SDS-CGE Method Development for Purified Protein Vaccine Antigens Michael Leach, Elena Newman and Bruce Carpick Sanofi Pasteur Analytical R&D, Biochemistry
Overview SDS-CGE method development for purified protein vaccine antigens Protein purity Relative antigen content (stability) Method development and optimization Examples: Tuberculosis H4 fusion protein antigen Bacterial multi-subunit protein antigen 9/24/2018 2
CGE: Capillary Gel Electrophoresis Purpose: To develop electrophoresis methods as replacements for SDS-PAGE methods for the purity release testing of vaccine antigen drug substances and evaluation of their stability Traditional SDS-PAGE with densitometry CE Instrument Sciex/Beckman Coulter PA800+ Sample Prep In-house SDS buffer Addition of reducing agent (2-ME) Heating Capillary gel electrophoresis General run conditions Pressure Injection 30 cm capillary length 50 µm ID, gel-filled capillary 30 min separation time 9/24/2018 3
Assay development approach for SDS-CGE Instrument settings Injection type Aperture selection Capillary temperature Separation voltage Injection conditions Sample preparation BME concentration Heat, time and temperature SDS concentration Sample stability Antigen concentration Integration robustness Integration optimization Mock impurity spike < 1% detection 3 lots tested Degraded samples tested Assay suitability Instrument-toinstrument comparison Stability indication Artifact peak investigation Specificity Assay parameters Assay validity criteria: Resolution Assay validity criteria: Peak height System suitability control: BSA LOD 2.5 µg/ml System suitability control: BSA 100 µg/ml MW range of antigen Performance attributes Mock impurity spike linearity & Accuracy Intermediate precision preliminary assessment 9/24/2018 4
Light absorbance at 220 nm (a.u.) Tuberculosis (TB) H4 antigen protein HyVac H4 antigen is a recombinant fusion of Mycobacterium tuberculosis antigens Ag85B and TB10.4 with molecular size of 41.3 kda H4 was developed by Statens Serum Institut, Denmark, and the H4 antigen produced by Sanofi Pasteur was used in a vaccine trial in South Africa in partnership with Aeras SDS-CGE methods were developed to assess H4 antigen protein purity and stability (relative content, intactness) Electropherograms of TB H4 antigen run under reducing and non-reducing conditions 0.030 IS 0.025 0.020 0.015 Non-reducing conditions H4 H4 oligomers 0.010 0.005 Reducing conditions 0.000 10 12 14 16 18 20 22 Migration time (min) 9/24/2018 5
SDS-CGE method development and optimization Design of Experiment (DoE) approach to optimize method parameters for antigen quantitation Separation electric field (230, 430 and 630 V/cm) Capillary temperature (20, 25 and 30 o C) Amount of SDS-MW sample buffer (30, 57 and 83% v/v) DoE approach to optimize method parameters for purity Injection time (10 and 40 sec) Electric field for electrokinetic injection (65 and 333 V/cm) Capillary temperature (20 o C and 30 o C) Separation electric field (330 V/cm and 1000 V/cm) Sample heating temperature (65 o C and 100 o C) Sample heating time (5 and 15 min) Optimization based on: Purity reportable value Reportable value precision Peak resolution BSA relative peak area 9/24/2018 6
BSA-normalized peak area of H4 Peak area normalization with BSA internal standard a 0.03 b 104 Light absorbance at 220 nm (a.u.) IS H4 0.02 BSA 40 g/ml 0.01 70 g/ml 100 g/ml 130 g/ml 160 g/ml 0.00 10 12 14 16 18 20 22 Migration time (min) Relative recovery of H4 (%) 102 100 98 96 94 92 90 BSA-corrected non-corrected IS-corrected 40 60 80 100 120 140 160 Concentration of H4 ( g/ml) 2.5 Using co-injected BSA as a normalization standard increases the robustness of the reportable value to the amount of antigen loaded onto the capillary 2.0 1.5 1.0 y = 0.0232 + 0.0196x R 2 = 0.9997 0.5 0.0 0 20 40 60 80 100 120 140 Concentration of H4 (µg/ml) 9/24/2018 7
TB H4: SDS-CGE relative antigen quantity for stability Method Performance Intermediate precision (%RSD) 2% Accuracy (recovery) 98% to 100% 99%-101% Accuracy, % Recovery LOQ to Target Linearity method (slope and r 2 ) 1.0073 0.9993 Dose Linearity (r 2 ) 0.9997 15-135 g/ml 9/24/2018 8
TB H4: Purity assay optimization Light absorbance at 220 nm (a.u.) 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 18 19 20 H4 BSA Optimized conditions H4 BSA 16 17 Non-optimized conditions 14 16 18 20 22 24 26 Migration time (min) Light absorbance at 220 nm (a.u.) 0.25 0.20 0.15 0.10 0.05 0.00 BSA g/ml H4 g/ml 0 650 7 643 13 637 33 617 67 583 100 550 133 517 167 483 H4 BSA 10 12 14 16 18 Migration time (min) 9/24/2018 9
Light absorbance at 220 nm (a.u.) TB H4 purity: SDS-CGE vs SDS-PAGE a 0.18 0.16 H4 b (kda) 75 Std 1 2 3 0.14 0.12 0.014 0.10 0.08 0.06 0.04 0.02 0.00 0.012 0.010 0.008 0.006 0.004 0.002 Impurities (11%) 0.000 12 14 16 10 12 14 16 18 Migration time (min) 50 37 25 H4 Impurities (7%) The SDS-CGE method provides a comparable %Purity reportable value and improved analytical performance to the SDS-PAGE method used for release testing of TB H4 GMP lots used in formulating H4-IC31 vaccine for clinical trial 9/24/2018 10
TB H4: SDS-CGE purity Method Performance Intermediate precision (%RSD) 0.7% at 81% Purity spike level Accuracy (recovery) 98% to 100% 99%-100% Accuracy, % Recovery LOQ to Target Linearity Slope method Linearity method (r 2 ) 1.15 0.995 9/24/2018 11
Bacterial multi-subunit antigen: SDS-CGE purity Capillary Gel Electrophoresis (CGE) separates based on SIZE S1 S4 S3 S5 S2 Typical result: 95% purity (sum of S1 to S5, not including S2 shoulder) 9/24/2018 12
µl used in sample preparation Bacterial multi-subunit antigen: SDS-CGE purity Increase antigen load to increase sensitivity of detecting impurities BME 1% SDS, Tris BME 1% SDS, Tris PBS BME 3% SDS, Tris PBS PBS Antigen Antigen Antigen Consistent detection Inconsistent detection Consistent detection 9/24/2018 13 RED_00093471
Bacterial multi-subunit antigen: SDS-CGE purity Mock impurity spike near LOD Antigen purity Injected antigen, µg/ml BSA % (w/w) BSA %CPA Observed BSA detected? 250 1.2 0.92 Yes 200 1.2 0.94 Yes 150 1.2 0.84 Yes 125 1.2 0.84 Yes 100 1.2 0.48 Yes 100 0.5 N/Ap No 42 0.5 N/Ap No Impurity detection robustness Robust purity reportable value when varying antigen concentration 9/24/2018 14
Bacterial multi-subunit antigen: SDS-CGE purity Method Performance Intermediate precision (%RSD) 0.30% Accuracy (recovery) 98% to 100% 99%-102% Accuracy, % Recovery LOQ to Target Method Linearity - Slope 1.05 Method Linearity (r 2 ) 0.998 9/24/2018 15
Bacterial multi-subunit antigen: Relative content 5 reportable values per injection: BSA-normalized peak area S1/BSA, S2/BSA, S3/BSA, S4/BSA, S5/BSA Antigen subunits BSA The relative antigen content for stability approach can be extended to more complex antigen drug substances, including multi-subunit antigen complexes 9/24/2018 16
Balancing sensitivity and resolution Optimized sample injection conditions (pressure, time) for purity and relative content methods Purity High Signal Impurities > LOD Relative content High Resolution Precise integration 2.6 psi, 300s 1.4 psi, 240s Higher pressure Long injection time Peak area High Resolution Low Injection psi, min High Sample plug Low Separation gel in capillary Low [antigen] Smaller sample plug = higher resolution No resolution change 9/24/2018 17
Bacterial multi-subunit antigen: CGE stability data Example of stability data set showing multiple storage temperatures and containers 9/24/2018 18
CGE relative antigen content: Assay performance Method performance ranges for 5 subunits, each treated as reportable value Intermediate precision (%RSD) 7.5-12% 98% Accuracy to (recovery) 100% 96%-106% Accuracy, % Recovery LOQ to Target Method Linearity - Slope 0.97-1.07 Method Linearity (r 2 ) 0.98-0.99 9/24/2018 19
Next Steps Bacterial multi-subunit antigen Qualification of Purity and Relative Antigen Content for use on GMP clinical lots CZE and icief analysis of intact multi-subunit complex Purity analysis of recombinant lipidated protein antigens with potential analytical challenges (lipid chain heterogeneity, detergent process residuals) CGE for protein purity CGE relative antigen content for stability Stability analysis of multivalent aluminum-adjuvanted protein antigens CGE for combined antigen content and %Adsorption CZE for separation of variants CZE-MS for analysis of intact mass 9/24/2018 20
Acknowledgements Sanofi Pasteur, Analytical R&D, Biochemistry Vivian (Wai Wan) Tam Nancy Quatela Jing Zhang Yukyung Ha Jeffery Gerard Sanofi Pasteur, Bioprocess R&D, Formulation & Stability Davinder Chawla Jalil Hakimi SGS Canada Samaneh Beheshti York University (Toronto) Sergey Krylov Lana Krylova Agnesa Shala-Lawrence 9/24/2018 21
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