Session 2 Global Harmonisation Part 2
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Transcript of Session 2 Global Harmonisation Part 2
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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
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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
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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.
G. Ronzoni Institute
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
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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
G. Ronzoni Institute
(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
G. Ronzoni Institute
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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.
G. Ronzoni Institute
Characterization of heparins: monosaccharides molar % contentdetermined via HSQC spectra
Guerrini M. et al ; 2001 Semin Thromb Hemost, 274, 100-123.
G. Ronzoni Institute
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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
G. Ronzoni Institute
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
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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
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G. Ronzoni Institute
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
G. Ronzoni Institute
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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
G. Ronzoni Institute
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)
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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
Contaminated heparin
House reference heparin
Mw determination through SEC/multiple detector approach
G. Ronzoni Institute
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%)
Contaminated heparin
House reference heparin
Mw determination through SEC/multiple detector approach
G. Ronzoni Institute
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92.02.53.03.54.04.55.05.5 ppm
heparin
Heparin 2 Contaminated ?
G. Ronzoni Institute
2.02.53.03.54.04.55.05.5 ppm
heparin 2 Contaminated 4%
heparin 2 contaminated 20%
G. Ronzoni Institute
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10
ppm
3.03.23.43.63.84.04.24.44.64.85.05.25.45.65.8 ppm
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Heparin + 4% of alginate sulfate
G. Ronzoni Institute
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
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Oversulphated chondroitin sulphate (OSCS)in heparin. NMR analyses.
IdentificationQuantificationLimits of detection (LOD)Some other topics
Heparin
Repeating disaccharide unit
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1H-NMR spectrum of Heparin
-CH3
1H-NMR spectra of Heparin (blue line),and of contaminated heparin (red line).
contaminant
-CH3 (heparin)
acetate
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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).
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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
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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).
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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.
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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
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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
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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.
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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]
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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
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23 Presentation Title / Name / Date
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.
4 Presentation Title / Name / Date
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
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35 Presentation Title / Name / Date
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)
6 Presentation Title / Name / Date
Sample of CAE instruction video
Full-length video will be shipped upon request pls. contact [email protected]
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47 Presentation Title / Name / Date
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.
8 Presentation Title / Name / Date
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
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59 Presentation Title / Name / Date
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.
10 Presentation Title / Name / Date
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
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611 Presentation Title / Name / Date
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
12 Presentation Title / Name / Date
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
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713 Presentation Title / Name / Date
Method Comparison: Overview
Heparin batchcontainingno detectableOSCS
Heparin batchcontainingapprox. 10 %OSCS
CE NMR (300 MHz) CA-ELPHO
Dermatan-sulfate
OSCS
14 Presentation Title / Name / Date
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
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815 Presentation Title / Name / Date
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
16 Presentation Title / Name / Date
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.
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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
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LEO PharmaManufacturing Heparin API
ESBJERGLEO Pharma
CORK WEXPORT LTD
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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
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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.)
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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
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Sample preparation: Hydrolysis into monomers
Heparin hydrolyses into Glucosamine (GluN) monomers Chondroitins hydrolyse into Galactosamine (GalN)
monomers HCl best for Glucosaminoglycans
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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
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HydrolysisGluN Profile
Practical Optimum at 6 hr
Heparin Sodium GluN Hydrolysis Trend
Sample EA9141
0
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10
15
20
25
30
0 2 4 6 8 10 12 14Time Points (Hrs)
Are
a n
C*m
in
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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
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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
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Validation - Linearity
Linearity of Galactosamine
y = 0.99x + 0.27
R2 = 1.00
0
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15
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30
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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
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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
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12
14
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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
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Analytical PerformanceAccuracy: Recovery > 99 %
Precision: 9 days
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Combination techniques
Use of Anti-factor IIa and complete depolymerisationusing Heparinase
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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.
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Complete Depolymerisation ofHeparin Sodium using heparinase
Heparinase will not depolymerise chondroitins
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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
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Results
Heparin assay (Ph.Eur.) method 59.8 IU/mg
Anti-factor IIa method
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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
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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.
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Thank you for listening!