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PhD. Emilio Medina-Rivero Principal Scientist Analytical Development R&D
for the development, characterization, and release of biopharmaceuticals
CE
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, 1133–1140
2. 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, 2754–2759
Comparability
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
Functional assays
Biological activity - CDC - ADCC - ADCP - Apoptosis Affinity - ITC - SPR
Analytical platforms for extended characterization
Capillary Zone Electrophoresis (CZE) for physicochemical characterization of mAbs
• High resolution • Analysis of charged molecules • Well-known technology • Nearly native condition (CZE) • Standard techniques for mAbs (CGE and CIEF) • Robust applications
Capillary Electrophoresis as an analytical platform
Method development BGE
• ionic strength • pH • Viscosity
Sample injection time Sample diluent
Validation tests
Capillary Zone Electrophoresis (CZE) for physicochemical characterization of mAbs
CZE method development (Rituximab) BGE ionic strength and pH
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
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
Current
CZE method development (Rituximab) Sample injection time and diluent
Water Tris buffer
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
CZE for charge heterogeneity analysis Application on other mAbs
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
CZE method development (Infliximab) BGE ionic strength and pH
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
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. 0.27 %a n.d.
n.d. 0.21 %b n.d.
n.d. 0.91 %c n.d. a Kikuzubam® b Trastuzumab´s biosimilar API c Infliximab´s biosimilar API n.d.: not determined
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
CZE method performance Specificity
Kikuzubam® + Trastuzumab-B
Trastuzumab-B Kikuzubam®
Sample matrix
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
Rituximab Trastuzumab
Rituximab
Acidic Basic
Mabthera® + Kikuzubam®
Kikuzubam®
Mabthera® Sample matrix
CZE method as an identity test
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
Trastuzumab
Acidic Basic
Herceptin® + Trastuzumab-B
Trastuzumab-B
Herceptin® Sample matrix
CZE method as an identity test
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
Orthogonal methods for physicochemical characterization
Chromatographic Electrophoretic
Rituximab
Trastuzumab
A B
M
P NP A B
M
B A
M
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 1133–1140
Rituximab Sample Batch †CDC relative potency (%)a
ADCC relative potency (%)b
Kikuzubam® 5445120812 118 98
5433110301 109 91
5433120914 102 103
Reditux® RIAV00910 81 -
RIAV00912 111 -
RIAV01312 108 -
Orthogonal methods for functional characterization
†Flores-Ortiz, et al. J. Liq. Chromatogr. Related Technol. 2013, Published Online
a Relative to Mabthera® batches B60450 and B60480 b Relative to Mabthera® batches B60711, B62101, and B60490
Capillary Gel Electrophoresis (CGE) for in-process control of recombinant proteins
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 2754–2759
rIFN-β 1b
10 KDa
Low Mr impurities High Mr impurities
CGE method for in-process control of recombinant proteins
rIFN-β 1b standard
10 KDa + rIFN-β 1b IBs
rIFN-β 1b IBs
10 KDa
Sample matrix
CGE method validation Repeatability
n = 6
Concentration of rIFN-β 1b standard (mg/mL)
Concentration of rIFN-β 1b IBs (mg/mL)
Purity of rIFN-β 1b (%)
Mean 0.26 0.18 33.70
RSD (%) 2.01 2.29 4.24
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 2754–2759
CGE method validation Accuracy
Level Concentration of rIFN-β 1b (mg/mL)
Mean = 3 RSD (%)
Recovered concentration (mg/mL)
Recovery (%)
1 0.21 0.19 92.06 5.97
2 0.27 0.25 93.83 2.28
3 0.31 0.29 93.55 10.34
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 2754–2759
rhGH
rLAP
rG-CSF
rIFN-β 1b
CGE for in-process control of recombinant proteins Application on other IB´s samples
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 2754–2759
CGE method development IBs´ solubilization solution
Espinosa-de la Garza, et al. Electrophoresis 2013, 34, 2754–2759
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.
Conclusion
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
Aknowledgements
