Session 2 Global Harmonisation Part 2

EDQMEDQM--USPUSP--NIBSCNIBSC

WWORKSHOP ON THEORKSHOP ON THE CCHARACTERISATION OF HARACTERISATION OF

HHEPARIN EPARIN PPRODUCTSRODUCTS

S ES S I O N 2SES S I O N 2

G LO B AL H AR MO N I S AT I O NG LO B AL H AR MO N I S AT I O N

1Advanced techniques suitable for commercial heparin Advanced techniques suitable for commercial heparin characterization characterization

Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Milano - Italy

Photo YOSHIE NISHIKAWA

2workshop on the characterization of heparin products19-20 June 2008, EDQM, Strasburg, France

Giangiacomo Torri

(ppm)20253035404550556065707580859095100105110

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

COCH3

A6S

A6OH

Heparin NMR spectra

1H

13C

I2OH

G. Ronzoni Institute

2Heparin origin trough NMR spectra run on pure heparin samples

Casu B, Guerrini M, Naggi A, Torri G, De-Ambrosi L, Boveri G, Gonella S, Cedro A, Ferro L, Lanzarotti E, Paterno` M, AttoliniM, Valle MG. Characterization of sulfation patterns of beef and pig mucosal heparins bynuclear magnetic resonance spectroscopy. Arzneim-Forsh (Drug Res) 1996; 46:472477.


Linkage Region Sequences of Heparins and HeparanSulfates: Detection and Quantification by NMR

SpectroscopyM. Iacomini, B. Casu, M. Guerrini, A. Naggi, A. Pirola, and G. Torri

Analytical Biochemistry 274, 5058 (1999)

Heparins

Heparan sulfates

3Heparin 13C NMR spectra: contaminants

ethanol

A

B

C

ppm20253035404550556065707580859095100105110

epoxide

OC H2 O H

N HAc

O

O

S O 3-

O

O H

C O 2-O HO

IdoA 1-3 GalNAc4SO3

dermatansulfate

OCO2-

O

OCH2OSO3-

NHSO3-

OHO O

-L-2,3-epoxy-GulA 1-4 GlcNAc6SO3

Semin Thromb Hemost, 274, 100-123 2001 M. Guerrini, A.Bisio, G. Torri, Characterization of Heparin Preparations Combined Quantitative 1H and 13C Nuclear Magnetic Resonance Spectroscopy for


(ppm)2030405060708090100110

heparin 1Commercial drug collected on 2002

*

*

(ppm)2030405060708090100110

heparin 2Commercial drug

L.R.L.R.DS

DS

ETOHETOH

DS

* EDTA

Heparin 13C NMR spectra: contaminants

Casu B, Naggi A, Oreste P, Torri G, Pangrazi J, Maggi A, Abbadini M, Donati M.B.Bleeding associated with heparin contaminanants Lancet Letter 21 march 1987

sample bleeding timeHeparin 0.75 mg/Kg 164Heparin + 1% EDTA 207Heparin + 3% EDTA 275 5% EDTA 176


4A6OH

H1 A3S

H1 I2S

H1 I

H2 ANS

(ppm) 5.2 4.8 4.4 4.0 3.6 3.2

104

96

88

80

72

64

56

(ppm)

A6S

H2 G

H2 A

3S

Advanced characterisation:quali- & quantitative

2D 1H-13C correlation spectrum(HSQC)

Guerrini M. et al 2001Semin Thromb Hemost, 274, 100-123.


Characterization of heparins: monosaccharides molar % contentdetermined via HSQC spectra

Guerrini M. et al ; 2001 Semin Thromb Hemost, 274, 100-123.


5G. Torri and M. Guerrini. Quantitative 2d nmr analysis of glycosaminoglycansin 'nmr spectroscopy in pharmaceutical analysis,Part III, chapter 4, ed

Holzgrabe; Integra, India; 2008, 407-427


6065707580859095100105110 ppm180 ppm 2426 ppm

**

*

*

***

Standard Heparin

Contaminated heparin

M Guerrini,D Beccati, Z Shriver, A Naggi, K Viswanathan, A. Bisio, I Capila, J C Lansing, S Guglieri, B Fraser, A Al-Hakim, N S Gunay, Z Zhang, L Robinson, L Buhse, M Nasr, J Woodcock, R Langer, G Venkataraman, R J Linhardt, B Casu, G Torri1 & R Sasisekharan; Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events; Nature Biotechnology 2008 Apr 23 [Epub ahead of print] G. Ronzoni Institute

6Red ChS-OSBlack contaminated heparin

3.23.43.63.84.04.24.44.64.85.05.25.45.65.8ppm

3.23.43.63.84.04.24.44.64.85.05.25.45.65.8 ppm

55

60

65

70

75

80

85

90

95

100

105

55

60

65

70

75

80

85

90

95

100

105


1.92.02.12.2 ppm

0,55%COCH3+

ChS-OS

*

COCH3 HepAcetyl region of 600 MHz proton spectra of heparinscontaminated with different amount of OCS

Normal 1H spectrum

1H spectrum, 13C decoupled

ChS-OS


71.92.02.12.2 ppm

Hep + 0.5% ChS-OS

Hep + 0.2% ChS-OS

Hep + 0.1% ChS-OS

Hep + 0.05% ChS-OS

Hep + 0.02% ChS-OS

1.92.02.12.2 ppm


Acetyl region of 400 and 600 MHz proton spectra of heparinscontaminated with different amount of OCS

Electropherograms on cellulose acetate (HCl/KCl) of different heparin samples: A, B, and C are recalled commercial heparin preparations in comparison with a house reference heparin. Only samples A and B show an extra component. G. Ronzoni Institute

Other techniques

heparin monography: not less than +35

OSCS** -15

*

* Courtesy of dr. Maggia (LDO SpA) ** courtesy of dr Mascellani (Opocrin SpA)

8G6242a_01.vdt: Mw 28,000 detector: Refractive Index13_03_08_01.vdt: Mw 18,000 detector: Refractive IndexG6196_01.vdt: Mw 20,100 detector: Refractive IndexG6243C_01.vdt: Mw 30,000 detector: Refractive Index

-150.00

34.62

80.77

126.92

173.08

219.23

265.38

311.54

357.69

403.85

450.00

Ref

ract

ive

Inde

x(m

V)

Retention Volume (mL)9.50 9.85 10.19 10.54 10.88 11.23 11.58 11.92 12.27 12.62 12.96 13.31 13.65 14.00


House reference heparin

Mw determination through SEC/multiple detector approach


synthetic OSCS sample

G6242a_01.vdt: Mw 28,000 detector: Refractive Index13_03_08_01.vdt: Mw 18,000 detector: Refractive IndexG6196_01.vdt: Mw 20,100 detector: Refractive IndexG6243C_01.vdt: Mw 30,000 detector: Refractive Index

-150.00

34.62

80.77

126.92

173.08

219.23

265.38

311.54

357.69

403.85

450.00

Ref

ract

ive

Inde

x(m

V)

Retention Volume (mL)9.50 9.85 10.19 10.54 10.88 11.23 11.58 11.92 12.27 12.62 12.96 13.31 13.65 14.00

extracted OSCS (purity >85%)


House reference heparin

Mw determination through SEC/multiple detector approach


92.02.53.03.54.04.55.05.5 ppm

heparin

Heparin 2 Contaminated ?


2.02.53.03.54.04.55.05.5 ppm

heparin 2 Contaminated 4%

heparin 2 contaminated 20%


10

ppm

3.03.23.43.63.84.04.24.44.64.85.05.25.45.65.8 ppm

50

55

60

65

70

75

80

85

90

95

100

105

110

50

55

60

65

70

75

80

85

90

95

100

105

110

Heparin + 4% of alginate sulfate


Informations coming from NMR analysis:13C spectra

The origin of a pure heparin preparationPossible contamination by other GAGs (quali- & quantitative data)The presence of other contaminants (qualitative data)Structural peculiarity (linkage region, sulfated versus unsulfated uronic acid)Structural damages (epoxides, N-desulfation)

1H spectraThe origin of a pure heparin preparationPossible contamination by other GAGs (quali- & quantitative data)The presence of other contaminants (quantitative data)Structural peculiarity (N-acetylation and N-sulfation degree, gluronic and iduronic acid)

Heteronuclear spectraFully quantitative evaluation of the components of heparin macromoleculeThe complete way to control molecular purity

Oversulphated chondroitin sulphate (OSCS)in heparin. NMR analyses.

IdentificationQuantificationLimits of detection (LOD)Some other topics

Heparin

Repeating disaccharide unit

1H-NMR spectrum of Heparin

-CH3

1H-NMR spectra of Heparin (blue line),and of contaminated heparin (red line).

contaminant

-CH3 (heparin)

acetate

Identification of the contaminant

Oversulphated chondroitin sulphate (OSCS) was one of the main suspects.

OSCS

Repeating disaccharide unit inchondroitin sulphate

Chemicallymodified

Identifying contaminants can be done by comparing with published data.1H-NMR spectra of Heparin (blue line), and of heparin contaminated with OSCS(red line). The spectrum at the bottom is a published 1H-NMR spectrum of OSCS.(T. Maruyama, et. al. Carbohydrate research 306, (1998), 35).

Advanced NMR experiments can be performed to identify the unknowncontaminant. In this example, a TOCSY spectrum (blue-green) and a NOESYspectrum (red) of contaminated heparin are superimposed.

To identify the unknown contaminant, one can also get hold of or synthesizea reference material. A HSQC spectrum (a 2-D NMR experiment) is shownof oversulphated chondroitin sulphate (OSCS).

1H-axis

13C-

axis

1H-NMR spectrum of heparin contaminated withOSCS (1 mol% on a disaccharide basis).

OSCS (-CH3) heparin (-CH3)

What is the limit of detection?

From different sources, a limit of 1% has been given.Our experience is that by increasing the signal to noise ratio(S/N), the limit of detection can be less than 0.1 mol% (on adisaccharide basis).

1H-NMR spectra (obtained with a 300 MHz instrument) of heparin (red line) and of heparinspiked with OSCS (blue line).When the size of the OSCS peak is similar to the size of one of the satellites, there isapproximately 0.05% OSCS in heparin (on a disaccharide basis).

N:B: In OSCS, every disaccharide is acetylated, but roughly every fifth disaccharide in heparin is acetylated..

OSCS (0.2%)

13 C satellites of the methyl protonsin heparin

S/N=800

S/N=600

S/N=450

S/N=300

S/N=200

13 C satellites of the methyl protonsin heparin

The limit of detection can be lowered by increasing the signal to noise ratio (S/N).This is done by increasing the number of scans (transients in NMR jargon) and/orby increasing the concentration of the solution.

Is the chemical shift value of 2.15 ppm 0.02 for OSCS inheparin reliable?

It seems to be acceptable for unfractionated heparin, but caremust be taken with low molecular heparins (LMMH). OSCS in atleast one LMMH "behaves" very differently.

In case of uncertainty, one should spike the sample beinganalyzed with OSCS.

1H-NMR spectra of OSCS (0.6 mg in 0.7 ml D2O) spiked with the low molecularheparin: nadroparin.The chemical shift of the methyl protons of OSCS moves from 2.12 ppm (greenline) to 2.18 ppm (red line). Both values outside the 2.15 0.02 ppm.

CH3OSCS

CH3nadroparin

1:15 ratio

1:2 ratio

100% OSCS

What NMR instruments can be used?

At least from 300 MHz and upwards (400, 500, 600 MHz).

At the Swedish MPA, we have two NMR instruments, a 300MHz, and a 600 MHz (with a CryoProbe).

1H-NMR spectra of the same contaminated low molecularheparin sample obtained with a 300 MHz (red line) and a 600MHz NMR instrument.

OSCS

OSCS

OSCS

OSCS13 C satellites

Number of heparin samples tested with NMR in our laboratories. APIand products. Unfractionated heparins and LMMH.

Samples from Sweden: 57 (2 contaminated).

Samples from five other countries: 116 (33 contaminated).

NMR spectroscopy for assessing heparin purity:

Very effective for the identification of unknown substances (no need forreference substances, although it helps).Very reliable method for the detection and quantification of impurities andcontaminants.Very fast analytical method. Less than an hour per sample, frompreparation to printed results.

In our endeavour to start a validating work on the detection and quantification ofimpurities / contaminants in heparin (unfractionated and LMMH) with NMR, weare working on:

Characterization and identification of heparins, natural impurites like dermatansulphate, and non-natural contaminants like OSCS.Quantitation and detection limits of impurities / contaminants: Integration ofpeaks; heparin as internal standard; other internal standard; etc.How to define impurity / contamination: weight %; mol % on a disaccharidebasis; area % (related to total methyl groups of present polysaccharides, or insteadrelated to the 13C satellites); etc.The influence on the proton chemical shifts of heparin, impurities andcontaminants by temperature, concentration, pH and intermolecular interactions.NMR running procedures to secure accuracy, precision, specificity, range, LOD,LOQ, and effectiveness.

Thank you for your attention!

Dr Ian McEwenNMR specialistSwedish Medical Products Agency (MPA)

E-mail: [email protected]

1a Novartis company

Electrophoretic method to separate andquantify contaminants in heparin

Dr. Thomas Freudemann

Head IPC Analytical & Tech. Process SupportLabs

Sandoz GmbH, Plant Schaftenau, Austria

2 Presentation Title / Name / Date

Contents

1 History & Introduction

2 Electrophoresis method

3 Comparison with 1H-NMR and CE methods

4 Conclusions


History & Introduction

1990s Development of analytical method feasible for quantificationof dermatan sulfate in heparin by Biochemie/Sandoz labs: One-dimensional cellulose acetate plate electrophoresis

Start of electrophoretic quality control of heparin by Biochemie/Sandoz Austria.

2006: First detection of new impurity in heparin sourced in China bySandoz electrophoretic method; contaminated material wasrejected. CAE Method provided to Chinese suppliers and

pre-shipment analysis of heparin raw material established.

2008: Oversulfated chondroitin sulfate (OSCS) identified as contaminant in heparin from Chinese sources.


Heparin on the market in early 2008

1, 6, 7 are heparin samples from the market in early 2008 withOSCS-contamination contents of up to 20%.

1 2 3 4 5 6 7

2 control sample; 3 - 5 dermatan sulfate standards 0,5%, 2,5% and 4,5%

Dermatan sulfate

OSCS impurity


Sandoz electrophoresis method in short

1 . Sample spots are applied to cellulose acetate electrophoresis plate

1 . Heparin is degraded by application of nitrous acid; this step may beomitted for separation of nitrous acid-degradable heparin fractions (withN-sulfated groups) themselves (fast- and slow-moving heparin)

2 . Residual (non nitrous acid degradable) GAGs are separated on plate byapplication of electric field

3 . Plate with separated spots is stained with Alcian blue 8GS: GAG spotsare permanently coloured, extra dye is washed off the plate

4 . Quantification of GAGs by means of comparision of optical density ofstained spots with reference standards (may be simplified to limit test)


Sample of CAE instruction video

Full-length video will be shipped upon request pls. contact [email protected]


Specifity of Sandoz electrophoresis method

1) Heparin sample; dermatan sulfate content < 0.5 %2) Control sample (with a known dermatan sulfate content of 2.0 %)3) Dermatan sulfate standard 0.5 %4) Dermatan sulfate standard 2.5 %5) Dermatan sulfate standard 4.5 %6) Chondroitin sulfate A&C 0.6 %7) Heparin sample with unknown impurity 1.9 %8) Mix: heparin sample with unknown impurity 1.9 % + dermatan sulfate 0.5 % + chondroitin sulfate A&C 0.6 %

Other non nitrous aciddegradable residues

Chondroitin sulfate A&C

Dermatan sulfate

OSCS

1) 2) 3) 4) 5) 6) 7) 8)

Heparin is degraded to greatextent by nitrous acid; residues donot interfere with other GAGs

Dermatan sulfate, OSCS andchondroitin sulfate A&C areclearly separated and can bequantified by means ofcomparison of optical density withthree-point dermatan sulfatestandard calibration

If exact quantification of GAGs isnot desired -> simplified limit testby optical comparison of samplewith 0,5% dermatan sulfatestandard is sufficient -> higherthroughput, no densitometricevaluation.


Electrophoresis without nitrous acid degradation

Chondroitin A/C

Hyaluronicacid

Heparan sulfate

Dermatan sulfate

Slow-moving heparin

Fast-moving heparin

OSCS impurity is covered byheparin bands

Dermatan sulfate cannot bequantified due to overlappingwith heparin bands

analysis of ratio of slow- vs.fast-moving heparin possible

1 Pure pharmaceutical heparin2 GAG-mixture


Densitometric evaluation

Electrophoresis plate isscanned and optical density ofthe spots is evaluated.

Optical densities of samplespots are compared to linearthree-point calibration functionprovided by dermatan sulfatestandards.


Other results of validation study

LOD: 0,4 %

LOQ: 0,5%

examined range: 0.4% to 4.8%

corr. coeff: 0,9997

y-axis intercept 7.049

slope 806.646

res. sum of squares 0.9995

repeatability: rel = 1,823 %rel.intermediate precision: rel = 2,610 %rel.

Linearity Plot

0

500

1000

1500

2000

2500

3000

3500

4000

0 1 2 3 4 5

Dermatan sulfate %

Are

a


1st FDA-Method: 1H-NMR

Some pros and cons

Orthogonal test for unknownsubstances in heparin with respectto separation techniques

Due to structural inhomogenities ofheparin and OSCS, signal intensityof additional feature may vary

Characteristic peak of dermatansulfate close to peak of additionalfeature

Expensive NMR-equipmentnecessary


2nd FDA-Method: Capillary Zone Electrophoresis

Some pros and cons

No baseline separation ofheparin and its impurity ->quantification of impurity may bedifficult Automated, high-throughputanalysis possible Expensive CE-equipmentnecessary No additional information withrespect to Sandozelectrophoresis method


Method Comparison: Overview

Heparin batchcontainingno detectableOSCS

Heparin batchcontainingapprox. 10 %OSCS

CE NMR (300 MHz) CA-ELPHO

Dermatan-sulfate

OSCS


Cons of Sandoz electrophoresis method

Throughput limited by size of electrophoresis plate

Automation not possible due to many manual steps

Skilled analyst necessary to successfully perform analysis

Plate electrophoresis is not (yet) a common method


Pros of Sandoz electrophoresis method

Sensitive: LOD: 0,4 % LOQ: 0,5%

Specific: distinct GAG spots visible, no interference with heparin

Either quantitative analysis or simplified limit test possible

Detection of various GAGs (e.g. dermatan sulfate, chondroitin sulfateA&C and oversulfated chondroitin sulfate (OSCS)) in a singleelectrophoretic run

Separation mechanism different from HPLC/GPC/CE

Analysis of fast/slow moving heparin fractions by small methodvariation (no nitrous acid degradation) possible

Only inexpensive equipment is needed

Validated, reliable method


Conclusions

Reliable methods for detection of GAG impurities in heparin are inurgent need by pharmaceutical industry and authorities.

Sandoz cellulose acetate plate electrophoresis method is validatedand feasible for this purpose and may be used to detect and quantifyvarious GAGs for other matters as well.

Analytical data delivered by Sandoz electrophoresis method issuperior to current CE methods; 1H-NMR is not a separationtechnique which therefore may yield additional valuable information.

11

research based, people driven

Method for determination ofGalactosamine

as part of total Hexosamines

Presentation at 2nd Workshop

on the Characterisation of Heparin Products

Rhonda Lecky, LEO Pharma20th June 2008, Strasbourg, France

2


LEO PharmaManufacturing Heparin API

ESBJERGLEO Pharma

CORK WEXPORT LTD

23


Agenda

Galactosamine as part of Hexosamines Method Overview

Heparin and Chondroitin Composition

Hydrolysis sample preparation

Method Principle HPIC analytical technique

Linearity

Combination techniques Plasma assay vs chromatogenic assay

Results

4


Method Overview and Background

LEO Pharma has analysed Galactosamine in HeparinSodium routinely since July 1992 using a GC Method

Recently we developed and validated a High PerformanceIon Chromatography method which is based on a DionexCorporation published method (Dionex technical note No.40.Analysis of carbohydrates by high performance anion exchangechromatography with pulsed amperometric detection. 2004.)

35


Glycosaminoglycans - Heparin and Chondroitin Sulphate

HEPARIN CHONDROITIN SULPHATE

Repeat unit of Chondroitin Sulphate

Glucosamine and uronic acid Galactosamine and uronic acid

Heparin and Chondroitin Composition

6


Sample preparation: Hydrolysis into monomers

Heparin hydrolyses into Glucosamine (GluN) monomers Chondroitins hydrolyse into Galactosamine (GalN)

monomers HCl best for Glucosaminoglycans

47


Hydrolysis GalN Profile

Practical Optimum at 6 hr

Heparin Sodium GalN Hydrolysis Trend

Sample EA9141

0

0.1

0.2

0.3

0 2 4 6 8 10 12 14

Time Points (Hrs)

Are

a n

C*m

in

8


HydrolysisGluN Profile

Practical Optimum at 6 hr

Heparin Sodium GluN Hydrolysis Trend

Sample EA9141

0

5

10

15

20

25

30

0 2 4 6 8 10 12 14Time Points (Hrs)

Are

a n

C*m

in

59


HPIC Instrument Conditions HPIC System:

High Performance Ion Chromatography system with PEEK(Polyetheretherketone) flow path from injector to detector

Detector: Pulsed Amperometric detector (PAD) with gold cell, Ag/Cl reference

electrode set to standard carbohydrate quad waveform setting

Column System: Column heater at 30 C. Dionex amino acid trap column BioLC

AminoTrap 3 x 30mm Dionex CarboPac PA20G 3 x 30 mm guardcolumn Dionex ion exchange column CarboPac PA-20, 3 x 150 mm

Run Time: 10 min

Injection Volume: 10 L

Mobile Phase: Degassed: 14 mM Potassium Hydroxide in P.W.

Flow rate: 0.5 ml per minute

10


Chromatograph of HeparinSodium Sample

0.16 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.03

-14.1

0.0

10.0

20.0

30.0

40.0

50.0

64.9 11 0107 # 7 T0.15 EA 9 141 1:250 d il ution ED_1nC

min

1 - 1 .034

2 - 1.134

3 - 5.900

4 - 7.15 0

nC

*m

in

Time (min)

Glucosamine

Galactosamine

611


Validation - Linearity

Linearity of Galactosamine

y = 0.99x + 0.27

R2 = 1.00

0

5

10

15

20

25

30

35

0 10 20 30 40

uM GluN

Area n

C*m

in

Linearity of Glucosamine

y = 0.91x + 0.13

R2 = 1.00

0

5

10

15

20

25

30

35

0 10 20 30 40

uM GluN

Area n

C*m

in

12


Linearity of USP and Ph. Eur Condroitin Sulphate Sodium Certified

Reference Standards analysed by HPIC Analysis SAM C237 method

R2 = 1.00

R2 = 1.00

0

2

4

6

8

10

12

14

16

18

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

CS Concentration (mg / L)

Pe

ak

Are

a R

es

po

ns

e (

nC

*min

)

Ph. Eur CS

USP CS

Linearity of USP and Ph.Eur. certifiedreference standards

Chondroitin Sulphate Sodium

713


Analytical PerformanceAccuracy: Recovery > 99 %

Precision: 9 days

14


Combination techniques

Use of Anti-factor IIa and complete depolymerisationusing Heparinase

815


Plasma assayvs

Anti IIa chromogenic assay

Plasma Assay (Ph.Eur. 2.7.5 and USP heparin Assay) The anticoagulant activity of heparin is determined in vitro by

comparing its ability in given conditions to delay the clottingof recalcified citrated sheep plasma with the same ability of areference preparation of heparin calibrated in InternationalUnits

Heparin Co-factor II mediated and AT-III mediated anti-IIa

Heparin and dermatan sulphate, as well as modifiedchondroitins i.e. OSCS, will give a response

Chromogenic Anti IIa Assay Similar to the Ph.Eur. Anti IIa method for LMW heparins or

the USP method for Anti Xa determination in heparin.

Antithrombin III mediated

Only heparin will give a response as thepentasaccharide is required.

16


Complete Depolymerisation ofHeparin Sodium using heparinase

Heparinase will not depolymerise chondroitins

917


Preparation of OSCS sample

A solution of Heparin Sodium and OSCS was treatedwith heparinase with the purpose of completelydepolymerising the content of Heparin Sodium.

A precipitation with 1.0Vol Ethanol was carried out.

The precipitated substance was characterised usingCE (one peak) and 1HNMR (clear OSCS peak; nodermatan sulphate peak) = purified OSCS obtained

18


Results

Heparin assay (Ph.Eur.) method 59.8 IU/mg

Anti-factor IIa method

10

19


Results

120

130

140

150

160

170

180

190

200

210

1.85 1.90 1.95 2.00 2.05 2.10 2.15

D32 Hep. Std.

OSCS

DC5491

OSCS

Heparin RS

Heparin

Slope/Rate

Log 2 Conc

Slope of OSCS: 60 % of Heparin Sodium

20


Discussion In the plasma assay response lines of different

substances will not be parallel = different slopes

The OSCS response line has a considerable differentslope than the line of Heparin Sodium

This method could possibly reveal anti-coagulantcontaminants other than OSCS in the future.

A combination of heparinase/chondroitinasetreatment could be interesting to investigate as theanti-coagulant activity of Dermatan Sulphate(naturally present in Heparin crude material) alsowould be eliminated i.e. If activity is found in theplasma assay a contaminant is present in theHeparin Crude material.

11

21


Thank you for listening!

Documents

Session 2 Global Harmonisation Part 2