CE Pharm 2009

Update on Heparin Contamination: Assessment of Optimized Capillary Electrophoresis, Proton NMR and Ion-Exchange HPLC Methodologies

Michel Girard*, Marc-André Joly, Barry Lorbetskie, Terry Cyr and Yves Aubin

Centre for Biologics Research, Health Canada, Tunney’s Pasture, Ottawa (ON), Canada K1A 0L2

Biologics and GeneticTherapies Directorate

Health CanadaHealth Products and Food Branch

CE Pharm 2009

CE Pharm 2008

CE Pharm 2009

Heparin - structureChronology of events

Early eventsJune-September 2008

Capillary ElectrophoresisHPLCNMRConclusion

Outline

CE Pharm 2009

What is Heparin?Highly sulfated polysaccharides ranging between 3 kDa and 50 kDa (Avg MW: 12-15 kDa)

Extracted from animals, mostly porcine origin

Highest charge density of known biological molecules

Widely used as anticoagulant in medical procedures, injectables, medical devices

CE Pharm 2009

Structures of major disaccharide repeat units of sulfated glycosaminoglycans (sGAG)

Chondroitin sulfate Dermatan sulfate (Chondroitin sulfate B)

Heparin

R = H, SO3H

For heparin 1 in 5 disaccharide repeat unit bears an acetyl group: absorbance at λ200 nm approx. 5 times less than DS or CS

CE Pharm 2009

Early Events (Nov 2007 – Apr 2008)

Nov 2007 – Feb. 2008: dramatic increase of severe adverse drug reactions (ADR) due to heparin in the USA.Feb 2008: Baxter USA recalls its heparin productsMar 2008: - US FDA requires CE and NMR tests for detection of unknown impurity in unfractionated heparin; - Health Canada requests CE and NMR tests and provides assistance for analysis due to lack of necessary instrumentation at most manufacturers- CE and NMR analyses at HC confirm 1 contaminated sample in B. Braun products; product is recalled within 24 hours Apr 2008:- unknown identified as oversulfated chondroitin sulfate

(OSCS)

CE Pharm 2009

FDA recommended CE method

Instrument: Agilent HP 3D-CE equipped with diode array detector or equivalent

Capillary: Bare fused silica, 50μm i.d., 64.5cm-total length, 56cm-effective length

Temp.: 25ºC Detection wavelength: 200nm (bandwidth 10nm) Polarity: Negative Voltage: 30 kV Injection: 50 mbar pressure for 10 seconds Filter: Cellulose acetate membrane filters (0.22μm) Separation Time: 15 minutes Electrolyte: 36mM Phosphate buffer (pH 3.5)Specification: The electropherogram of test solution

does not exhibit a sharp distinguishable peak in front of the main heparin peak. The migration time of heparin in the test solution is about 5.7 min. See attached for examples.

Reference:1. Private communication, Baxter study number 41010 2. R.P. Patel, C. Narkowica, J.P. Hutchinson, E.F. Hilder, G.A. Jacobson, A

simple CE method for the rapid separation and determination of intact low molecular weight and unfractionated heparins, J. Pharm. Biomed. Anal. 46 (2008) 30-35

PASS

FAIL

CE Pharm 2009

Minutes

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

AU

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

AU

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

HeparinOSCS DS

OSCS

Heparin

OSCS-contaminated Heparin

CE Pharm 2009

Summary of heparin analysis at Health Canada, Mar-Jun 2008170 heparin samples received from 8 manufacturers between March and June 2008Unfractionated heparin (120 samples) analyzed by CE and 1H-NMRLow molecular weight heparin (50 samples) analyzed by 1H-NMRSeveral samples showed variability in content of dermatan sulfate (DS)/chondroitin sulfate(CS)1 sample with OSCS was detected and confirmed (CE + NMR)1 sample contained another unidentified impurity

CE Pharm 2009

Events Jun-Sept 2008

Workshops between authorities and manufacturers are heldEP and USP revise heparin monographs to include CE and NMR tests (Jun-Jul) with new revisions soon to followOn-going discussions in regulatory agencies on limits for DS and other sGAGMethods to improve separation / quantification of OSCS are reported and discussed with regulatory agencies (Jun-Sept)

Capillary electrophoresis – Baxter, Utrecht Univ. SAX-HPLCNMR

CE Pharm 2009

Improved CE methods

2 new CE methods developed:Utrecht Univ. (UU)Baxter (BX)

Both based on high molarity background electrolytesLower limit of detection for OSCS Quantification of DS/CS

CE Pharm 2009

CE Conditions

UU Method

Bare-fused silica capillary850 mM TRIS phosphate, pH 3.0, 35ºC-30 kV (reverse polarity)60 cm total length, 25 μm i.d.Developed on PACE MDQ CE with regular capillaries

Somsen G.et al., J Chromatogr A. 2009 May 1;1216(18):4107-4112.

BX Method

Bare-fused silica capillary600 mM lithium phosphate, pH 2.5, 20ºC-15 kV (reverse polarity)30 cm total length, 25 μm i.d.Developed on Agilent CE with bubble cell capillaries

Wielgos, T., et al.,. J. Pharm. Biomed. Anal., 49 (2009) 319-326.

CE Pharm 2009

UU CE method

Minutes0 1 2 3 4 5 6 7 8 9 10

AU

-0.0075

-0.0050

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

UU method (30 cm cap), -15 kV

Minutes1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

AU

0.000

0.001

0.002

0.003

0.004

0.005

UV - 200nmBraun 60910839heparin braun 60910839 50mg-ml 850mm trisphosphate ph3 30kv 35c 50cm-rep1

UV - 200nmHeparin Organon L00027442heparin organon l00027442 50mg-ml 850mm trisphosphate ph3 30kv 35c 50cm-rep2

UU method (60 cm cap), -30 kV

CE Pharm 2009

Comparison of UU and BX methods on MDQ

Minutes

0 1 2 3 4 5 6 7 8 9 10

AU

-0.0075

-0.0050

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175 UU method

Minutes

0 1 2 3 4 5 6 7 8 9 10

AU

-0.0075

-0.0050

-0.0025

0.0000

0.0025

0.0050

0.0075

0.0100

0.0125

0.0150

0.0175

BX method

Minutes0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

UU method

BX method

CE Pharm 2009

Selectivity of BX and UU methods for reference samples

Minut

0 2 4 6 8 10

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Heparin

CS bovine

CS shark

DS

Minutes

0 2 4 6 8 10

AU

0.00

0.01

0.02

0.03

0.04

0.05

1

Heparin

CS bovine

CS shark

DS

UU methodBX method

CE Pharm 2009

Separation of DS - UU methodUU CE method: Dermatan Sulfate Linearity

y = 12770x - 267.04R2 = 0.9855

0

10000

20000

30000

40000

50000

60000

70000

0 1 2 3 4 5 6Concentration (mg/ml)

Cor

rect

ed A

rea

Uni

ts

Minutes0 2 4 6 8 10

AU

0.001

0.002

0.003

0.004Heparin

DS

M inutes

5 .0 5 .5 6 .0 6 .5 7 .0 7 .5

AU

0 .00075

0 .00100

0 .00125

0 .00150

0 .00175

0 .00200

0 .00225

0 .00250

1

LOQ = 0.3 mg/mL

Dermatan Reference solution 1.25mg/ml : Heparin Sodium BRP solution 8.75mg/ml

N=3

CE Pharm 2009

Comparative Evaluation of UU and BX Methods

Both methods perform similarlyLinear, precise and sensitiveProvide better separation and quantification of OSCS (LOD < 0.1%) than original methodProvide quantification of DS/CS (LOQ < 0.5%)

CE Pharm 2009

SAX-HPLC (Initial EP method)

Strong-anion exchange HPLC (SAX)Developed by heparin manufacturerQuantification of OSCS and DS/CSDS and CS co-elute

CE Pharm 2009

DS-CS

Heparin

OSCS

CS marine

SAX-HPLC / Initial EP methodColumn: Dionex IonPac AS11 2 x 250 mm

with pre-column Dionex IonPac AG11 2 x 50 mm or equivalent

Mobile phases: (A) 2.5mM NaH2PO4, pH 3.0; (B) 2.5mM NaH2PO4 + 1M NaClO4 pH 3.0

Detection at 202 nm

Column temperature 40°C

Flow rate 0.22ml/min

Equilibration + Run time 75 min

Gradient composition

Time (min) % mobile phase B

0 20

60 90

61 20

75 20

CE Pharm 2009

Comparison of SAX and UU CE for Sample J

Minutes0 2 4 6 8 10

AU

0.000

0.005

0.010

0.015

0.020 1

2

3

Hep

DS

OSCS

CE Pharm 2009

Comparison of SAX and CE OSCS/hep

SampleSAX EParea ratio

CE UU area ratio

CE Baxterarea ratio

B 1.37 0.93 0.91

E 0.58 0.55 0.55

F ‐ ‐ ‐

G 1.37 1.05 0.94

H 1.21 1.05 1.15

J 0.46 0.44 0.45

DS‐CS/hep

SampleSAX EParea ratio

CE UU area ratio

CE Baxterarea ratio

B 0.02 0.07 0.07

D 0.37 0.29 0.29

F 0.45 0.41 0.41

G 0.02 0.09 0.09

H 0.03 0.08 0.08

J 0.02 0.05 0.05

CE Pharm 2009

Comparative Evaluation of SAX and CE Methods

SAX and CE provided results that were consistent for a series of samples containing various levels of OSCS and DS/CSCE provides better peak shapes for OSCS and heparin but not for DS/CSSAX method was not used with depolymerization as proposed

CE Pharm 2009

Outcome of 1st EP coll. Study

SAX-HPLC – difficult to adequately quantify DS Concentration sensitivity of CE not as good as HPLCNext collaborative study to require 2-steps procedure for optimized SAX-HPLC:

identificationquantification after depolymerization

CE Pharm 2009

SAX-HPLC / Optimized EP method

DS/CS

Heparin

OSCS

CS marine

DS-CS

Heparin

OSCS

CS marine

CE Pharm 2009

SAX-HPLC / Optimized EP method: reference sample

CE Pharm 2009

SAX-HPLC / Optimized EP method: sample 2

After depolymerization

CE Pharm 2009

SAX-HPLC / Optimized EP method: sample 5

After depolymerization

CE Pharm 2009

Optimized SAX-HPLC

Shorter analysis time for identification stepBetter peak shape for DSResolution between DS and heparin not as goodDepolymerization

worked well but increases overall analysis time (2 steps)potential for artefacts

CE Pharm 2009

CE analysis of Test samples 1-6 from 2nd EP collaborative study

Minutes

0 2 4 6 8 10

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016OSCS

Heparin

DS

DS

CE Pharm 2009

Optimized 1H-NMR method

Heparin + 1% OSCS withoutcarbon decoupling

Heparin with carbondecoupling

Heparin without carbondecoupling

At 600 MHz, the low field signal from the carbon-13 satellites of the methyl from the acetyl group overlaps with the signal of OSCS. To ensure the absence of OSCS, (<0.2 %w/v), a weak carbon decoupling field was applied at the methyl carbon chemical shift (17 ppm) to collapse the proton signals arising from the carbon satellites

CE Pharm 2009

Conclusion

Rapid setup of CE and NMR methods provided results in timely fashion and prevented potential problems from the presence of contaminated product.Improved CE methods have been developed and performed well CE and SAX-HPLC results were compatibleOptimized SAX-HPLC has been retained by EP for inclusion in revised heparin monograph

CE Pharm 2009

Team and Collaborators

CE - Marc-André Joly HPLC - Barry Lorbetskie1H-NMR – Dr. Yves AubinCoordination – Dr. Terry CyrCollaborators

Dr. G.Rautmann (EP) Dr. G. Somsen (Utrecht Univ.)Dr. R. Weinberger (CE Technol.)Dr. T.Wielgos (Baxter)Collaborative study team from CE Pharm