrument

Average( %) GXII GXII Touch

LMW 2.6 2.8

NGHC 4.7 4.7

Purity 91.7 91.5

n = 6 preps

Comparability study between the GXII and GXII Touch

• GXII model being superseded by the GXII Touch HT

• Examined 10 separate molecules

• Any differences were ≤ 0.8%, which is within the

established assay variability (SD%).

0.2

71

0.2

82

0.2

84

0.2

86

0.3

12

0.3

21

0.3

24

0.3

29

0.3

35

0.3

58

0.3

86

0.3

92

RF

U

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00

Minutes

0.260 0.265 0.270 0.275 0.280 0.285 0.290 0.295 0.300 0.305 0.310 0.315 0.320 0.325 0.330 0.335 0.340 0.345 0.350 0.355 0.360 0.365 0.370 0.375 0.380 0.385 0.390 0.395 0.400

GXII Touch

0.2

75 0

.285

0.2

87

0.2

90

0.3

17

0.3

24

0.3

27

0.3

32

0.3

40

0.3

62

0.3

91

0.3

97

RF

U

-20.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

200.00

220.00

240.00

260.00

280.00

300.00

Minutes

0.260 0.265 0.270 0.275 0.280 0.285 0.290 0.295 0.300 0.305 0.310 0.315 0.320 0.325 0.330 0.335 0.340 0.345 0.350 0.355 0.360 0.365 0.370 0.375 0.380 0.385 0.390 0.395 0.400 0.405

LC

GXII

HC

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ALKYLATING AGENT AND PH

RF

U

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

2200.00

2400.00

2600.00

2800.00

3000.00

3200.00

Minutes

0.360 0.362 0.364 0.366 0.368 0.370 0.372 0.374 0.376 0.378 0.380 0.382 0.384 0.386 0.388 0.390 0.392 0.394 0.396 0.398 0.400 0.402 0.404 0.406 0.408 0.410

RF

U

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

2200.00

2400.00

2600.00

2800.00

3000.00

3200.00

Minutes

0.360 0.362 0.364 0.366 0.368 0.370 0.372 0.374 0.376 0.378 0.380 0.382 0.384 0.386 0.388 0.390 0.392 0.394 0.396 0.398 0.400 0.402 0.404 0.406 0.408 0.410

RF

U

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

2200.00

2400.00

2600.00

2800.00

3000.00

3200.00

Minutes

0.360 0.362 0.364 0.366 0.368 0.370 0.372 0.374 0.376 0.378 0.380 0.382 0.384 0.386 0.388 0.390 0.392 0.394 0.396 0.398 0.400 0.402 0.404 0.406 0.408 0.410

pH

4.4 6.1 8.8

Total area increases with higher

pH while the concentration of

the alkylating agent is robust

across a wide range

Non-Reduced

DETERMINATION OF OPTIMUM SAMPLE PREPARATION

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Non-Reduced

Reduced

DOE study previously performed and temperature confirmed for material from new cell line

STABILITY OF PLATED SAMPLES AT 5C

1448

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

75.00

80.00

85.00

90.00

95.00

100.00

0 60 120 180 240

Perc

ent

Minutes

Reduced LC+HCPurity

LMW

HMW

NGHC

Prepared samples are stable up to 4 hours at 5C

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

75.00

80.00

85.00

90.00

95.00

100.00

0 30 60 90 120150180210240

Perc

ent

MInutes

Non ReducedMP+NGMP

MP Purity

LMW

HMW

NGMP

PRELIMINARY COMPARISON OF CE AND MCE

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Non-Reduced Reduced

Non-Reduced Reduced

Purity LMW NGMP Purity LMW NGHC

MCE 90.09 3.85 6.05 88.99 2.62 7.81

CE-SDS 92.4 2.23 5.43 87.3 3.54 8.55

Difference -2.31 1.62 0.62 1.69 -0.92 -0.75

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COMPARISON OF STABILITY SAMPLES – 3M AT 40C

Non-Reduced

Method %area

CE (Purity) 83.7

MCE (Purity) 84.4

CE (LMW) 7.75

MCE (LMW) 9.73

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COMPARISON OF STABILITY SAMPLES – 3M AT 40C

Reduced

Method %area

CE (Purity) 84.2

MCE (Purity) 85.3

CE (LMW) 6.94

MCE (LMW) 5.67

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LINEARITY

Total Area

Consistency of Analysis

Consistency of Analysis

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LOQ/LOD DETERMINATION

Product Level

(ppm)0.5 1 2 2.5

Average Total

Time

Corrected

Area

239443 316124 905277 1444274

SD 98620 28588 30929 42300

RSD 41.2 9.0 3.4 2.9

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MCE METHOD ACCURACY

Non-reduced

Nominal

Concentration

(mg/mL)

Recovered

Concentration

(mg/mL)

% Recovery

0.35 0.30 85.1

0.40 0.44 109.4

0.50 0.55 109.5

0.60 0.58 96.8

0.65 0.64 106.0

Reduced

Nominal

Concentration

(mg/mL)

Recovered

Concentration

(mg/mL)

% Recovery

0.35 0.36 102.2

0.40 0.40 101.2

0.50 0.46 92.7

0.60 0.64 106.6

0.65 0.63 97.6

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MCE ASSAY PRECISION

Reduced

Prep LMW NGHC HMW Purity

1 2.46 7.6 0.53 89.4

2 2.89 7.5 0.68 88.9

3 2.54 7.4 0.73 89.3

4 2.51 7.7 0.75 89.1

5 2.65 7.3 0.76 89.3

6 2.53 7.6 0.65 89.2

Average 2.60 7.5 0.69 89.2

SD 0.16 0.13 0.08 0.16

RSD 6.03 1.71 12.34 0.18

Non-Reduced

Prep LMW NGMP MP HMW

1 3.82 5.32 90.9 0

2 3.88 5.53 90.6 0

3 3.77 6.14 90.1 0

4 3.60 6.14 90.3 0

5 3.56 5.69 90.8 0

6 3.88 5.50 90.6 0

Average 3.75 5.72 90.5 0

SD 0.14 0.35 0.29 0

RSD 3.76 6.04 0.32 0

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MCE INTERMEDIATE PRECISION

Non-reduced

Analyst % LMW % NGMP % HMW

1 3.85 6.4 0.13

2 3.75 5.7 ND

Average 3.80 6.1 0.07

SD 0.07 0.47 0.09

RSD 1.76 7.69 141.42

Reduced

Analyst % LMW % NGHC % HMW % Purity

1 2.62 8.1 0.5 88.8

2 2.60 7.5 0.7 89.2

Average 2.61 7.8 0.6 89.0

SD 0.02 0.43 0.12 0.31

RSD 0.62 5.47 20.55 0.35

Two different analysts were

utilized using separate

instruments, reagent kits and

microchips

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PERFORMANCE AND TRENDING

mean UCL LCL

CE Purity 94.7 95.8 93.7

MCE

Purity

94.0 95.4 92.7

CE LMW 1.68 2.45 0.91

MCE

LMW

2.52 3.57 1.48

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PERFORMANCE AND TRENDING

mean UCL LCL

CE Purity 96.8 97.8 95.7

MCE

Purity

94.8 96.3 93.2

CE LMW 2.09 3.05 1.11

MCE

LMW

3.35 4.42 2.27

TROUBLESHOOTING

CASE STUDIES

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TROUBLESHOOTING ATYPICAL PROFILE – FRONTING ON LIGHT CHAIN

Issue

Analyst noted atypical fronting of the light chain peak

Root Cause

After thorough investigation, it was determined that

the O-rings were soiled. The O-rings were replaced

and subsequent eletropherograms were typical

Remediation

O-rings are now visually inspected and replaced more

often

27

CELL LINE CHANGE FOR A MONOCLONAL ANTIBODY

Issue

Analyst noted shoulder on the tail of the main peak in

new cell line

Root Cause

After thorough investigation with supporting

post-translational and mass spec analysis, dye was

conjugating more in the hinge region of the cell line 2

more so than the cell line 1 material

Comparison of Non-Reduce MCE Electropherograms

• MCE analysis using GXII has demonstrated to provide quality data comparable to CE with comparable method

qualification performance

• Robust operating ranges have been developed and qualified for use in QC testing

• Data trending between the assays is similar however the number of total invalid assays thus far by MCE is

about half that of CE

• Instrument downtime is nearly non-existent for MCE with regular preventative maintenance and daily cleaning

• Careful cleaning and training of analysts is necessary to reduce abnormal profiles and maintain instrument

performance

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CONCLUSIONS AND NEXT STEPS

IOPS QC and Analytical Sciences

• Timothy Riehlman

• Gabriel Carreau

• Nicole Nall

• Anu Rambhadran

• Matthew Allen (intern)

• Paul Bigwarfe

IOPS management

• Daniel Van Plew

• Rong Wang

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ACKNOWLEDGEMENTS