low molecular weight heparin
DESCRIPTION
Description of heparin productsTRANSCRIPT
-
EDQMEDQM--USPUSP--NIBSCNIBSC
WWORKSHOP ON THEORKSHOP ON THE CCHARACTERISATION OF HARACTERISATION OF
HHEPARIN EPARIN PPRODUCTSRODUCTS
S ES S I O N 3SES S I O N 3
LO W MO LE C ULA R WE I G HT HE PAR I NLO W MO LE C ULA R WE I G HT HE PAR I N
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1Heparin Characterization
Use of PCR to control origin of species
ANGER PascalJune, 20th 2008
Introduction
Enoxaparin monographSpecifies that enoxparin is derived from heparin of porcine origin
Why control origin?To ensure product consistencyTo ensure product safetyTo monitor good supplier practice2nd safeguard (in addition to technical agreement, audits,
process validated for viral & prion reduction)QPCR is currently the only routine method implemented which controls the species of origin
-
2Exclusive porcine origin by Q-PCR
Although already degraded by the process, DNA is present at crude Heparin stageDNA can be amplified and quantified by Inhibitory Controlled Quantitative Polymerase Chain Reaction (IC Q-PCR)
Fully validated methodAlready made public:
Poster: FASEB, Washington DC, April 28th May 2nd 2007Presentations: NIBSC 2007 workshop, EDQM April 2008 Accepted for publication in Clinical and Applied Thrombosis/Homeostasis
primer 1
Fragment to amplify
DNAprimer 2
probe
Q-PCR preliminary treatment
Crude Heparin mainly contains heparin (>50%) and also many uncontrolled impurities (heparan, chondroitins, proteins, nucleic acids) which are potential inhibitors
Heparin is a strong PCR inhibitor which is degraded in situ by heparinasesDevelopment and validation on all heparin sourcesSimultaneous (duplex PCR) control of the absence of inhibition by an Internal Positive Control amplification of an exogenous DNA fragment, using specific primers and probe (IC-QPCR)
Non compliant sample: contamination
Positive control
wLOD
Compliant sample:
NO contamination
Detection of contaminant DNA
Non compliant sample: contamination
Positive control
wLOD
Compliant sample:
NO contamination
Detection of contaminant DNA
-
3Q-PCR run in QC & justification
Specifications (amplifiable DNA)Porcine DNA 0.1 g/mg of crude HeparinRuminant DNA working LOD or
Bovine: 7pg/mg (or less than 0.007%)Ovine: 70pg/mg (or less than 0.07%)
Used in routine since Jan 2007 on every crude batch used for Lovenox manufacturing (replaces previous les reliable and sensitive extraction + qualitative PCR)Benchmarking 2007: on 7 potential (non approved) suppliers / 16 batches, 7 batches (4 suppliers) were contaminated
conclusion
Status in sanofi-aventisSystematic Q-PCR on every crude heparin batch by QC (more than 400 batches analyzed)No pure Chinese heparin used (only crude)Method proposed to the EDQM and USP
LimitationsWorks on crude heparin onlyCurrently looks for porcine, bovine, ovine and caprine
-
4Input in current crisis
UnableTo detect introduction of a DNA-free product, typically hemisynthetic product
ButAvoids mixture of heparin (or natural chondroitin) from different species due to possible porcine heparin shortageEnsures accuracy of NMR results
NMR accuracy and heparin origin
NMR principle% N-acetyl (HSCS)/ N-acetyl (heparin)
% N-acetyl (heparin) variabilityspecies porcine porcine porcine bovine ovine
quality pure pure crude pure pure
origin s-a suppliers
non s-a suppliers
non s-a suppliers
/ /
# batches 12 6 16 3 6
Nacetyl/NSO4 % 12.9 15.3 21.6 7.5 8.9
min-max 12.2-14.2 14.4-17.1 20.1-22.9 6.8-8.9 8.0-10.0
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11
Low molecular weight heparincharacterisation: past and current views
Christian Viskov, Ph D EDQM June 20, 2008
2
Low Molecular Weight Heparins: LMWH
Some basic aspectsLMWH are the result of the cleavage of the heparinmacromolecule (chemical or enzymatical)The usual average MW is between 3500 and 7000 DaBelow 3500 Da, the products are called Ultra Low MolecularWeight Heparins (ULMWH).
Aim of the depolymerisation:Improve the therapeutic margin of the productImprove pharmacokinetic propertiesReduce bleeding riskReduce non-specific binding to proteins, example PF4
-
23
Preparation of LMWH and ULMWH
How to depolymerise this heterogeneous macromolecule?
Does this depolymerization only relates to a size reduction?
O
O
O
NHAc
OH
OSO3Na
COONa
OH
OH
O
O
O
OSO3Na
OSO3Na
NHSO3Na OSO
3Na
COONaOH
O
OSO3Na
OH
NHSO3Na
O
O
OSO3Na
COONaOH
O
O
O
OSO3Na
OH
NHSO3Na OH
COONaOH
OCOONaOH
OSO3Na
O
O
OSO3Na
OH
NHSO3Na
O
O OOO
O
A HD
Minimal ATIII binding domain
2000HBPM
5000 10000 15000 20000 25000 30000
MolecularWeight
UFHUFH
4
Origin of LMWH
Industrial Manufacturing processes for LMWH preparation
Nitrous depolymerization:Fragmin, Fraxiparin, Reviparin, Certoparin.
Oxidative cleavage:Parnaparin.
Enzymatic - elimination:Tinzaparin
Chemical - elimination:Enoxaparin, Bemiparin,
-
35
Differentiation of LMWH: The concept
Modifications induced by the depolymerization process
Modifications at the cleavage pointFirst levelFirst level of LMWH of LMWH differentiationdifferentiation
Modification of the endogenous backbone (Sidereactions)Second levelSecond level of LMWH of LMWH differentiation: chemical fingerprint of differentiation: chemical fingerprint of LMWHLMWHss..
Cleavage selectivities vs. antithrombin binding site.Third levelThird level of LMWH of LMWH differentiationdifferentiation
Specific mixtures of ATIII binding and non-anticoagulantoligosaccharides depend on the process used
Each specific mixture leads to distinct biological properties
6
Structural differences : First level
Structural modification occurs around the cleavage point of heparinChemical or Enzymatic - elimination reactions
Enoxaparin, Bemiparin, Tinzaparin
O
COONa
OH
OSO3Na
O
OSO3Na
OH
NHSO3Na
OH
O
OSO3Na
OH
NHSO3Na
O
O
COONa
OH
O
O
O
O
OH
NH
O
O
COONa
OH
OSO3Na
SO3Na
SO3Na
O
SO3Na
O
n
(
( ( ))
)
X
Y Z
Heparin cleavage by nitrous acidFraxiparin, Fragmin
O
COONa
OH
OSO3Na
O
OSO3Na
OH
CH2OH
O
OSO3Na
OH
NHSO3Na
O
O
COONa
OH
O
O
O
O
OH
NH
O
O
COONa
OH
OSO3Na
SO3Na
SO3Na
O
SO3Na
O
n
(
( ( ))
)
X
Y Z
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47
Structural differences : Second level
Depolymerization processes modify endogenous disaccharidicbackbone and induce specific fingerprints through side reactions
Generation of non-natural disaccharides moieties
LMWH Enoxaparin Bemiparin Tinzaparin
Conditions class Chemical !- elimination :
Basic media
Chemical !- elimination :
Basic media
Enzymatic !- elimination :
Neutral media
Depolymerization reaction
Heparin benzyl ester
by NaOH
Depolymerisation of
Hepari n
benzethonium salt
by CTA+,OH
-
Heparin
by Heparinase I
Main side reactions
! 2-O desulfation
!1,6 anhydro ring
! Epimerization in
manosamine
! 2-O desulfation
! Epimerization in
manosamine
No side reactions
i.e: 1,6 anhydro ring is specific of Enoxaparin
8
Structural differences : Third level
Positions 1 are not cleaved during lovenox processesRespective cleavages of 2 and 3 positions depend on
Strength of the base and its steric hindranceReaction conditions (temperature, solvent ..)
Different -eliminative processes generate distinct ATIII binding oligosaccharides inprocess-dependent proportionsThe process can modify the chemical structure of a given AT binding site and eitherincrease or decrease the initial affinity.
O
O
O
NHAc
OH
OSO3Na
COONa
OH
OH
O
O
O
OSO3Na
OSO3Na
NHSO3Na OSO
3Na
COONa
OH
O
OSO3Na
OH
NHSO3Na
O
O
OSO3Na
COONa
OH
O
O
O
OSO3Na
OH
NHSO3Na OH
COONa
OH
O
COONa
OH
OSO3Na
O
O
OSO3Na
OH
NHSO3Na
OO OO
O
O
A HDATIII "core binding domain"
IsiduIIaidu IIsglu Isidu IsiduIsidu
1 22 3
1 3
Macromolecule cleavage selectivity
-
59
Mechanistic considerations on -eliminative process
DepolymerizationDepolymerizationmechanismmechanism::The E1cb The E1cb pathwaypathway
O
O
O
NHSO3Na
OH
OSO3Na
OH
OSO3Na
O OO O
O
OH
OH
O
O
O
NHSO3Na
OH
OSO3Na
OH
OSO3Na
O O
H
O O
O
OH
OH
O
O
O
NHSO3Na
OH
OSO3Na
OH
OSO3Na
O ONaO O
O
OH
OHO
O
O
NHSO3Na
OH
OSO3Na
OH
OSO3Na
O O
H
NaO O
O
OH
OH
O
OH
NHSO3Na
OH
OSO3NaNaO O
O
OH
OH
O
OH
OSO3Na
O O
O
O
O
NHSO3Na
OH
OSO3Na
OH
OSO3Na
NaO O
H
NaO O
O
OH
OH
O
OH
NHSO3Na
OH
OSO3NaNaO O
O
OH
OH
O
OH
OSO3Na
NaO O
O O
-OH
-OH
O-
Base
Elimination
OO +
E1cb
O
O +
1
2
35
4
10
Mechanistic considerations on -eliminative process
Aim of the fundamental studies: understand why the ATIII binding siteis not cleaved
Depolymerization of benzyl heparinate is selective of iduronic acidmoieties
Selectivity is controlled by steric and electronic repulsion control; i.e.:highly sulfated domains are usually avoided by basesThis favor the preservation of ATIII binding sequence and roughly theanti Xa activity
O
O
O
OH
NH
OSO3-
O
O
R
HOSO
3-
OH
O
O
OH
NH
OSO3-
R O
O
O
HOH
OH
O
O
O
OH
NH
OSO3-
O
O
R
HOH
OH
Iduronic acid residue Glucuronic acid residue
e
a
a
aa
a
2-O sullfate iduronic acid residue
Trans diaxial Trans diaxial Cis axial / equatorial
-
611
How to accurately analyze this complexmixture generated by depolymerisationprocess?
12
Disaccharides building block analysis
Exhaustive enzymatic digestion by Heparinases mixture followed byHPLC analysis may give access to the building block content in LMWH
Nevertheless such analysis does not give a clue how to reassemblethem in discrete oligosaccharides compounds
OO
OH
O
OH
CO2Na
OH
Ac
O
CH2
OH
NH
OH
OH
O
OH
CO2Na
OH
SO3Na
O
CH2
OH
NH
OH O
OH
O
OH
CO2Na
OSO3Na
Ac
O
CH2
OH
NH
OH
!IVa !IVs
!IIa
OO
O
OH
O
OH
CO2Na
OSO3Na
SO3Na
O
CH2
OH
NH
OH
OH
Ac
O
CH2
OH
NH
OH
OH
SO3Na
O
CH2
OH
NH
OH
OSO3Na
O
OH
CO2Na
OSO3Na
O
OH
CO2Na
!IIs!IIIa
!IIIs
OO
OSO3Na
O
OH
CO2Na
OSO3Na
O
OH
CO2Na
OSO3Na
Ac
O
CH2
OH
NH
OH
OSO3Na
SO3Na
O
CH2
OH
NH
OH
!Ia !Is
Example of tetrasaccharidic ATIIIbinding fragment resistant to lyases
OSO3Na
OSO3Na
SO3Na
O
CH2
NH
OHO
OH
OSO3Na
O
O
OH
Ac
O
CH2
OH
NH
CO2Na
O
OH
O
OH
CO2Na
! UA-GlcNAc-GlcA-GlcNS(3,6S) or ! IIa-IIsglu
-
713
ATIII binding sequence analysis ?
Such analysis of LMWH do not accurately reflect either:a) the real amount of ATIII-binding sequences an their structuraldiversityb) the wide range of ATIII-affinity caused by the particularmonosaccharides flanking the classical ATIII-binding sequences
Classical ATIII Binding Sequences
Tetrasaccharide part analysed after lyases I,II, III digestion
? ?
14
A more accurate view of ATIII bindingsequences complexity
min0 10 20 30 40 50 60 70 80
mAU
0
20
40
60
80
100
120
!IIs-IIsglu-Is id
!Is-IIsglu-Is id
!IIa-IVsglu-Is id
!IIa-IIs glu-IIIs id !IIs-IIs glu-Is idIsid
!IIa-IIsglu-Is id-Is id
!IIa-IIs glu-Is id-Is id-Is id
!IIa-IIs glu-Is id-Is id-Is id-Is id
min0 10 20 30 40 50 60 70 80
mAU
0
20
40
60
80
100
120
!IIs-IIsglu-Is id
!Is-IIsglu-Is id
!IIa-IVsglu-Is id
!IIa-IIs glu-IIIs id !IIs-IIs glu-Is idIsid
!IIa-IIsglu-Is id-Is id
!IIa-IIs glu-Is id-Is id-Is id
!IIa-IIs glu-Is id-Is id-Is id-Is id
tinzaparin
enoxaparin
bemiparin
AT affinity column followed by CTA-SAX HPLC analysis: even if the peaks are notseparated, the wide diversity in at binding compounds across the LMWH can beseen.
-
815
ATIII binding affinity of oligosaccharides
The process can strongly impact the ATIII binding by denaturatingthe original sequence (side reaction of E1cb mechanism)
O
OH
OH
CO2Na
O
OSO3Na
OSO3Na
NHSO3Na
O
O
OSO3Na
OH
CO2Na
O
O
OH
NHSO3Na
OSO3Na
OHO
O
OH
NHAC
OSO3Na
O
O
OH
OH
CO2Na
O
O
OH
OSO3Na
NHSO3Na
O
O
OH
OSO3Na
NaO2C
O
Normal ATII binding sequence
Kd : ~100nM
Modified ATIII binding sequence
Kd : ~1,2 nM
Strong increase of the ATIII binding affinity of this modifiedoctasaccharide (Kd pentasaccharide:~26 nM)
O
OH
OH
CO2Na
O
OSO3Na
OSO3Na
NHSO3Na
O
O
OSO3Na
OH
CO2Na
O
O
OH
NHSO3Na
OSO3Na
OHO
O
OH
NHAC
OSO3Na
O
O
OH
OH
CO2Na
O
O
OH
OSO3Na
NHSO3Na
O
O
OH
OSO3Na
NaO2C
O
Iduronic acid
glucuronic acid
16
A more accurate view of ATIII bindingsequences complexity
AT affinity chromatography of enoxaparin decasaccharide fraction
0 50 100 150 200 250 300 ml1 2 3 4 6 7 8 9 1
01112131415161718192021222324F325262728293031323334353637383940414243444
54647 N5N6
-
917
A more accurate view of ATIII bindingsequences complexity
!"#0 10 20 30 40 50 60 70
!$%
- 2
0
2
4
6
8
10
12
AS11 - 1 \ 20 - 04 - 06.D - C
22
S11 - 1 \20 - 04 - 07.D
26
S11 - 1 \20 - 04 - 08.D
28
\ 2 0 -0 4 - 2 3.D
21
1- 1\ 20 - 04 -24.D -
23
1- 1 \20 - 04 - 25.D -
25
1 \ 20- 04 - 09.D
30
1\ 20 - 04- 28.D -
31
1- 1\ 20 - 04 - 29.D
33
11 - 1\ 20 - 04 -11.D -
34
X \ AS11 - 1\ 20 - 04- 12.D -
36
!"#0 10 20 30 40 50 60 70
!$%
- 2
0
2
4
6
8
10
12
AS11 - 1 \ 20 - 04 - 06.D - CAS11 - 1 \ 20 - 04 - 06.D - C
22
S11 - 1 \20 - 04 - 07.DS11 - 1 \20 - 04 - 07.D
26
S11 - 1 \20 - 04 - 08.D S11 - 1 \20 - 04 - 08.D
28
\ 2 0 -0 4 - 2 3.D \ 2 0 -0 4 - 2 3.D
21
1- 1\ 20 - 04 -24.D -1- 1\ 20 - 04 -24.D -
23
1- 1 \20 - 04 - 25.D -1- 1 \20 - 04 - 25.D -
25
1 \ 20- 04 - 09.D1 \ 20- 04 - 09.D
30
1\ 20 - 04- 28.D -1\ 20 - 04- 28.D -
31
1- 1\ 20 - 04 - 29.D 1- 1\ 20 - 04 - 29.D 1- 1\ 20 - 04 - 29.D
33
11 - 1\ 20 - 04 -11.D -11 - 1\ 20 - 04 -11.D -
34
X \ AS11 - 1\ 20 - 04- 12.D -X \ AS11 - 1\ 20 - 04- 12.D -
36
NaCl conc.
AT Affine Fraction
+
-
18
A more accurate view of ATIII bindingsequences complexity
!"#0 10 20 30 40 50 60 70
!$%
- 3
- 2
- 1
0
1
2
3
4
\ 20 - 04 - 17.D
46
1 - 1 \20 - 04 - 18.D -
48
X \AS11 - 1 \ 20 - 04 - 19.D -
50
\20 -04 -35.D -
45
\20 -04-37.D -
47
D ATA \ 20 - 04 - 36.D -
\ AS11 - 1 \ 20 - 04 - 16.D -
43
\20 -04 -33.D -
41
\20-04 -32.D
39
DATA \20 -04 -31.D -
37
!"#0 10 20 30 40 50 60 70
!$%
- 3
- 2
- 1
0
1
2
3
4
\ 20 - 04 - 17.D \ 20 - 04 - 17.D
46
1 - 1 \20 - 04 - 18.D -1 - 1 \20 - 04 - 18.D -
48
X \AS11 - 1 \ 20 - 04 - 19.D -X \AS11 - 1 \ 20 - 04 - 19.D -
50
\20 -04 -35.D -\20 -04 -35.D -
45
\20 -04-37.D -\20 -04-37.D -
47
D ATA \ 20 - 04 - 36.D -D ATA \ 20 - 04 - 36.D -
\ AS11 - 1 \ 20 - 04 - 16.D -\ AS11 - 1 \ 20 - 04 - 16.D -
43
\20 -04 -33.D -\20 -04 -33.D -
41
\20-04 -32.D\20-04 -32.D
39
DATA \20 -04 -31.D -DATA \20 -04 -31.D -
37
NaCl conc.
AT Affine Fraction
+
-
-
10
19
ATIII binding affinity of oligosaccharides
OCO
2-
OH
OSO3-
OSO3-
R1
SO3-
O
CH2
NH
O
O
O
OH
OSO3-
O
O
CO2-
OH
R
O
CH2
OH
NH
O
OO
R1
R
O
CH2
R2
NH
O
CO2-
OH
R
O
CO2-
OH
R1
O
O
R
R
O
CH2
OH
NH
OSO3-
R1
O
CO2-
OH
O
SO3-
O
CH2
OH
NH OSO3-
OH
SO3-
Ac
OSO3-
OH
OSO3-
OHSO
3-
Ac
OSO3-
OH
OSO3-
OH
OSO3-
OH
SO3-
Ac
OH
OSO3-
idu
Glucu idu
Glucu
Gluc
Mann
C D E F G HA B I J
++
The isoforms present in a given LMWH are depending on the process
About 40 different ATIII binding ranging from hexasaccharides to decasaccharideshave been isolated from Enoxaparin and tested up to now:
The affinity (Kd) of the ~40 ATIII binding compounds is varying from 1nM to 11000 nM range!
The aXa activity as well as the aIIa activity is mediated by a oligosaccharide bindingdomain which has multiple isoforms, different from the pentasaccharide.Representation of the current SAR and knowledge of the binding domain:
20
Conclusions
Those data demonstrate the major impact of the depolymerization process on theLMWH mixture
The interaction with AT is not only mediated by one pentasaccharidesequence but by various isoformsFlanking disaccharides units can play a major role in the modulation of theinteraction with AT
Therefore Lovenox is unique mixture when properly looked intoLovenox biological profile as well as other LMW are process dependent:
Modulation of AT affinityModification of aXa activityPharmacokinetic profile of the constituentsDifferent non-specific protein interaction
Those results summarizes 10 years of analytical research effort in order to betterunderstand the structure activity relationship of this complex but fascinatingmixture
-
2ndWORKSHOP ON THE CHARACTERIZATION OF HEPARIN PRODUCTS, 19-20 June 2008, EDQM, Strasbourg, FRANCE
LASER LIGHT SCATTERING CHARACTERIZATION OF LOW MOLECULAR
WEIGHT HEPARINS: COMPARISON OF MOLECULAR WEIGHT
DISTRIBUTION METHODS
Gyngyi S. Gratzl, Ph.D.
MOLECULAR WEIGHT DETERMINATION OF LOW MOLECULAR WEIGHT HEPARINS
Narrow standard calibration
- Relative heparin standards preparation, characterization, daily gel permeation column calibration. Many assumptions are taken and extrapolation.
Broad standard calibration:
- 1st International Reference Preparation Low Molecular Weight Heparin for Molecular Weight Calibration standard used as a broad integral standard B. Mulloy et.al. Thrombosis and Haemostasis 77 (4) 1997.
Absolute Molecular Weight Distribution of LMWH by Multi-Angle-Laser Light Scattering
- No calibration standards needed, rapid precise and accurate.
The molecular weight determination and molecular weight distribution are very important characteristic of the polydisperse mixture of LMMH oligosaccharide chains:
-
What is light scattering?
How does light scatter?
When light interacts with matter, it causes charges to polarize.
The oscillating charges radiate light.
How much the charges move, and hence how much light radiates, depends upon the polarizability of the matter.
-
Index of refraction n
The polarizability of a material is directly
related to its index of refraction n.
The index of refraction is a measure of
the velocity of light in a material.
e.g., speed of light
For solutes, the polarizability is expressed as the specific
refractive index increment, dn/dc.
n
vacuumliquid
vv =
dcdnE scattered
2
scattered
dcdnI
How light scattering measures M
22total 4 EEEI =+
2
scattered
dcdnMcI
coherent: incoherent:222
total 2 EEEI =+
-
Basic light scattering principles
Principle 1
The amount of light scattered is directly proportional to the product of the polymer molar mass and concentration.
2
scattered
dcdnMcI
Principle 2
The angular variation of the scattered light is directly related to the size of the molecule.
Running an experiment 1: Calibration
Why?
The detectors output voltages proportional to the light scattering intensities. The voltages must be converted to meaningful units.
How? 1. Flow pure, filtered (0.02 m) toluene through the flow cell.2. ASTRA software measures the voltages from the 90 and lasermonitor photodiodes with the laser on and off (dark voltages).
3. ASTRA then computes the calibration constant.
-
Running an experiment 2: Normalization
Why?detector sensitivities vary.each detector views a different scattering volume.scattered light is refracted.only the 90 detector is calibrated.
How?
1. Fill flow cell with isotropic scatterer in actual solvent to be used.2. ASTRA software measures voltages for each angle and:
a. Determines refraction angle from solvent index of refraction.b. Determines angle and scattering volume corrections.c. Normalizes each corrected detector voltage signal to the 90detector.
Plots
Zimm Plot
Measure light scattering at
multiple angles and
concentrations.
Retrieve M, rg, and A2.
Debye PlotMeasure angular variation of light scattering for low concentrations.Retrieve M and rg.
C 1
-
Laboratory Set Up
SEC-MALS Conditions
MALS detector: DAWN EOS and miniDAWN from Wyatt
Concentration detector: Optilab rEX from Wyatt and Agilent RI detector
HPLC system: Agilent 1100
Column: Shodex OH Pak SB-G guard column and Shodex OH Pak SB- 803 HQ aqueous GPC column
Mobile phase: 100 mM NaNO3 with 0.1 % Sodium Azide
Flow rate: 0.5 mL/min
dn/dc: 0.130 mL/g
Sample concentration: 6.4 -20.0 mg/mL
Injection volume: 100 L
-
A Typical Chromatograms
-2.0
0.0
2.0
4.0
6.0
10.0 12.0 14.0 16.0 18.0 20.0
De
tect
or:
AU
X1
Time (min)
0.16
0.20
0.24
0.28
0.32
De
tect
or:
11
Strip Chart - Hep7_100uL_f_filt_01
LSLS@
90
LS@90
LS@90
Processing, collection
information:
Cell Type: K5
Laser Wavelength: 690.0 nm
Solvent RI: 1.33 water
Calibration constants: DAWN and
AUX1
Smoothing, spiking: Heavy
Fit method: Zimm Model
Calculation method: dn/dc + AUX constant
Using fitted data:
First order Exponential for ASTRA 5.1.9.1
MM fit and Radius fit.
3-Dimensional Overlay of LS Detectors Ranging from 25 to 155
25 3D Plot - Hep7_100uL_f_filt_01
17 1615
1412
1110
98
7 65
4
155
-
A Debye Plot of one Data SliceMolar mass and root-mean square radius (Rg, if >10nm) are
determined from the respective intercept and slope for each dataslice
90 & AUX detector
Peak, Slice : 1, 944 Time : 15.733 min Fit degree : 0 Conc. : (7.805 0.000)e-4 g/mL Mw : (4.693 0.008)e+3 g/mol Radius : 0.0 0.0 nm
-41.80x10
-42.00x10
-42.20x10
-42.40x10
0.0 0.2 0.4 0.6 0.8 1.0
K*c/
R(th
eta)
sin(theta/2)
Debye Plot - Hep7_100uL_f_filt_01
A Typical Molar Mass vs. Elution Time Plot
31.0x10
41.0x10
51.0x10
61.0x10
13.0 14.0 15.0 16.0 17.0 18.0
Mo
lar
Ma
ss (g/
mo
l)
Time (min)
Molar Mass vs. Time Hep7_100uL_f_filt_011st orderHep7_100uL_f_filt_021st order
Data scattering at the ends of the peak is due to low singal-to-noise
ratio, which can be addressed by results fitting in ASTRA.
-
SEC-MALS
Minimum Amount of Sample
Required*
4
Mw [kD] x (dn/dc)2g
* for s/n = 5, noise = 3 mV, with a monodisperse sample using
one standard SEC column.
Example: 80 g for 3 kD oligosaccharide
MOLECULAR WEIGHT DISTRIBUTION OF DALTEPARIN SODIUM
-
SEC-LC Broad LMM Heparin Calibrator Method
Conditions:
Mobile Phase: 0.1 M Ammonium Acetate
Flow Rate: 0.5 mL/min
Column: TSK Gel G 3000 SW XL + TSK Gel G 2000 SWXL columns
Injection volume: 20 L
Detection: Refractive index
Column temperature: Ambient
Assay concentration: 10 mg/mL
Data processing: Polymer Laboratories Cirrus GPC software with broad range calibration and third order polynomial fitting
The molecular weight profiles of low molecular weight heparin samples measured by high-performance gel permeation
chromatography using the heparinase-degraded 1st International Reference Preparation as Broad standard
-
DALTEPARIN SODIUM CROSS-VALIDATION
RESULTS
84.920.6170.369.0361134939GPC
86.5819.176.624.2861345174WYATT MALLS
FRAGMIN 74068B51 Exp. 08/07
85.4219.9170.819.2860174850GPC
86.0019.3075.705.0061205104WYATT MALLS
FRAGMIN 96144A51 Exp. 09/07
85.7619.6071.828.5859824862GPC
85.2820.2274.165.6262025130WYATT MALLS
FRAGMIN MB0619 Exp. 07/07
85.5419.8470.989.1960294854GPC
84.8220.8872.906.2462615071WYATT MALLS
FRAGMIN LI1114 Exp. 04/07
2,000-9000> 8,0003,000 - 8000< 3,000MwMnSAMPLE
80.0-100.0 %14.0 - 26.0 %65.0 - 78.0 %3.0 - 15.0 %
5,600-
6,400
About
5,000Spec
System Suitability Requirements:
6002 + 1613,536 + 82Literature value
5,9973,678
5,9903,706
1.22RSD % Mw6,0063,644
5,8903,738
6,0203,521
2.34RSD % Mn5,9613,759
6,1133,697
MwMnWHO LMWH MOLECULAR WEIGHT
CALIBRATOR
-
ENOXAPARIN SODIUM PRODUCTS
Enoxaparin sodium is obtained by alkaline depolymerization of heparin benzyl ester derived from porcine intestinal mucosa. Its structure is characterized by a 2-O-sulfo-4-enepyranosuronic acid group at the non-reducing end and a 2-N,6-O-disulfo-D-glucosamine at the reducing end of the chain. About 20% (ranging between 15% and 25%) of the enoxaparin structure contains an 1,6 anhydro derivative on the reducing end of the polysaccharide chain. The drug substance is the sodium salt. The average molecular weight is about 4500 daltons. The molecular weight distribution is:< 2000 daltons 20%2000 to 8000 daltons 68% > 8000 daltons 18%
ENOXAPARIN SODIUM CROSS-VALIDATION
RESULTS
6.977.315.83994WYATT MALLS LOVENOX MDV 41E010 Exp. 07/07
42.7143.612.14,160GPC LOVENOX MDV 41E010 Exp. 07/07 Average
7.677.914.54094WYATT MALLS LOVENOX 15060 Exp. 07/07 Ave
9.575.814.84421GPC LOVENOX 15060 Exp. 07/07 Ave
6.777.815.53983WYATT MALLS LOVENOX 15049 Exp. 12/06 Ave
9.375.715.04399GPC LOVENOX 15049 Exp. 12/06 Ave
7.077.515.54021WYATT MALLS LOVENOX 12076 Exp. 06/07 Ave
9.375.615.14395GPC LOVENOX 12076 Exp. 06/07 Ave
6.778.415.03990WYATT MALLS LOVENOX 89488 Exp. 08/08 Ave
8.974.916.24313GPC LOVENOX 89488 Exp. 08/08 Ave
7.478.514.14129WYATT MALLS LOVENOX 7182 Exp. 05/08 Ave
9.374.815.94362GPC LOVENOX 7182 Exp. 05/08 Average
SPECIFICATION Mw about 4,500 Da < 2,000 68% > 8,000 < 18 %
-
CONCLUSIONS:
1. The SEC/MALLS technique provides similar results to the Broad column calibration GPC method on molecular weight determination of Low Molecular Weight Heparins.
2. The SEC/MALLS method does not use relative heparin standards for daily column calibration.
3. The molecular weights are calculated directly from the angular dependence of scattered light intensity as a function of concentration by light scattering equations.
4. The System suitability requirements assure the comparable results of the1st IRP Low Molecular Weight Heparin for Molecular Weight Calibration with GPC column calibration vs. SEC/MALLS method.
-
1The Standard of QualityTM
USP LMWH Monographs: Current and FuturePerspectives
Anita Szajek, Ph.D.
Session 3 LMWH2nd Workshop on the Characterization of Heparin Products
19-20 June 2008EDQM, Strasbourg, France
The Standard of QualityTM
Talk Outline
1. Acknowledgements USP Heparin Ad hoc Advisory Panel
2. Issues and Challenges3. Monograph update4. Associated General Chapters update
-
2The Standard of QualityTM
1. USP Heparin Ad Hoc Advisory Panel
Reports to B&B Blood and Blood Products ExpertCommittee (BBP EC)
Co-Chairs: Wesley Workman, Ph.D., KristianJohansen, Ph.D.
Liaison: Anita Szajek, Ph.D. Members: Neil Desai, Ph.D. Elaine Gray, Ph.D. Gyngyi Gratzl, Ph.D. Hester Hasper-van Heusden, Ph.D. Craig Jackson, Ph.D. Robert Linhart, Ph.D. Barbara Mulloy, Ph.D. Zachary Shriver, Ph.D. Jeanine Walenga, Ph.D.
The Standard of QualityTM
Major Activities of USP Heparin Panel
June 11, 2006 Inception of Heparin Ad Hoc AdvisoryPanel
3 Face-to-Face Meetings 8 Teleconferences 1 Web Meeting Next Face-to-Face Meeting scheduled for August,
2008. Since the Heparin crisis, the Heparin Panel met
three times: 1 telecon, 1 F2F & 1 web meeting Publication of revised Heparin Sodium and
Heparin Calcium monographs (Target June 16,2008)
-
3The Standard of QualityTM
2. Issues and Challenges
Quality control of UFH used in manufacture of LMWH Sufficiently defining quality attributes of LMWH that
are not innovator exclusive, yet sufficient to controlthe quality of all products on the market
LMWH Method harmonization Are potency assignments based on the LMWH IS
appropriate for the entire product group?
The Standard of QualityTM
Quality control of heparin used in manufacture ofLMWH
Stage 1 Monograph Revision Revise LMWH monograph to include the following:
Heparin source material used in the manufacture ofLMWH (Dalteparin or Enoxaparin) complies with thecompendial requirements stated in the Heparin Sodiummonograph
Stage 2 Monograph Revision Is it sufficient to control the quality of starting
heparin or should additional testing be included inthe LMWH monographs?
The depolymerization processes for LMWHgenerally do not eliminate oversulfatedchondroitin sulfate.
-
4The Standard of QualityTM
USP monograph for Crude Heparin?
Crude heparin is raw materialUSP General Notices
Articles listed herein are official and the standards set forth in themonographs apply to them only when the articles are intended orlabeled for use as drugs, as nutritional or dietary supplements, or asmedical devices and when bought, sold, or dispensed for these purposesor when labeled as conforming to this Pharmacopeia or NationalFormulary.
Very much process dependentDifferent grades of crude heparinNo universal specifications for acceptance criteria
No meaningful analytical characterization can beperformed
Point of enforcement?Supplier/ point of entry/ manufacturers
The Standard of QualityTM
3. Monograph Update: Enoxaparin Sodium andEnoxaparin Sodium Injection Monographs
Initial publication PF29(6) Revision publication PF 33(1) Official publication Second Supplement of USP31-NF26 Official date of December 1, 2008
-
5The Standard of QualityTM
USP Reference Standards for Enoxaparin SodiumMonographs
In development/qualification Enoxaparin Sodium (ES) RS (13C NMR, MW) ES Solution for Bioassays RS (Anti-factor Xa and
IIa activity assays) ES Molecular Weight Calibrant A RS ES Molecular Weight Calibrant B RS
Available Benzyl Alcohol RS Endotoxin RS
The Standard of QualityTM
Definition of Quality Attributes
1,6-Anhydro method for Enoxaparin Sodium Issues raised by public comment Validated method protocol is needed if the 1,6-
anhydro content is defined in the monograph Is this really a product-defining quality attribute? Can the method be described sufficiently to be
applicable to the broader industry?
USP proposal Test for 1,6-Anhydro Derivative for
Enoxaparin Sodium method chapter
-
6The Standard of QualityTM
1,6-Anhydro Derivatives
Alkaline depolymerization ofEnoxaparin sodium in waterproduces a partial butcharacteristic conversion ofglucosamines at the reducingtermini of oligosaccharide chainswhose terminal glucosamine is 6-O sulfated :1. Conversion is epimerization
of glucosamine intomannosamine.
2. 6-O desulfatation ofglucosamine ormannosamine yielding1,6anhydro derivatives :
n = 0 to 20 R , R = H or SO3- X, Y, Z = 0 or 1 When Z and/or Y = 0, sulfated
group is replaced by the radical H When X = 0, sulfated group is
replaced by the radical COCH3
OO
O
CH2
OR'
NH
O
OR
O
CO2-
OH
OSO3-
SO3-
z
x
OH
SO3-
O
CH2
NH
O
x
O
OSO3-
O
CO2-
OH
SO3-
O
CH2
OH
NH
O
O
CO2-
OH
OSO3-
OSO3-
n
y
The Standard of QualityTM
Test for 1,6-Anhydro Derivative for Enoxaparin
Sodium method chapter
Published in PF 34(1), Jan/Feb 2008 Public comments received Not sufficiently defined for routine use Difficulty meeting system suitability requirements Difficulty assigning peaks using relative retention
times only (0-24% difference in rrt has beenobserved)
1,6-anhydro standards are recommended
Official publication deferred until the comments canbe fully addressed
-
7The Standard of QualityTM
LMWH Method Harmonization
Molecular Weight Distribution and Weight-AverageMolecular Weight Mulloy method EP method Innovator methodUSP Proposal: Low Molecular Weight Heparin
Molecular Weight Determinations test chapter
Activity Assays USP EP InnovatorsUSP Proposal: Anti-factor IIa and Anti-factor Xa
Assays for Low Molecular Weight Heparins test chapter
The Standard of QualityTM
MW Measurements of LMWH by GPC
ES MW RS (1,500-11,000)
Low-molecular-mass heparin CRS
1st IRP LMWHeparin(90/686)
Calibrant
20 L of 10mg/mL25 L of 10mg/mL20 L of10mg/mL
Injectionamount
RI detectorRI/UV234 nmRI at 234 nmDetection
0.6 mL/min0.5 mL/min0.5 mL/minFlow Rate
0.5M lithium nitrate28.4 g/L anhydroussodium sulfate R,pH 5
0.1M ammoniumacetate
MobilePhase
7.8-x 300mm TSKG3000 SWXL TSKG2000 SWXL
7.5-x 300mmporous silica beads(5m)
TSK G3000SWXL-TSK G2000SWXL
Column
InnovatorEPMulloy
90/686 CRS
-
8The Standard of QualityTM
Potency Assignment
Are Potency assignment based on the LMWHInternational Standard appropriate for theentire product group?
Enoxaparin Sodium & Dalteparin Sodium EP applies the same potency standard
Ultra LMW heparin does not exhibit consistentparallelism when assayed against the currentIS
The Standard of QualityTM
-
9The Standard of QualityTM
-
1Are current PhEur. monographs specification of LMW heparins sufficient tocontrol the quality of both innovator and biosimilar products?
Peter Jongen PharmDRIVM, Centre for Biological Products and Medical technology
2nd workshop on Characterization of heparin products,Strasbourg, 19-20 June 2008
Low Molecular Weight (LMW)Heparin
- partially depolymerisedderivative 3-7 kDa ofunfractionated heparin (15-20kDa)
- widely used to prevent and treatthrombosis.
- Anticoagulant activities of LMWHeparin depend on molecularweight
- Each LMW heparin ischemically (and clinically)unique
-
2 LMW-Heparins in Europeanregulatory perspective
LMW-Heparins in PhEurmonographs
LMW heparin = biological substance
A biological medicinal product is a product, the activesubstance of which is a biological substance. A biologicalsubstance is a substance that is produced by or extractedfrom a biological source and for which a combination ofphysico-chemical-biological testing and the production processand its control is needed for its characterisation and thedetermination of its quality.
Regulatory approach differs from chemical DS. For abridgedapplications Article 10(4) of Directive 2001/83/EC applies!
Consequently for generic applications: compliance with PhEurand demonstration of bioequivalency are not enough!
-
3Article 10 (4) DIRECTIVE 2001/83/EC
Where a biological medicinal product which is similar to areference biological product does not meet the conditions inthe definition of generic medicinal products, owing to, inparticular, differences relating to raw materials ordifferences in manufacturing processes of the biologicalmedicinal product and the reference biological medicinalproduct, the results of appropriate pre-clinical tests orclinical trials relating to these conditions must be provided.
The type and quantity of supplementary data to be providedmust comply with the relevant criteria stated in the Annexand the related detailed guidelines.
LMW heparins are biologicals not chemicals Biological source material DS is complex mixture of glycosaminoglycans of different
sizes that can be characterised with difficulties by state ofthe art analytical methods.
Manufacturing process defines the characteristics of the drugsubstance.
The complexity of LMWH results from the nature of thestarting material, the extraction, the fractionation and theproduction processes.
When the process defines the product : Variations within aprocess and between processes should be carefullyconsidered for clinical consequences(Comparability/Biosimilarity)
-
4Another important message:For abridged applications:
Compliance with Pharmacopoeial monographs of LMW-heparins will never be sufficient to demonstrate biosimilarity
Additional chemical, biological and/or clinical may beneeded.
EU Regulatory guidance to be established:
-
5Why is similarity difficult to establish LMW-heps are complex due to
source molecule LMW are a mixture consisting on
many chemical entities It is not known which subfractions
contribute to efficacy (/safety) Mode of action not completely
understood Uncertain whether PD markers
(=anti XDa, anti IIa)representative for clinicaloutcome
-
6Aim of pharmacopoeial monograph
Quality standard for substances- (often from biological-regulators perspective minimal requirements
for MA)
And NOT: criteria to demonstrate biosimilarity of a preparation to the
innovator product- Biosimilarity characterization will be far more extensive than EP
monograph
LMW-heparin monographs
heparins, low-molecular-mass (0828)
specific monographs: Dalteparin sodium (1195) Enoxaparin sodium (1097) Nadroparin calcium (1134) Parnaparin sodium (1252) Tinzaparin sodium (1271)
-
7Cross references between monographs:Heparins, Low-molecular-mass (0828), production: Low molecular mass heparins are obtained by fractionation
or depolymerisation of heparin of natural origen thatcomplies with the monograph on Heparin sodium (0333) orHeparin calcium (0333),
Substance specific monographs: parin complies with the monograph Low-molecular
mass heparins (0828) with the modifications and additionalrequirements below.
PhEur requirements for LMW-heparins Definition of structure and production process Specification Anti Xa activity and ratio Anti Xa/ Anti IIa Molecular masses mean and dispersion 13C NMR Molar ratio Sulphate / Carboxylate Nitrogen Sodium or Calcium Process related impurities (heavy metals, endotoxins)
And for individual substances: link
-
8Are current Monographs still state-of-the-art?
New analytical tools available New insights in biological properties
To be debated!
New analytical targets: Oligosaccharide mapping/sequencing (many possibilties) Measurement ATIII binding pentasaccharide Measurement specific structural features of depolymerisation
process (for each process different)
(conventional) fix anti Xa/anti IIa ratio, Mw dispersion,SO4/CO3 -ion molar ratio
. .
-
9Biosimilar preparations
Comply with Definition Normally also comply with all PhEur specifications Evolving science and development of biosimilars will
probably give us more insight in critical quality attributes More insight may give us improved monographs
Answer to the question:Is current monograph specification of LMW heparins sufficient
to control the quality of both innovator and biosimilarproducts?
Control Quality: maybe, but standard can be improved(evolving science)
Characterise Substance: by no means (additional physico-chemical and biological tests needed)
-
10
Thanks for your attention!
-
1Low Molecular Mass Heparinsin the Japanese Pharmacopoeia
National Institute of Health SciencesNana Kawasaki
2nd Workshop on the Characterization of Heparin Products
Current situationJP official monograph
Scientific approaches to characterization of -parins
JAN Approved JP
Parnaparin Sodium
Dalteparin Sodium
Reviparin Sodium
Enoxaparin Sodium
Tinzaparin Sodium
Nadroparin Calcium
Bemiparin Sodium
Certoparin Sodium
Deligoparin Sodium
Minolteparin Sodium
Ardeparin Sodium
Livaraparin Calcium
Low molecular mass heparins in Japan
INN
2008
2011 ?1990s
-
2Official Monograph of parnaparin Sodium(1)
Description: Color, Solubility, Hygroscopicity
Identification
1) Saccharides: Tritoluidine blue reaction
2) Sodium salt quantitative test: Flame Coloration test
pH
Purity
1) Clarity and color of solution
2) Heavy metal limit test
JP XV 2006
Loss on drying
Molecular mass; Distribution of molecular mass:
Size exclusion HPLC/UV
The degree of sulfate ester: Potentiometric titration
Total nitrogen
Anti-factor IIa activity
The ratio of anti-factor Xa activity to anti-factor IIa
activity
Official Monograph of parnaparin Sodium(2)
-
3In Japan, LMM heparin products have been manufacturedby several companies.
Parnaparin products: 2 manufacturers Dalteparin products: >10 manufacturers
- How should we assess comparability of dalteparin products (parnaparin products) ?- How should we discriminate among the four different LMM heparins?
Challenges (1)
In the near future JP heparin sodium monograph wouldcontain purity tests of OSCS and DS by NMR and/or CE.
- Is it adequate to ensure the purity of LMM heparin
products?
Challenges (2)
-
4Depoly-merization
H
H
OH
CO2H
H
HHO
H O
OSO3H
OR1
H
OH H
H
O
OSO3H
OR1HO2C
H
HO
R1O
H
HO3SO
H
HOH
HO
H
H O
NH
R2
OH
H
H
HO3SO
R1O
SO3H
R3
H
H O
NH
O
OH
H
H
R1O
SO3H
H
DalteparinSodium
ReviparinSodium
ParnaparinSodium
EnoxaparinSodium
HNO2
HNO2
H2O2/Cu2+
Benzylester/OH-
5,600 6,400(6,000)
3,150 5,150(4,150)
4,000 6,000(5,000)
3,500 5,500(4,500)
2.0 2.5
2.1
2.0 2.6
Approx.2
Molecularmass range
SO3/di-saccharidesINNNon-reducingend
Reducingend
H
H O
NH
R2
OH
H
H
HO3SO
R1O
SO3H
R3
Definitions of LMM heparins (-Definitions of LMM heparins (-parinparin))
Approaches to theApproaches to theidentification/comparability and purity testsidentification/comparability and purity tests
for for prinsprins, including LMM heparins, including LMM heparins
Molecular mass range/sulfate group: - LC/ESI MS
Structure of reducing end/nonreducing end: - Monosaccharide composition analysis by HPAEC-PAD - Glycan mapping by enzyme digestion/HPLC
-
510 20 30 40 50 60 70 80 90 100 110Time (min)
0
Rel
ativ
e In
tens
ity
100
2
The number ofresidues
3456789
1011121314151617
LC/MS of parnaparin (MW: 4,000 6,000)Oligosaccharides consisting of 2-17 monosaccharides and 2-24sulfates (molecular mass: 5,735.38 Da) were identified.
LC/ESI-MS of LMM Heparins
2N TFA, 100 oC
% o
f con
trol
0
25
50
75
100
0 4 8 12 16 20 24hr
Monosaccharide analysis of heparin and Monosaccharide analysis of heparin and chondroitinchondroitin sulfate sulfate
PAD
resp
onse
GalN
GlcN
0 4 8 12 16 20 24hr
FucGalN
GlcNGal
XylGlcA
IdoA
Heparin
Chondroitin sulfate C
Monosaccharides
GlcN
GalN
0 5 10 15 20 25 30 35 40 45 50 55min
PAD
resp
onse
GalN
GlcN
HPAEC-PAD of GAG
-
60 5 10 15 20 25 30 35 40 45 50 55
Parnaparin-B
Parnaparin-A
Dalteparin-B
Dalteparin-A
Reviparin
Enoxaprin
min
PAD
resp
onse
HPAEC-PAD elution profiles of LMM heparinhydrolysis products
GalNGlcN
22.5
12.4
13.1
Elution profiles of heparinase-digestsed LMM heparins by SAX-HPLC
0 5 10 15 20 25 30 35 40 50 55 6045
Parnaparin B
Enoxaprin
Parnaparin A
Dalteparin B
Dalteparin A
Reviparin
min
UV
(232
nm
)
Ion-exchange chromatography of LMM heparins
-
7 Four LMM heparins are marketed in Japan.
JP has contained parnaparin sodium monograph.
Dalteparin sodium monograph is being developed.
MS, HPAEC-PAD and Ion-exchange HPLC
might be useful for identification/comparability
and purity tests of -parins.
Conclusion