capillary electrophoresis in pharmacopoeial...

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Capillary Electrophoresis in

Pharmacopoeial Monographs

Current Situation, Requirements and Obstacles

Stefan Christians, Immunochemistry

CE-Pharm 2011; Amelia Island

Stefan Christians, PEI NASA/CXC/Univ. of Maryland/A.S. Wilson et al.

The Big Bang in Astronomy

Spiral galaxy M106

Stefan Christians, PEI cIEF of a stressed monoclonal antibody (zoomed view)

The Big Bang in Separation Science

10

.0

9.5

5.5

4.1

8.6

5

8.5

1

8.3

3

8.0

7

7.8

8

8.7

7

9.0

3

14 * 10^6 19 * 10^6

1 * 10^6

4 * 10^6

3 * 10^6

plate numbers (Ph. Eur.)

Stefan Christians, PEI

Interstellar Noise ???

Albumin

α-Globulin

ß-Globulin

γ-Globulin

Purity by Cellulose Acetate Gel Electrophoresis (PH. Eur.)


What is a Ph. Eur. Monograph?


Sample dilution

Electrophoresis

Staining

Densitometry

Comparing to reference

What is a Ph. Eur. Monograph?


Problems of Gel Electrophoresis

Staining

Densitometry


Standard Electrophoresis 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.

30600

FS

Somatropin Protein

Identification &

Test for charged variants

22125 FS

CE in the European Pharmacopoeia


Identity Testing of Erythropoietin by CZE


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.

How binding are tests in monographs?

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.


General principles

Capillary Zone Electrophoresis

Capillary Gel Electrophoresis

Capillary Isoelectric Focussing

Micellar Electrokinetic

Chromatography (MEKC)

Quanitification

System Suitability

General Chapter: 2.2.47. Capillary Electrophoresis


Human Serum

SST Sample

Capillary Zone Electrophoresis

Interstellar Noise revisited

Cellulose Acetate Gel

Protein Composition of Immunoglobulins by CAGE & CZE


Validation of CE methods against classical gel tests

Test Result

Precision

Linearity

LOQ

Range

Accuracy

Specificity

Sensitivity

Robustness


Accuracy

Electrophoresis

• Separation efficiency

Staining

• Selectivity of protein stains

Densitometry

• Manual Setting of Integration limits

Gel:

CE:

Electrophoresis

• Separation efficiency

UV-Detection

• Selectivity of detection WL

Processing

• Defined Integration parameters


Specificity I: Separation Efficiency

(Isoelectric Focussing of Mabs)

10

.0

5.5

4.1

7.1

3

7.0

1

7.2

8

Agarose IEF

Capillary IEF


Specificity (II): Influence of detection (CZE of serum proteins)

SPE-01

Sorbit (50 g/L)Saccharose (100 g/L)

Mannit (10 g/L)

Glucose (50 g/L)

Glycin (25g/L)

Acetyltryptophan (4 g/L)

Maltose (100 g/L)

SPE-01

Sorbit (50 g/L)Saccharose (100 g/L)

Mannit (10 g/L)

Glucose (50 g/L)

Glycin (25g/L)

Acetyltryptophan (4 g/L)

Maltose (100 g/L)

Stefan Christians, PEI 18

Sensitivity

Lambert-Beer law: A = ε x c x d

+ - Typical internal diameter: 50 µm

Standard analytical flow cell

CE:

HPLC:


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)


Validation of CE against Standard Electrophoresis

Standard Gel Electrophoresis Capillary Electrophoresis


CE in Pharmaocopoeia


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 21st century.

CE is not sensitive enough.

… has to be tested in every case.


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.

What can we do to enforce CE methods into monographs?


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.

Problems


To discuss the need and the obstacles of CE methods in

Pharmacopoeia monographs please join our workshop !

Workshop


Thank you

for your attention

capillary electrophoresis in pharmacopoeial...

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