Session 2 Global Harmonisation Part 2

EDQM
EDQM
-
-
USP
USP
-
-
NI BSC
NI BSC

W
W
ORKSHOP ON THE
ORKSHOP ON THE

C
C
HARACTERISATI ON OF
HARACTERISATI ON OF
H
H
EPARI N
EPARI N
P
P
RODUCTS
RODUCTS

SESSI ON 2
SESSI ON 2

GLOBAL HARMONI SATI ON GLOBAL HARMONI SATI ON

1
Advanced 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 products
19-20 June 2008, EDQM, Strasburg, France
Giangiacomo Torri
(ppm)
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
COCH
3
A
6S
A
6OH
Heparin NMR spectra
1
H
13
C
I
2OH
G. Ronzoni Institute
2
Heparin 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 by
nuclear magnetic resonance spectroscopy. Arzneim-Forsh (Drug Res) 1996; 46:472477.
Linkage Region Sequences of Heparins and Heparan
Sulfates: Detection and Quantification by NMR
Spectroscopy
M. Iacomini, B. Casu, M. Guerrini, A. Naggi, A. Pirola, and G. Torri
Analytical Biochemistry 274, 5058 (1999)
Heparins
Heparan sulfates
3
Heparin
13
C NMR spectra: contaminants
ethanol
A
B
C

ppm
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110
epoxide
O
CH
2
OH
NHAc
O
O
SO
3
-
O
OH
CO
2
-
OH
O
IdoA 1-3 GalNAc4SO
3
dermatansulfate
O
CO
2
-
O
O
CH
2
OSO
3
-
NHSO
3
-
OH O O
-L-2,3-epoxy-GulA 1-4 GlcNAc6SO
3
Semin Thromb Hemost, 274, 100-123 2001 M. Guerrini, A.Bisio, G. Torri,
Characterization of Heparin Preparations Combined Quantitative
1
H and
13
C Nuclear Magnetic Resonance Spectroscopy for
(ppm)
20 30 40 50 60 70 80 90 100 110
heparin 1
Commercial drug collected on 2002
*
*
(ppm)
20 30 40 50 60 70 80 90 100 110
heparin 2
Commercial drug
L.R.
L.R.
DS
DS
ETOH
ETOH
DS
* EDTA
Heparin
13
C 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 time
Heparin 0.75 mg/Kg 164
Heparin + 1% EDTA 207
Heparin + 3% EDTA 275
5% EDTA 176
4
A
6OH
H1 A
3S
H1 I
2S
H1 I
H2 A
NS
(ppm)
5.2 4.8 4.4 4.0 3.6 3.2
104
96
88
80
72
64
56
(ppm)
A
6S
H
2

G
H
2

A
3
S
Advanced characterisation:
quali- & quantitative
2D
1
H-
13
C correlation spectrum
(HSQC)
Guerrini M. et al 2001
Semin Thromb Hemost, 274, 100-123.
Characterization of heparins: monosaccharides molar % content
determined via HSQC spectra
Guerrini M. et al ; 2001 Semin Thromb Hemost, 274, 100-123.
5
G. Torri and M. Guerrini. Quantitative 2d nmr analysis of glycosaminoglycans
in 'nmr spectroscopy in pharmaceutical analysis,Part III, chapter 4, ed
Holzgrabe; Integra, India; 2008, 407-427
60 65 70 75 80 85 90 95 100 105 110 ppm 180 ppm 24 26 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]
6
Red ChS-OS
Black contaminated heparin
3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8
ppm
3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 ppm
55
60
65
70
75
80
85
90
95
100
105
55
60
65
70
75
80
85
90
95
100
105
1.9 2.0 2.1 2.2 ppm
0,55%COCH
3
+
ChS-OS
*
COCH
3
Hep
Acetyl region of 600 MHz proton spectra of heparins
contaminated with different amount of OCS
Normal
1
H spectrum
1
H spectrum,
13
C decoupled
ChS-OS
7
1.9 2.0 2.1 2.2 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
1.9 2.0 2.1 2.2 ppm
Acetyl region of 400 and 600 MHz proton spectra of heparins
contaminated 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.
Other techniques
heparin monography:
not less than +35
OSCS** -15
*
* Courtesy of dr. Maggia (LDO SpA) ** courtesy of dr Mascellani (Opocrin SpA)
8
G6242a_01.vdt: Mw 28,000 detector: Refractive Index
13_03_08_01.vdt: Mw 18,000 detector: Refractive Index
G6196_01.vdt: Mw 20,100 detector: Refractive Index
G6243C_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
R
e
f
r
a
c
t
iv
e
I
n
d
e
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 Index
13_03_08_01.vdt: Mw 18,000 detector: Refractive Index
G6196_01.vdt: Mw 20,100 detector: Refractive Index
G6243C_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
R
e
f
r
a
c
t
iv
e
I
n
d
e
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
9
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 ppm
heparin
Heparin 2
Contaminated ?
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 ppm
heparin 2
Contaminated 4%
heparin 2
contaminated 20%
10
ppm
3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 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:
13
C spectra
The origin of a pure heparin preparation
Possible 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)
1
H spectra
The origin of a pure heparin preparation
Possible 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 spectra
Fully quantitative evaluation of the components of heparin macromolecule
The complete way to control molecular purity
Oversulphated chondroitin sulphate (OSCS)
in heparin. NMR analyses.
Identication
Quantication
Limits of detection (LOD)
Some other topics
Heparin
Repeating disaccharide unit
1
H-NMR spectrum of Heparin
-CH
3
1
H-NMR spectra of Heparin (blue line),
and of contaminated heparin (red line).
contaminant
-CH
3
(heparin)
acetate
Identication of the contaminant
Oversulphated chondroitin sulphate (OSCS) was one of the main suspects.
OSCS
Repeating disaccharide unit in
chondroitin sulphate
Chemically
modied
Identifying contaminants can be done by comparing with published data.
1
H-NMR spectra of Heparin (blue line), and of heparin contaminated with OSCS
(red line). The spectrum at the bottom is a published
1
H-NMR spectrum of OSCS.
(T. Maruyama, et. al. Carbohydrate research 306, (1998), 35).
Advanced NMR experiments can be performed to identify the unknown
contaminant. In this example, a TOCSY spectrum (blue-green) and a NOESY
spectrum (red) of contaminated heparin are superimposed.
To identify the unknown contaminant, one can also get hold of or synthesize
a reference material. A HSQC spectrum (a 2-D NMR experiment) is shown
of oversulphated chondroitin sulphate (OSCS).
1
H-axis
1
3
C
-
a
x
i
s
1
H-NMR spectrum of heparin contaminated with
OSCS (1 mol% on a disaccharide basis).
OSCS (-CH
3
)
heparin (-CH
3
)
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 a
disaccharide basis).
1
H-NMR spectra (obtained with a 300 MHz instrument) of heparin (red line) and of heparin
spiked with OSCS (blue line).
When the size of the OSCS peak is similar to the size of one of the satellites, there is
approximately 0.05% OSCS in heparin (on a disaccharide basis).
N:B: In OSCS, every disaccharide is acetylated, but roughly every fth disaccharide in heparin is acetylated..
OSCS (0.2%)
13
C satellites of the methyl protons
in heparin
S/N=800
S/N=600
S/N=450
S/N=300
S/N=200
13
C satellites of the methyl protons
in 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/or
by increasing the concentration of the solution.
Is the chemical shift value of 2.15 ppm 0.02 for OSCS in
heparin reliable?
It seems to be acceptable for unfractionated heparin, but care
must be taken with low molecular heparins (LMMH). OSCS in at
least one LMMH "behaves" very differently.
In case of uncertainty, one should spike the sample being
analyzed with OSCS.
1
H-NMR spectra of OSCS (0.6 mg in 0.7 ml D
2
O) spiked with the low molecular
heparin: nadroparin.
The chemical shift of the methyl protons of OSCS moves from 2.12 ppm (green
line) to 2.18 ppm (red line). Both values outside the 2.15 0.02 ppm.
CH
3
OSCS
CH
3
nadroparin
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 300
MHz, and a 600 MHz (with a CryoProbe).
1
H-NMR spectra of the same contaminated low molecular
heparin sample obtained with a 300 MHz (red line) and a 600
MHz NMR instrument.
OSCS
OSCS
OSCS
OSCS
13
C satellites
Number of heparin samples tested with NMR in our laboratories. API
and products. Unfractionated heparins and LMMH.
Samples from Sweden: 57 (2 contaminated).
Samples from ve other countries: 116 (33 contaminated).
NMR spectroscopy for assessing heparin purity:
Very effective for the identication of unknown substances (no need for
reference substances, although it helps).
Very reliable method for the detection and quantication of impurities and
contaminants.
Very fast analytical method. Less than an hour per sample, from
preparation to printed results.
In our endeavour to start a validating work on the detection and quantication of
impurities / contaminants in heparin (unfractionated and LMMH) with NMR, we
are working on:
Characterization and identication of heparins, natural impurites like dermatan
sulphate, and non-natural contaminants like OSCS.
Quantitation and detection limits of impurities / contaminants: Integration of
peaks; heparin as internal standard; other internal standard; etc.
How to dene impurity / contamination: weight %; mol % on a disaccharide
basis; area % (related to total methyl groups of present polysaccharides, or instead
related to the
13
C satellites); etc.
The inuence on the proton chemical shifts of heparin, impurities and
contaminants by temperature, concentration, pH and intermolecular interactions.
NMR running procedures to secure accuracy, precision, specicity, range, LOD,
LOQ, and effectiveness.
Thank you for your attention!
Dr Ian McEwen
NMR specialist
Swedish Medical Products Agency (MPA)
E-mail: ian.mcewen@mpa.se
1
a Novartis company
Electrophoretic method to separate and
quantify contaminants in heparin
Dr. Thomas Freudemann
Head IPC Analytical & Tech. Process Support
Labs
Sandoz GmbH, Plant Schaftenau, Austria
2 Presentation Title / Name / Date
Contents
1 History & Introduction
2 Electrophoresis method
3 Comparison with
1
H-NMR and CE methods
4 Conclusions
2
History & Introduction
1990s Development of analytical method feasible for quantification
of 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 by
Sandoz electrophoretic method; contaminated material was
rejected. 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 with
OSCS-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
3
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 be
omitted for separation of nitrous acid-degradable heparin fractions (with
N-sulfated groups) themselves (fast- and slow-moving heparin)
2. Residual (non nitrous acid degradable) GAGs are separated on plate by
application of electric field
3. Plate with separated spots is stained with Alcian blue 8GS: GAG spots
are permanently coloured, extra dye is washed off the plate
4. Quantification of GAGs by means of comparision of optical density of
stained 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 thomas.freudemann@sandoz.com
4
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 %
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 acid
degradable residues
Chondroitin sulfate A&C
Dermatan sulfate
OSCS
1) 2) 3) 4) 5) 6) 7) 8)
Heparin is degraded to great
extent by nitrous acid; residues do
not interfere with other GAGs
Dermatan sulfate, OSCS and
chondroitin sulfate A&C are
clearly separated and can be
quantified by means of
comparison of optical density with
three-point dermatan sulfate
standard calibration
If exact quantification of GAGs is
not desired -> simplified limit test
by optical comparison of sample
with 0,5% dermatan sulfate
standard is sufficient -> higher
throughput, no densitometric
evaluation.
Electrophoresis without nitrous acid degradation
Chondroitin A/C
Hyaluronic
acid
Heparan sulfate
Dermatan sulfate
Slow-moving heparin
Fast-moving heparin
OSCS impurity is covered by
heparin bands
Dermatan sulfate cannot be
quantified due to overlapping
with heparin bands
analysis of ratio of slow- vs.
fast-moving heparin possible
1 Pure pharmaceutical heparin
2 GAG-mixture
5
Densitometric evaluation
Electrophoresis plate is
scanned and optical density of
the spots is evaluated.
Optical densities of sample
spots are compared to linear
three-point calibration function
provided by dermatan sulfate
standards.
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 %
A
r
e
a
6
1
st
FDA-Method:
1
H-NMR
Some pros and cons
Orthogonal test for unknown
substances in heparin with respect
to separation techniques
Due to structural inhomogenities of
heparin and OSCS, signal intensity
of additional feature may vary
Characteristic peak of dermatan
sulfate close to peak of additional
feature
Expensive NMR-equipment
necessary
2
nd
FDA-Method: Capillary Zone Electrophoresis
Some pros and cons
No baseline separation of
heparin and its impurity ->
quantification of impurity may be
difficult
Automated, high-throughput
analysis possible
Expensive CE-equipment
necessary
No additional information with
respect to Sandoz
electrophoresis method
7
Method Comparison: Overview
Heparin batch
containing
no detectable
OSCS
Heparin batch
containing
approx. 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
8
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 sulfate
A&C and oversulfated chondroitin sulfate (OSCS)) in a single
electrophoretic run
Separation mechanism different from HPLC/GPC/CE
Analysis of fast/slow moving heparin fractions by small method
variation (no nitrous acid degradation) possible
Only inexpensive equipment is needed
Validated, reliable method
Conclusions
Reliable methods for detection of GAG impurities in heparin are in
urgent need by pharmaceutical industry and authorities.
Sandoz cellulose acetate plate electrophoresis method is validated
and feasible for this purpose and may be used to detect and quantify
various GAGs for other matters as well.
Analytical data delivered by Sandoz electrophoresis method is
superior to current CE methods;
1
H-NMR is not a separation
technique which therefore may yield additional valuable information.
1
1
research based, people driven
Method for determination of
Galactosamine
as part of total Hexosamines
Presentation at 2
nd
Workshop
on the Characterisation of Heparin Products
Rhonda Lecky, LEO Pharma
20
th
June 2008, Strasbourg, France
2
LEO Pharma
Manufacturing Heparin API
ESBJERG
LEO Pharma
CORK
WEXPORT LTD
2
3
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 Heparin
Sodium routinely since July 1992 using a GC Method
Recently we developed and validated a High Performance
Ion Chromatography method which is based on a Dionex
Corporation published method (Dionex technical note No.40.
Analysis of carbohydrates by high performance anion exchange
chromatography with pulsed amperometric detection. 2004.)
3
5
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
4
7
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)
A
r
e
a

n
C
*
m
i
n
8
Hydrolysis
GluN 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 14
Time Points (Hrs)
A
r
e
a

n
C
*
m
i
n
5
9
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 guard

column 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 Heparin
Sodium 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
11 010 7 # 7 T0.15 EA 9 1411:2 5 0d i l ution ED_1
nC
min
1-1 . 0 34
2-1.134
3-5.90 0
4-7.15 0
n
C
*
m
i
n
Time (min)
Glucosamine
Galactosamine
6
11
Validation - Linearity
Linearity of Galactosamine
y = 0.99x + 0.27
R
2
= 1.00
0
5
10
15
20
25
30
35
0 10 20 30 40
uM GluN
A
r
e
a

n
C
*
m
i
n
Linearity of Glucosamine
y = 0.91x + 0.13
R
2
= 1.00
0
5
10
15
20
25
30
35
0 10 20 30 40
uM GluN
A
r
e
a

n
C
*
m
i
n
12
Linearity of USP and Ph. Eur Condroitin Sulphate Sodium Certified
Reference Standards analysed by HPIC Analysis SAM C237 method
R
2
= 1.00
R
2
= 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)
P
e
a
k

A
r
e
a

R
e
s
p
o
n
s
e

(
n
C
*
m
i
n
)
Ph. Eur CS
USP CS
Linearity of USP and Ph.Eur. certified
reference standards
Chondroitin Sulphate Sodium
7
13
Analytical Performance
Accuracy: Recovery > 99 %
Precision: <3 % Relative Variation (% RSD)
Detection Limit: 0.24 % Galactosamine of total Hexosamines
(actual concentration 3 ppb Galactosamine)

Quantitation Limit: 0.70 % Galactosamine of total Hexosamines
(actual concentration 7 ppb Galactosamine)

Range: 0.7 to 50 % Galactosamine to total Hexosamines
Linearity: Correlation Coefficient R
2
= 1.00 over the range
Robustness: Robust to 10 % deliberate changes in
Temperature, Mobile phase concentration and flow rate.
Stability: Standards and samples stable for > 9 days
14
Combination techniques
Use of Anti-factor IIa and complete depolymerisation
using Heparinase
8
15
Plasma assay
vs
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 clotting
of recalcified citrated sheep plasma with the same ability of a
reference preparation of heparin calibrated in International
Units
Heparin Co-factor II mediated and AT-III mediated anti-IIa
Heparin and dermatan sulphate, as well as modified
chondroitins 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 the
pentasaccharide is required.
16
Complete Depolymerisation of
Heparin Sodium using heparinase
Heparinase will not depolymerise chondroitins
9
17
Preparation of OSCS sample
A solution of Heparin Sodium and OSCS was treated
with heparinase with the purpose of completely
depolymerising the content of Heparin Sodium.
A precipitation with 1.0Vol Ethanol was carried out.
The precipitated substance was characterised using
CE (one peak) and 1HNMR (clear OSCS peak; no
dermatan sulphate peak) = purified OSCS obtained
18
Results
Heparin assay (Ph.Eur.) method 59.8 IU/mg
Anti-factor IIa method <2 IU/mg
Anti-factor Xa method 2.6 IU/mg
Results confirm that a complete depolymerisation of
Heparin Sodium has occured and that OSCS only has
heparin co-factor II activity.
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
S
l
o
p
e
/
R
a
t
e
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 different
slope than the line of Heparin Sodium
This method could possibly reveal anti-coagulant
contaminants other than OSCS in the future.
A combination of heparinase/chondroitinase
treatment could be interesting to investigate as the
anti-coagulant activity of Dermatan Sulphate
(naturally present in Heparin crude material) also
would be eliminated i.e. If activity is found in the
plasma assay a contaminant is present in the
Heparin Crude material.
11
21
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