E VA L U AT I O N O F N E W T E C H N O L O GY F O R T H E Q U A L I T Y C O N T R O L L A B O R ATO R I E S JEFF SCHNEIDERHEINZE, PH.D. 1
OUTLINE Background on Microchip CE (MCE) Extended Qualification and Robustness of MCE Troubleshooting Case Studies 2
EVOLUTION OF ELECTROPHORESIS Technology Resolution Maximum Throughput PAGE Low ~ 20 3.5 hours Run Time Schematic Instrument Example Result (Depends on gel size) CE High 24 35 mins MCE High 384 40 s per sample (16 mins for 24 samples) 3
MCE USING PERKINELMER LABCHIP INSTRUMENTS GXII GXII Touch Protein PICO Assay Reagent Kit WHT_LabChip_GXII_Touch_Antibody_Analysis.pdf 4
PRINCIPLE OF THE PICO MCE ASSAY Alkylating agent Dye LDS Heat 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 5
COMPARISON OF CE-SDS RESULTS TO MCE USING PICO 6
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 7
EXTENDED QUALIFICATION AND ROBUSTNESS
WORKFLOW OF PICO MCE ASSAY Samples Add Buffers Denature Add Dye Integration parameters Analyst integration Identity ph Ionic strength Concentration of alkylating agent Detergent (SDS vs. LDS) Humidity Temperature Temperature Volume Pooled vs. unpooled Cooling time Sitting time Sitting location Heated vs. unheated lid Cooling time Incubate to Attach Dye Electropherograms Run Assay Load Plate and Chip Add Stop Solution 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 9 9
GXII AND GXII TOUCH COMPARABILITY STRATEGY 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 10
0.271 0.282 0.286 0.312 0.321 0.335 0.358 0.386 0.392 0.324 RFU 0.284 0.329 0.275 0.290 0.317 0.324 0.340 0.362 0.391 0.397 0.285 0.327 RFU 0.287 0.332 COMPARABILITY BETWEEN INSTRUMENT MODELS 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%). 300.00 280.00 260.00 240.00 220.00 200.00 180.00 160.00 140.00 120.00 LC HC GXII 100.00 80.00 60.00 40.00 20.00 Instrument Average( %) GXII GXII Touch LMW 2.6 2.8 NGHC 4.7 4.7 Purity 91.7 91.5 n = 6 preps 0.00-20.00 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 Minutes 400.00 350.00 300.00 250.00 200.00 150.00 100.00 50.00 GXII Touch 0.00 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 Minutes 11
RFU ALKYLATING AGENT AND PH 3200.00 3000.00 2800.00 2600.00 2400.00 2200.00 2000.00 1800.00 1600.00 Non-Reduced ph 4.4 6.1 8.8 1400.00 1200.00 1000.00 800.00 600.00 400.00 200.00 0.00 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 Minutes Total area increases with higher ph while the concentration of the alkylating agent is robust across a wide range 12
DETERMINATION OF OPTIMUM SAMPLE PREPARATION Non-Reduced Reduced DOE study previously performed and temperature confirmed for material from new cell line 13
Percent Percent STABILITY OF PLATED SAMPLES AT 5C Non Reduced Reduced 100.00 95.00 90.00 85.00 10.00 9.00 8.00 7.00 6.00 5.00 4.00 MP+NGMP MP Purity LMW HMW 100.00 95.00 90.00 85.00 4.00 3.50 3.00 2.50 2.00 1.50 LC+HC Purity LMW HMW NGHC 80.00 75.00 0 30 60 90 120150180210240 MInutes 3.00 2.00 1.00 0.00 NGMP 80.00 75.00 0 60 120 180 240 Minutes 1.00 0.50 0.00 Prepared samples are stable up to 4 hours at 5C 48 14
PRELIMINARY COMPARISON OF CE AND MCE 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 15
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 16
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 17
LINEARITY Consistency of Analysis Total Area Consistency of Analysis 18
LOQ/LOD DETERMINATION Product Level (ppm) Average Total Time Corrected Area 0.5 1 2 2.5 239443 316124 905277 1444274 SD 98620 28588 30929 42300 RSD 41.2 9.0 3.4 2.9 19
MCE METHOD ACCURACY Non-reduced Nominal Concentration Recovered Concentration % Recovery (mg/ml) (mg/ml) 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 Recovered Concentration % Recovery (mg/ml) (mg/ml) 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 20
MCE ASSAY PRECISION 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 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 21
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 22
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 23
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 24
TROUBLESHOOTING CASE STUDIES
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 26
CELL LINE CHANGE FOR A MONOCLONAL ANTIBODY Comparison of Non-Reduce MCE Electropherograms 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 27
CONCLUSIONS AND NEXT STEPS 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 28
ACKNOWLEDGEMENTS 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 29