for the development, characterization, and release of biopharmaceuticals PhD. Emilio Medina-Rivero Principal Scientist Analytical Development R&D

Transcription

1 CE for the development, characterization, and release of biopharmaceuticals PhD. Emilio Medina-Rivero Principal Scientist Analytical Development R&D

2 Agenda 1. Capillary Zone Electrophoresis for physicochemical characterization of mabs Espinosa-de la Garza, C. E., Perdomo-Abúndez, F. C., Padilla-Calderón, J., Uribe-Wiechers J. M., Pérez, N. O., Flores-Ortiz, L. F., Medina-Rivero, E. Electrophoresis 2013, 34, Capillary Gel Electrophoresis for in-process control of recombinant proteins Espinosa-de la Garza, C. E., Perdomo-Abúndez, F. C., Campos-García, V. R., Pérez, N. O., Flores-Ortiz, L. F., Medina-Rivero, E. Electrophoresis 2013, 34,

3 Analytical platforms for extended characterization Physicochemical characterization Charge - HIC - CEX - CZE Glycation - SEC FL - Elisa pi - cief Degradation - CGE NR Purity - CGE R - SEC Identity - MS - Peptide mapping Physical characterization Size - SEC Mass/Radii - SEC-MALS-QELS-RI - MS Stability - DSC Structure - Fluorescence - CD - HDX MS Aggregation - Subvisible particles Comparability Functional assays Biological activity - CDC - ADCC - ADCP - Apoptosis Affinity - ITC - SPR

4 Capillary Zone Electrophoresis (CZE) for physicochemical characterization of mabs

5 Capillary Electrophoresis as an analytical platform High resolution Analysis of charged molecules Well-known technology Nearly native condition (CZE) Standard techniques for mabs (CGE and CIEF) Robust applications

6 Capillary Zone Electrophoresis (CZE) for physicochemical characterization of mabs Method development BGE ionic strength ph Viscosity Sample injection time Sample diluent Validation tests

7 CZE method development (Rituximab) BGE ionic strength and ph Current 10.5 µa 10.8 µa 12.1 µa 13.8 µa 29.2 µa 26.0 µa 33.4 µa 35.0 µa 28.0 µa Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

8 CZE method development (Rituximab) Sample injection time and diluent Tris buffer Water Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

9 CZE for charge heterogeneity analysis Application on other mabs Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

10 CZE method development (Infliximab) BGE ionic strength and ph Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

11 CZE method performance Repeatability Parameter Result Basic Main Acidic RSD of Area % 1.21 % a 0.89 % a 1.12 % a 1.90 % b 0.25 % b 0.38 % b 0.15 % c 0.12 % c 0.31 % c RSD of MT n.d % a n.d. a Kikuzubam b Trastuzumab s biosimilar API c Infliximab s biosimilar API n.d.: not determined n.d % b n.d. n.d % c n.d. Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

12 CZE method performance Specificity Rituximab Trastuzumab Kikuzubam + Trastuzumab-B Trastuzumab-B Kikuzubam Sample matrix Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

13 CZE method as an identity test Rituximab Basic Acidic Mabthera + Kikuzubam Kikuzubam Mabthera Sample matrix Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

14 CZE method as an identity test Trastuzumab Basic Acidic Herceptin + Trastuzumab-B Trastuzumab-B Herceptin Sample matrix Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

15 Orthogonal methods for physicochemical characterization Chromatographic Electrophoretic Rituximab M M M P NP A B B A B A Trastuzumab Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

16 Orthogonal methods for functional characterization Rituximab Sample Batch CDC relative potency (%) a Kikuzubam Reditux RIAV RIAV RIAV a Relative to Mabthera batches B60450 and B60480 b Relative to Mabthera batches B60711, B62101, and B60490 ADCC relative potency (%) b Flores-Ortiz, et al. J. Liq. Chromatogr. Related Technol. 2013, Published Online

17 Capillary Gel Electrophoresis (CGE) for in-process control of recombinant proteins

18 CGE method for in-process control of recombinant proteins rifn-β 1b Low Mr impurities High Mr impurities 10 KDa rifn-β 1b standard 10 KDa + rifn-β 1b IBs rifn-β 1b IBs 10 KDa Sample matrix Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

19 CGE method validation Repeatability n = 6 Concentration of rifn-β 1b standard (mg/ml) Concentration of rifn-β 1b IBs (mg/ ml) Mean RSD (%) Purity of rifn-β 1b (%) Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

20 CGE method validation Accuracy Level Concentration of rifn-β 1b (mg/ml) Mean = 3 RSD (%) Recovered concentration (mg/ml) Recovery (%) Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

21 CGE for in-process control of recombinant proteins Application on other IB s samples rg-csf rlap rhgh rifn-β 1b Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

22 CGE method development IBs solubilization solution Espinosa-de la Garza, et al. Electrophoresis 2013, 34,

23 Conclusion CE is a suitable alternative to evaluate physicochemical properties of a biopharmaceutical product in each stage of its lifecycle, however, the use of orthogonal techniques is required for extended characterization and to attain a reliable comparability.

24 Aknowledgements Luis Flores Research Associate Analytical Development Jesús Padilla Research Associate Analytical Development Néstor Pérez Principal Scientist Up-Stream Process Development Rodolfo Salazar Principal Scientist Down-Stream Process Development Analytical Development Specialists Alexis Romero Antonio Hernández Carlos Espinosa Erika García Esmeralda Ramírez Francisco Perdomo Laura Juárez Lilia Acosta Maribel Jardón Mariana Bolívar Nancy Ramírez Nelly Piña Víctor Campos Víctor Pérez