Knock! Knock! Who s there? Maurice Who? SungAe Suhr Park, Mee Ko, Eleanor Le, Janice Chen Amgen Inc.

Transcription

1 Knock! Knock! Who s there? Maurice Who? SungAe Suhr Park, Mee Ko, Eleanor Le, Janice Chen Amgen Inc.

2 Abstract SungAe Suhr Park, Mee Ko, Eleanor Le and Janice Chen Amgen Inc. Applications of Capillary Electrophoresis (CE) Technique have been increasingly utilized as a key analytical tool to support various areas in academic and industry. The advantages of capillary electrophoresis are ease in quantification, rapid analysis time, enhanced resolution, and automation. In order to meet the increased demand, platform assay and high throughput enhancement formats have been requested. The applications of CE-sodium dodecyl sulfate (CE-SDS) and Capillary iso-electric focusing (CIEF) methods are required by agencies to release Drug substances and Drug products to replace gel electrophoresis. In this talk, the new capillary electrophoresis system Maurice will be evaluated for suitability as a high throughput and platform system due to ease of use, reproducibility and precision for CE-SDS and CIEF method.

3 Our Wish for New CE Instrument Ease of Use Ease of Sample Preparation Ease of Method Transfer with GMP Compliance Ease of Maintenance No Error in Sequence Analysis Good Baseline Good Sensitivity Good Reproducibility High Throughput

4 Why Maurice is here? The advantages of capillary electrophoresis compared to Gel Electrophoresis Ease in quantification Rapid analysis time Enhanced resolution Automation Need platform assay and high throughput enhancement formats Maurice was evaluated for suitability as a high throughput and platform system for CE-SDS and CIEF method

5 Who is Maurice? Named from Grandma and Grandpa: From ice to More-iCE: Maurice Grandma & Grandpa Mom & Dad Maurice CIEF CIEF CIEF &CE-SDS

6 What is considered in this study? Reproducibility of r-ce-sds, nr-ce-sds and CIEF Simplicity of method Sensitivity Comparison with Historical Data

7 CE-SDS Vial Set-Up: 10 Vials for 48 Injections 9 Vials in Reagent Tray 1 Vial in Cartridge Default run method: 20 sec injection, 4600 Volts 25 minute separation, 5750 Volts Code Reagent Volume CAP Position Orange pressure C1 Condition Solution ml cap P1 C2 Condition Solution ml Orange pressure cap P2 Orange pressure Water DI Water 1.5 ml cap P3 Sep Separation Matrix 1.0 ml Orange pressure cap P4 Wash Wash solution 1.0 ml Orange pressure cap P5 Air Empty vial for Waste Orange pressure cap P6 Wash Wash solution 1.5 ml Clear Screw cap N1 Wash Wash solution 1.5 ml Clear Screw cap N2 Run Running Buffer- Bottom 1.0 ml Clear Screw cap N4

8 Reduced CE-SDS Sample Preparation Sample Concentration: 1 mg/ml Sample Preparation Amount 1x sample buffer 25 µl Sample (1 mg/ml) 25 µl Beta mercaptoethanol (2.5 µl, 14.2 M) 2.5 µl 25x Internal standard 2 µl 70 C for 10 min Ice for 5 min Transfer 50 µl to 96 well plate

9 Reproducibility of Proteinsimple IgG Standard by r-ce- SDS: %CV is less than 5 % Injection: 25, 26, 27, 28, 29, 30 IS LC NGHC HC 10 Absorbance (mau) Relative Migration Time %LC RMT %HC RMT %NGHC RMT Average std dev % cv N=6

10 Reproducibility: Reduced CE-SDS: Amgen IgG Sample %CV is less than 5% 3 Injection: 13, 14, 15, 16, 17, Absorbance (mau) Relative Migration Time %LC RMT %MMW1 %MMW2 %NGHC %HC RMT %Post HC %HMW1 %HMW2 Average std dev % CV

11 Sensitivity: Compared to Historical Data Maurice Data %LMW %LC %MMW1 %MMW2 %NGHC %HC %Post HC %HMW1 %HMW2 ND Historical Data %LMW %LC % MMW1 %MMW2 %NGHC %HC %Post HC %HMW1 %HMW ND R-CE-SDS Evaluation of Amgen mab sample using Protein Simple Preparation Profiles are consistent with historical data for mabs tested

12 Can Detect Small species for Degraded Amgen IgG Sample:%CV is less than 5% for Major Peaks IS Injection: 19, 20, Absorbance (mau) AMG 785, 25C (I19) AMG 785, 25C (I20) AMG 785, 25C (I21) Relative Migration Time %LMW1 %LMW2 %LC %MMW1 %MMW2 %NGHC %HC %post HC %HMW Average std dev % CV

13 Non-Reduced (nr)-ce-sds Sample Preparation Sample Concentration: 1 mg/ml Sample Preparation Amount 1x sample buffer 25 µl Sample (1 mg/ml) 25 µl Iodoacetamide (IAM, 250 mm: 46 mg/ml) 2.5 µl 25x Internal standard 2 µl 70 C for 10 min Ice for 5 min Transfer 50 ul to 96 well plate

14 Reproducibility: nr-ce-sds: Proteinsimple IgG Standard: n=5: Relative Migration Time issue 40 Injection: 11, 12, 13, 14, 15 Absorbance (mau) Injection RMT % Main RMT % Main Average std dev % CV Relative Migration Time RMT of sample is changed (2.31 to 2.23) because of Capillary conditioning every 12 samples

15 Reproducibility of nr-ce-sds: Amgen IgG Sample: % CV of RMT and % Main is less than 1% Injection: 16, 17, 18, 19, 20, 21, 22 Absorbance (mau) Dip Relative Migration Time Issue: Dip in the baseline Injection RMT % Main RMT % Main Average std dev % CV

16 Reproducibility: nr-ce-sds: Degraded Amgen IgG Sample % CV of RMT and % Main is less than 1% but Dip in baseline Absorbance (mau) Injection: 23, 24, 25, 26, 27 Dip Relative Migration Time Injection RMT % Main RMT % Main Average std dev % CV

17 How Proteinsimple Solve the DIP in the baseline problem After we reported DIP in the baseline, Proteinsimple created new sample running buffer. Running Buffer 2 Top Separation Matrix 2 Running buffer 2 Bottom Dip is Gone with new Running Buffer with Amgen IgG Absorbance (mau) Injection: 16, 18, 20 No Dip Relative Migration Time

18 Assessment and Conclusion for CE-SDS Advantages More samples can be run at one time (48 total samples) Ease of use (No cartridge preparation and less wash vials) Reduced and non-reduced samples can be run in one sequence in one sample plate Drawbacks Noise on Maurice unit: baseline and dip problem: New buffer solved this problem No manual integration at this time; need to export data for integration and system suitability calculations (noise, asymmetry, etc.) Require method development for new modalities May be useful as a screening tool or for a quick readout for new modalities

19 mab Platform CIEF Method Separation Conditions 1 Minute 1500 Volts 6 Minutes 3000 Volts Sample Composition (Per 200 µl Sample Volume) 0.35 % MC = 70 µl 1% MethylCellulose 2M Urea = 40 µl 10 M Urea 2.5 mm Arginine = 1 ul 500 mm Arg 1.25% 5 to 8 = 2.5 µl Pharmalyte 5 to % 8 to 10.5 = 2.5 µl Pharmalyte 8 to mm H3PO4 = 20 µl Maurice Anolyte pi 5.85 Marker = 2 µl pi 5.85 Marker pi Marker = 2 µl pi 9.99 Marker DI H20 = 20 µl DI Water 200 µg/ml mab = 40 µl 1 mg/ml mab Solution

20 CIEF Vial Set up Code Reagent Volume CAP Position MC 0.5% Methyl Cellulose 2.0 ml Blue pressure cap P1 FI Cal Fluorescence Calibration Standard 500 ul Blue pressure cap P2 Water DI Water 2.0 ml Blue pressure cap P3 Air Empty vial for Waste Blue pressure cap P6 Water DI water 2.0 ml Clear Screw cap N1 Separation Method: 1.0 min: 1500 Volts 6.0 min 3000 Volts Detection: 5 exposures Sample Load: 55 sec pi markers: 5.85, 9.99

21 Can Separate Neutral and Basic mab Injection: 3, 4 Mkr 5.85 Mkr Absorbance (mau) pi

22 Native Fluorescence showed less noise in baseline Injection: 3 Absorbance (mau) Mkr 5.85 Mkr Absorbance ,000 20, pi Injection: 3 Mkr 5.85 Mkr 9.99 Native Fluorescence Fluorescence 15,000 10,000 5, pi

23 Reproducibility of Basic mab cief: %CV is less than 5% Percent Composition Acidic Main Basic Sample Sample Sample Sample Sample Sample Average Std Dev %CV 2.28% 0.82% 2.34% Main Acidic Basic

24 Assessment and Conclusion for CIEF Advantages Native fluorescence detection in CIEF mode for higher sensitivity Ease of use (No cartridge preparation) Shorter runtime for CIEF method: 10 min. vs. 60 min Faster method development for new modalities Drawbacks Need simple sample preparation such as automatic dilution No manual integration at this time; need to export data for integration and system suitability calculations (noise, asymmetry, etc.) Require method development for new modalities

25 What Can Maurice Do? Analyze by CIEF and CE-SDS methods Simple Cartridge format: No cartridge preparation and Easy Installation Smaller footprint with an integrated autosampler with 96 well plate 48 samples in one sequence Native fluorescence detection in CIEF mode for higher sensitivity Analyze reduced and Non-reduced CE-SDS in one sample plate

26 Acknowledgement Jeremy Primack Richard Wu Matt Fesinmeyer Sugu Patro Sharel Figueredo Tufan Aydogdu Scott Mack Craig Magee Jiang Xin