characterization of currently marketed heparin products ... · 1 characterization of currently...

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Characterization of currently marketed heparin products: Analysis

of heparin digests by mass spectrometry.

David KeireFDA/DPA

09/13/2012

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DisclaimerThe findings and conclusions in this seminar have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency

determination or policy.

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“What will really improve drug safety is for us to improve the science of safety, and we are working on that. We are working

on better ways to detect safety signals, and we’re working on the mechanistic side to figure out what causes drug safety problems

and how they could be prevented.”Dr. Janet Woodcock

Director of the Center for Drug Evaluation and Research from an interview with Life Science Leader

Knowledge gained from CDER science and research increases the certainty and consistency of regulatory decisions, and contributes to the

development of regulatory guidance documents and best practice standards for pharmaceutical companies.

CDER Scientific Research

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Heparin

G2OH-(1,4)-ANAc

I2S-(1,4)-ANS,6S

Epimerization andSulfation activity in ER and Golgi

~70% of heparin

Heparin starts out as

~15% of heparin~20% of heparin

Mw~17kDa

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Low Molecular Weight Heparin (LMWH)• Dalteparin is produced through controlled nitrous

acid depolymerization of UFH heparin• Tinzaparin is obtained by controlled enzymatic

depolymerization of UFH heparin using heparinase from Flavobacterium heparinum

• Enoxaparin is obtained by alkaline depolymerization of UFH heparin benzyl ester

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Dalteparin

O

OCH2OH

O3SOH2C

O

O2C

HO

OSO3

OH

OH

Dalteparin: Nitrous Acid Deaminative Cleavage

2,5-anhydromannitol(AM.ol6S)

13C: C4-87.9 ppm,C2-85.9 ppmC5-82.3 ppm

245

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Tinzaparin (Enoxaparin) Ends

O

OHO

NHSO3

O3SO

O

CO2

HO

OSO3

Tinzaparin: (beta-elimination by heparinase)

U2S: 2-O-sulfo-4-deoxy-alpha-L-threo-hex-4-enopyranosil uronic acid

ANS- -reducing end

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3

OH

ANSred: 5.47/93.9ANAcred: 5.23/93.5

U2S: C1 C2 C3 C45.53/100.1 4.63/77.3 4.32/65.6 6.01/108.7

U2OH: C1 C45.18/103.9 5.83/110.4

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Enoxaparin Ends

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Marketplace Heparin Samples• 19 heparin sodium (a.k.a. unfractionated

heparin, UFH) samples from 6 DMF holders of heparin sodium and 10 samples of LMWH (dalteparin, tinzaparin and enoxaparin).

• Plus the USP Heparin Sodium and Enoxaparin identification standards.

• These samples were collected in the summer of 2009 (after the “heparin crisis”).

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A well characterized sample set• Brustkern, A.M., Buhse, F.L., Nasr, M., Al-Hakim, A. and Keire, D.A.,

“Characterization of currently marketed heparin products: Reverse-phase Ion-pairing liquid chromatography mass spectrometry of heparin digests.” Anal. Chem., 82(23), 9865-9870 (2010).

• Keire D.A., Ye H., Trehy M.L., Ye W., Kolinski R.E., Westenberger B.J., BuhseL.F., Nasr M., and Al-Hakim A., “Characterization of currently marketed heparin products: key tests for quality assurance.” Anal. Bioanal. Chem., 399(2), 581-591(2011).

• Sommers C.D., Ye H., Kolinski R.E., Nasr M., Buhse L.F., Al-Hakim A. and KeireD.A. “Characterization of currently marketed heparin products: Analysis of molecular weight and heparinase-I digest patterns.” Anal. Bioanal. Chem., 401(8), 2445-54, (2011).

• Sommers C.D., Adam A., Montpas N. and Keire D.A., “Characterization of currently marketed heparin products: adverse event relevant bioassays.” J. Pharm. Biomed. Anal., 67-68, 28-35, (2012).

• Wang B., Buhse L.F., Al-Hakim A., Boyne M.T. II, and Keire D.A., “Characterization of currently marketed heparin products: Analysis of heparin digests by RPIP-UHPLC-QTOF-MS.” J. Pharm. Biomed. Anal., 67-68, 42-50(2012).

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Intact heparin• We were unable to resolve mixture of

heparin sodium chains present in the drug on high resolution mass spectrometers.

– 12 Tesla FT-MS system at Wash Univ.-NCRR– Orbitrap systems in our lab.

– Issues were sulfate decomposition (loss of SO3) and the multiple acidic groups leading to adducts with metal cations.

• Thus we used a “bottom up” approach by digesting heparin to the disaccharide level.

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Reverse phase ion paring liquid chromatography-mass spectrometry (RPIP-LC-MS)

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The heparin family of possible disaccharides:

R1 R2 R3I-S SO3

- SO3- SO3

-

I-A Ac SO3 SO3

I-H H SO3- SO3

-

II-S SO3- SO3

- H

II-A Ac SO3- H

II-H H SO3- H

III-S SO3- H SO3

-

III-A Ac H SO3-

III-H H H SO3-

IV-S SO3- H H

IV-A Ac H H

IV-H H H H

I-P COEt SO3- SO3

-

--

N-sulfated glucosamine species

Internal standard

N-Acetyl glucosamine species

Amine glucosamine species

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UHPLC of UFH disaccharidesx106

0

0.4

0.8

1.2

1.6

2

2.4

0

0.2

0.6

1

1.4

1.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Retention Time (min)

I‐S

I‐A

III‐SII‐S

IV‐A III‐A

II‐AIV‐S

III‐H

II‐H

IV‐H

**

II-S and III-S are isobaric and only differ in the position of a SO4 group.

UFH digest products

Standard mixture of 12 possible disaccharides

Chromatography: Agilent 1290 system using a Waters BEH C18 column (2.1 x 100 mm). Buffer A: 30 mM HA in water at a pH of 5.3 adjusted with formic acid, Buffer B: 75% ACN, 25% water with 30 mM HA

Dynamic range: I-S vs. I-A

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Enzymatic Digestion Optimizationx106

1

2

2

1

2

2

1

2

2

3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

Retention Time (min)

A

B

C

D

E

FIV‐A IV‐S II‐A II‐S III‐S I‐A

III‐A

I

II

III

I + III

II + III

I + II + III

Mainly I-S, no X-A

No I-S; good for IV-A

I-S+good yield of other species

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II-S in {+} vs. {-} modes

x105

012345

519.1923

620.3133

700.2712

418.0716 750.8327

x105

0

1

2

3

4

416.0530496.0101

m/z

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

{+}

{-}

[M+HA-SO3]+

[M+3HA-2H]+

[M+2HA-H]+[M+HA]+

[M-H]-[M-H-SO3]-

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Response {+} vs. {-}y = 37951x + 1E+06

R2 = 0.9769

y = 13295x + 19613R2 = 0.9997

0.0E+00

1.0E+07

2.0E+07

0 200 400 600 800 1000 1200

I-S standard (pmol)

Ion

Peak

Are

a

{-} Better dynamic range and greater linear working range

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Response factors• Disaccharide standard mixture solutions

containing 12 disaccharides and an internal standard (IP) ranging from 1.1 to 50 µM in 5 steps were analyzed to obtain individual response factors.– EIC chromatograms for observed disaccharide

masses were obtained, summed and divided by the EIC I-P response and plotted vs. pmol injected.

• In general, RF values are higher for species with more sulfate groups.

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LMWH Digestsx106

0

0.5

1

1.5

0

0.5

1

1.5

Retention Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

12

*3

*

x106

Enoxaparin or tinzaparindigests: look like UFH.

Dalteparin digest:Has other disaccharide components.

1 2 3

I-S

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LMWH Composition

0

10

20

30

40

50

60

70

80

I‐S II‐S III‐S IV‐S I‐A II‐A III‐A IV‐A

% Com

positio

n

Disaccharides from heparin digest

Dalteparin: Lots #1-4Tinzaparin: Lots #5-7Enoxaparin: Lots #8-10

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RPIP-LC-MS Heparin Metrics

2 ± 01 ± 05 ± 02 ± 02 ± 05 ± 013 ± 170 ± 1Dalteparin(n=4)

7 ± 11 ± 05 ± 01 ± 06 ± 09 ± 012 ± 060 ± 1Tinzaparin(n=3)

5 ± 01 ± 04 ± 01 ± 05 ± 08 ± 013 ± 062 ± 1Enoxaparin(n=4)

4 ± 1

II-A

14 ± 2

II-S

7 ± 21 ± 01 ± 05 ± 18 ± 061 ± 3Heparin sodium(n=20)

IV-AIII-AI-AIV-SIII-SI-SType

• Here we use the same MS instrument and method across a large sample set to establish a set of percent relative composition values.

• Use for specification or surveillance purposes.

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Robust?8-5-2011: After 44 samples

8-23-2011: After 35 samples

8-27-2011: Before 60 samples



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Robust?6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

-ESI TIC Scan Frag=175.0V 09-08-2011-StdMix-2-negative.d

1 1 2

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

One month with a total of 157 injections.

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Manufacturer Signature?

7.0 ± 0.30.9 ± 0.14.1 ± 0.21.0 ± 0.04.5 ± 0.27.8 ± 0.211.2 ± 0.163.6 ± 0.820 (USP Reference Standard)

4.8 ± 0.30.8 ± 0.13.4 ± 0.01.0 ± 0.04.4 ± 0.27.5 ± 0.214.8 ± 1.563.3 ± 1.5Manufacturer #6 (lots #17-19)





3.6 ± 0.10.7 ± 0.13.6 ± 0.11.0 ± 0.03.8 ± 0.07.9 ± 0.117.6 ± 0.161.7 ± 0.2Manufacturer #2 (lot #4)


IV-AIII-AII-AI-AIV-SIII-SII-SI-SDisaccharides

Samples

• Lots of heparin sodium made by manufacturer #3 have more total N-acetyl content than lots from other manufacturers: mean total N-Acetyl 12.5 ± 2.3% vs. #3 16.2%

• Heparin begins as G2OH-ANAc

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OSCS Contaminated Heparin Digests?

n.o.n.o.n.o.n.o.n.o.100 ± 0MP#1+10.0% OSCS

n.o.n.o.n.o.2.0 ±0 .33.3 ± 0 .694.6 ± 0.4MP#1+5.0% OSCS

0.2 ± 0.00.2 ± 0.00.3 ± 0.02.0 ± 0.29.9 ± 0.182.8 ± 0.1MP#1+2.0% OSCS

0.2 ± 0.00.2 ± 0.00.4 ± 0.02.0 ± 0.215.2 ± 1.182.0 ± 0.9MP#1+1.0% OSCS

0.3 ± 0.00.6 ± 0.01.0 ± 0.02.1 ± 0.022.7 ± 0.673.3 ± 0.4MP#1+0.5% OSCS

0.2 ± 0.00.8 ± 0.01.4 ± 0.03.0 ± 0.227.5 ± 0.467.1 ± 0.3MP#1+0.1% OSCS

1.2 ± 0.02.3 ± 0.12.6 ± 0.12.7 ± 0.125.6 ± 0.265.6 ± 0.2MP#1

IV-AII-A/III-AI-AIV-SII-S/III-SI-SSample

Brustkern, A.M., Buhse, L.F., Nasr, M., Al-Hakim, A. and Keire, D.A., “Characterization of currently marketed heparin products: Reverse-phase Ion-pairing liquid chromatography mass spectrometry of heparin digests.” Anal. Chem., 82(23), 9865-9870 (2010).

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• How accurate are these values?– 2D-NMR as an

orthogonal assay.

NMR of intact heparin

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Orthogonal assay

3074132081MS-Heparin Sodium

2478 ± 2%15 ± 2%19 ± 2%81 ± 2%NMR-Heparin Sodiuma

I2OHI2SANAcANX,6OHANX,6SMonosaccharide

aKeire et al. unpublished results on the analysis of 2D-NMR data of intact heparins.

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Conclusions1. NMR and MS are information-rich assays for surveillance of

complex drug structure and composition:• Extra peaks or intensity changes are indications of

impurities, contaminants or structure alterations.• Important to establish the normal range of variability for

each drug with a marketplace survey.• Information-rich data sets from a robust method are

amenable to pattern recognition analysis that can make non-subjective pass/fail judgments for complex drugs.

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People that did the work!• Bo Wang (ORISE)

• Adam Brustkern (ORISE)• Supported by:

– Michael Boyne– Lucinda Buhse

– Ali Al-Hakim

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Thank You

[email protected]

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