Capillary Electrophoresis in Pharmacopoeial Monographs

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1 Capillary Electrophoresis in Pharmacopoeial Monographs Current Situation, Requirements and Obstacles Stefan Christians, Immunochemistry CE-Pharm 2011; Amelia Island

2 The Big Bang in Astronomy Spiral galaxy M106 NASA/CXC/Univ. of Maryland/A.S. Wilson et al.

9.03 8.07 7.88 8.77 8.33 8.51 9.5 5.5 4.1 10.0 8.

3 The Big Bang in Separation Science 1 * 10^6 plate numbers (Ph. Eur.) 3 * 10^6 4 * 10^6 14 * 10^6 19 * 10^6 cief of a stressed monoclonal antibody (zoomed view)

4 Interstellar Noise??? Albumin α-globulin ß-Globulin γ-globulin Purity by Cellulose Acetate Gel Electrophoresis (PH. Eur.)

5 What is a Ph. Eur. Monograph?

6 What is a Ph. Eur. Monograph? Sample dilution Electrophoresis Staining Densitometry Comparing to reference

7 Problems of Gel Electrophoresis Staining Densitometry

8 Standard Electrophoresis in the European Pharmacopoeia

9 CE in the European Pharmacopoeia Substance Structure Test MW Capillary Glutathione Related substances 307,3 FS Galantamine hydrobromide Enantiomeric purity 368,3 FS Levocabastine hydrochloride Related substances 457,0 FS Ropivacaine hydrochloride monohydrate Enantiomeric purity 328,9 FS Erythropoietin concentrated solution Protein Identification app FS Somatropin Protein Identification & Test for charged variants FS

10 Identity Testing of Erythropoietin by CZE

approved provided that they are validated against the pharmacopoeia test.

11 How binding are tests in monographs? The products have to comply with the requirements when tested according to the monograph Alternative tests may be approved provided that they are validated against the pharmacopoeia test. On the other hands this implies that the pharmacopoeia methods are considered as validated. but only if they are described in sufficient detail, which is usually not the case if they are only described in the general methods chapter.

12 General Chapter: Capillary Electrophoresis General principles Capillary Zone Electrophoresis Capillary Gel Electrophoresis Capillary Isoelectric Focussing Micellar Electrokinetic Chromatography (MEKC) Quanitification System Suitability

13 Interstellar Noise revisited Human Serum SST Sample Cellulose Acetate Gel Capillary Zone Electrophoresis Protein Composition of Immunoglobulins by CAGE & CZE

14 Validation of CE methods against classical gel tests Test Precision Linearity LOQ Range Result Accuracy Specificity Sensitivity Robustness

15 Accuracy Electrophoresis Staining Densitometry Gel: Separation efficiency Selectivity of protein stains Manual Setting of Integration limits Electrophoresis UV-Detection Processing CE: Separation efficiency Selectivity of detection WL Defined Integration parameters

16 Specificity I: Separation Efficiency (Isoelectric Focussing of Mabs) Agarose IEF Capillary IEF

17 Specificity (II): Influence of detection (CZE of serum proteins) Glucose (50 g/l) Maltose (100 g/l) Mannit (10 g/l) Acetyltryptophan (4 g/l) Glycin (25g/L) Saccharose (100 g/l) Sorbit (50 g/l) SPE-01

18 Sensitivity Lambert-Beer law: A = ε x c x d CE: + - Typical internal diameter: 50 µm HPLC: Standard analytical flow cell 18

19 Robustness (Typical parameter in capillary electrophoresis) Column lots, Column age CE-System Detector (e.g.: PDA vs. UV) Standing time in autosampler Electrolyte exhaustion Derivatisation time, temperature, reagents Buffer preparation (MEEKC)

20 Validation of CE against Standard Electrophoresis Standard Gel Electrophoresis Capillary Electrophoresis

21 CE in Pharmaocopoeia

22 Common Issues against CE and answers CE Equipment is very expensive and would restrict tests to very well equipped companies. Hardware costs compared to HPLC are usually balanced by less costs for consumables. CE requires highly trained personnel. Yes, but this is also the case for many tests (HPLC, GC, Cell Based Assays). CE is not as reliable and robust as HPLC. welcome to the 21 st century. CE is not sensitive enough. has to be tested in every case.

sufficient detail Publishing of data. Make the test procedures available.

23 What can we do to enforce CE methods into monographs? Develop CE methods as alternative to Standard Electrophoresis and HPLC Validation in sufficient detail Publishing of data. Make the test procedures available. Interlaboratory studies to establish the precision between different companies. Make pharmacopoeial groups aware of alternatives.

Problems Companies might not want to share data on the tests Many of the tests especially for biotechnological products are supplied as test kits which include proprietary

24 Problems Companies might not want to share data on the tests Many of the tests especially for biotechnological products are supplied as test kits which include proprietary reagents. Coated capillaries may be available only by a few or even by only one company. Upon shortage of proprietary reagents or capillaries products may lack of legally binding tests.

25 Workshop To discuss the need and the obstacles of CE methods in Pharmacopoeia monographs please join our workshop!

26 Thank you for your attention

Comparison of Different Brands of Carrier Ampholytes for Monoclonal Antibody Charge Heterogeneity Analysis by Capillary Isoelectric Focusing

Comparison of Different Brands of Carrier Ampholytes for Monoclonal Antibody Charge Heterogeneity Analysis by Capillary Isoelectric Focusing Application Note Biotherapeutics & Biosimilars Author Christian