title: Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies.
abstract: Within pharmaceutical industry charge heterogeneity testing of biopharmaceuticals has to be reproducible and fast. It should pass method validation according to ICH Q2. Classical approaches for the analysis of the charge heterogeneity of biopharmaceuticals are ion exchange chromatography (IEC) and isoelectric focusing (IEF). As an alternative approach, also capillary zone electrophoresis (CZE) was expected to allow reliable charge heterogeneity profiling by separation according to the analyte's net charge and hydrodynamic radius. Aim of this study was to assess if CZE possesses all of the required features. Therefore, beside lab internal validation of this method also an international cross company study was organized. It was shown that CZE is applicable across a broad pI range between 7.4 and 9.5. The coefficient of correlation was above 0.99 which demonstrated linearity. Precision by repeatability was around 1% (maximum relative standard deviation per level) and accuracy by recovery was around 100% (mean recovery per level). Accuracy was further verified by direct comparison of IEC, IEF and CZE, which in this case showed comparable %CPA results for all three methods. However, best resolution for the investigated MAb was obtained with CZE. In dependence on sample concentration the detection limit was between 1 and 3%. Within the intercompany study for CZE the same stressed and non-stressed samples were analyzed in each of the 11 participating labs. The finally obtained dataset contained more than 1000 separations which provided an extended dataset for further statistical evaluation. Among the different labs no significant differences between the peak profiles were observed. Mean driver for dropouts in quantitative evaluation was linked to the performance of some participating labs while the impact of the method performance was negligible. In comparison to a 50cm capillary there was a slightly better separation of impurities and drug substance related compounds with a 30cm capillary which demonstrates that an increased stability indicating potential can be combined with the increased separation velocity and high throughput capability of a shorter capillary. Separation can be performed in as little as approx. 3min allowing high throughput applications. The intercompany study delivered precise results without explicit training of the participating labs in the method prior to the study (standard deviations in the range of 1%). It was demonstrated that CZE is an alternative platform technology for the charge heterogeneity testing of antibodies in the pharmaceutical industry.
authors: F Hoffmann-La Roche Ltd, Grenzacher Str. 124, 4070 Basel, Switzerland. Electronic address: Bernd.Moritz@roche.com.
authors: Bernd
authors: B
authors: Moritz
authors: F Hoffmann-La Roche Ltd, Grenzacher Str. 124, 4070 Basel, Switzerland. Electronic address: Volker.Schnaible@roche.com.
authors: Volker
authors: V
authors: Schnaible
authors: F Hoffmann-La Roche Ltd, Grenzacher Str. 124, 4070 Basel, Switzerland. Electronic address: Steffen.Kiessig@roche.com.
authors: Steffen
authors: S
authors: Kiessig
authors: F Hoffmann-La Roche Ltd, Grenzacher Str. 124, 4070 Basel, Switzerland. Electronic address: Andrea.Heyne@roche.com.
authors: Andrea
authors: A
authors: Heyne
authors: F Hoffmann-La Roche Ltd, Grenzacher Str. 124, 4070 Basel, Switzerland. Electronic address: Markus.Wild@roche.com.
authors: Markus
authors: M
authors: Wild
authors: F Hoffmann-La Roche Ltd, Grenzacher Str. 124, 4070 Basel, Switzerland. Electronic address: Christof.Finkler@roche.com.
authors: Christof
authors: C
authors: Finkler
authors: Paul-Ehrlich Institut, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany. Electronic address: Stefan.Christians@pei.de.
authors: Stefan
authors: S
authors: Christians
authors: Boehringer Ingelheim, Birkendorfer Straße 65, 88397 Biberach/Riß, Germany. Electronic address: kerstin_2.mueller@boehringer-ingelheim.com.
authors: Kerstin
authors: K
authors: Mueller
authors: Boehringer Ingelheim, 6701 Kaiser Dr., Fremont, CA 94555 USA. Electronic address: li_2.zhang@boehringer-ingelheim.com.
authors: Li
authors: L
authors: Zhang
authors: Boehringer Ingelheim, 6701 Kaiser Dr., Fremont, CA 94555 USA. Electronic address: kenji.furuya@boehringer-ingelheim.com.
authors: Kenji
authors: K
authors: Furuya
authors: Novartis, Fabrikstrasse 2, 4056 Basel, Switzerland. Electronic address: marc.hassel@novartis.com.
authors: Marc
authors: M
authors: Hassel
authors: Merck, 770 Sumneytown Pike, West Pt, PA, USA. Electronic address: melissa_hamm@merck.com.
authors: Melissa
authors: M
authors: Hamm
authors: Merck, 770 Sumneytown Pike, West Pt, PA, USA. Electronic address: richard_rustandi@merck.com.
authors: Richard
authors: R
authors: Rustandi
authors: Pfizer, 700 Chesterfield Pkwy, St Louis, MO, USA. Electronic address: Yan.He2@pfizer.com.
authors: Yan
authors: Y
authors: He
authors: Seattle Genetics, 21823 30th Dr SE, Bothell, WA 98021, USA. Electronic address: osalas@seagen.com.
authors: Oscar Salas
authors: OS
authors: Solano
authors: Seattle Genetics, 21823 30th Dr SE, Bothell, WA 98021, USA. Electronic address: cwhitmore@seagen.com.
authors: Colin
authors: C
authors: Whitmore
authors: Amgen, One Amgen Center Drive Thousand Oaks, CA 91320-1799, USA. Electronic address: spark@amgen.com.
authors: Sung Ae
authors: SA
authors: Park
authors: AB Sciex, Landwehrstr. 54, 64293 Darmstadt, Germany. Electronic address: Dietmar.Hansen@absciex.com.
authors: Dietmar
authors: D
authors: Hansen
authors: AB Sciex, Brea, 250 South Kraemer Boulevard Brea, CA 92821, USA. Electronic address: Marcia.Santos@absciex.com.
authors: Marcia
authors: M
authors: Santos
authors: AB Sciex, Brea, 250 South Kraemer Boulevard Brea, CA 92821, USA. Electronic address: Mark.Lies@absciex.com.
authors: Mark
authors: M
authors: Lies
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal
keywords: Antibody; Capillary zone electrophoresis; High throughput; Intercompany study; Pharmaceuticals; Reproducibility