Updating the comparison between bovine lung and porcine mucosal heparins. USP & NIBSC 6th Workshop...

Updating the comparison between bovine lung and porcine mucosal heparins.

USP & NIBSC 6th Workshop on the Characterization of Heparin Products , São Paulo, Brazil August 6-7, 2015

Carlos Alaez,1 Giangiacomo Torri2, Annamaria Naggi2, Marco Guerrini2, Andreu

Franco1, John Hogwood3.

3National Institute for Biological Standards and Control South Mimms, UK.

1Bioiberica, Barcelona, Spain.

2Ronzoni Institute, Milano, Italy.

Outline

• Background and Objective.

• Sample Preparation

• Sample Characterization

• Summary

Background

2014

Proposal to reintroduce bovine heparin in the

USA market.

1916 Discovery of heparin

1947 1st International Standard

Lungs

Mid-to late 50s Beginning of a gradual shift

to porcine mucosa

1939 1st commercialization in USA (Roche-Organon)

1959 2nd International Standard

1933 Scott &Charles bovine lungs as

raw material

~ 20 YEARS

1935 1st clinical

trials in humans90s

Outbreak of BSE Bovine lung heparin

withdrawn from the market.

Evolution of bovine lung heparin (BLH).

1970 3rd International Standard

(Porcine)

Background

• More and higher sensitive analytical tools combined in orthogonal approaches.

• Conditions of the manufacturing process have evolved to better preserve the molecular integrity.

• Tighter control of the identity and quality of heparin.

The world of heparin has changed significantly during the last 20 years…

How would BLH look if it were produced today?

ConcernsReintroduction

BLHDifferences BLH vs PMH

Available Information: How BLH looked in the past.

Aim of the Presentation

This presentation provides preliminary data on the characterization of new samples of bovine lung heparin in comparison to porcine mucosa heparin.

How would BLH look if it were produced today?

Preparation of BLH Samples

Hashed bovine lungs

Enzymatic digestion

Recovery by anion exchange resins

Crude Heparin

Fractionation with Organic Solvent /Dry

Crude Heparin

Oxidative Treatment

Bleaching with H2O2

BLH

Fractionation with Organic Solvent /Dry

Heparin Number of Samples

150 Kg of SM

600 Kg of SM

Overall Yield (‰)

BLH 5 2 3 0.08-0.13

PMH 10 -- -- 0.15-0.20

Pilot Plant Laboratory

USP Parameters BLH 1 BLH 2 BLH 3 BLH 4 BLH 5

Nucleotidic Impurities NMT 0.1% (w/w)

0.03 0.03 0.03 0.03 0.03

Protein Impurities NMT 0.1% (w/w)

0.05 0.05 0.04 0.03 0.06

Galactosamine in Total Hexosamine

NMT 1.0%

<LOQ <LOQ 1.01 <LOQ 0.82

• All the BLH samples comply the acceptance criteria of the USP monograph.

Characterization: Impurity Profile

USP Parameters

Mw(15,000-19,000 Da)

M24,000

(NMT 20%)

M8,000-16,000

/ M16,000-24,000

(NLT 1.0)

PMH 2 15,462 8.9 1.8

PMH 3 15,586 9.0 1.7

PMH 4 15,472 8.6 1.8Mean PMH

(n=3) 15,507 8.8 1.8

SD 69 0.2 0.1

BLH 1 13,461 8.3 2.6

BLH 2 14,216 9.9 2.4

BLH 3 13,780 9.9 2.5

BLH 4 13,529 8.9 2.6

BLH 5 14,351 11.0 2.4

Mean BLH (n=5)

13,867 9.6 2.5

SD 401 1.0 0.1• Mw BLH < Mw PMH

• BLH do not meet the USP acceptance criterion for Mw.

Characterization: Molecular Weight

• Mw BLH 1980 Mw BLH 2015m < Mw PMH

Characterization: Molecular Weight

• Polydispersity BLH > PMH

PMH2 PMH3 PMH4 G46 G65 G121 BLH1 BLH2 BLH3 BLH4 BLH50

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

HP-SEC-TDA Mn Mw Mp

PMH BLH 1980 BLH 2015

USP

Anti-Xa (95% CL)

USP Anti-IIa

(95%CL)

Anti-Xa/Anti-IIa

EP Sheep Plasma(95% CL)

APTT Human Plasma (95% CL)

HCII dependent

Anti-IIa (95% CL)

PMH (USP)

-- NLT 180 0.9-1.1 -- -- --

BLH 1 132(129-137)

116(107-124)

1.14 147(143-151)

124(121-129)

202(188-216)

BLH 2 136(132-141)

119(106-134)

1.14 152(148-158)

130(128-132)

206(196-225)

BLH 3 131(127-136)

131(125-135)

1.00 142(136-151)

119(117-121)

212(198-229)

BLH 4 144(138-148)

145(141-150)

0.99 158(155-162)

136(126-146)

250(233-270)

BLH 5 130(123 – 135)

141(133 – 151)

0.92 145(141 – 148)

110(101 – 119)

279(255 – 306)

Potencies (IU/mg) were estimated against 07/328, 6th IS unfractionated heparin (porcine). Values are expressed

on dry basis.

• BLH do not meet the USP criterion for potency.

Characterization: Anticoagulant Activity

Characterization: HSQC Spectrum

1H/13C Anomeric Region

G+Gal1

G-ANAcG-(ANS,6X)

I-(A6OH)

ANS,6X-(G)

ANS,6X-(I2S)+ANAc-(G)

ANS,6X-(I)

Epox

ANAc,6X-(I)

ANS,6Xred

ANAcred

I-(A6S)

ANS,3S,6X

I2S-(ANS,6X)

Gal2

G-(ANS,3S,6X)

Xyl-(Ser,ox)

Porcine Mucosa HP

ANX,6S

ANX,6OH

2ANS,3S,6X

2ANH2

2ANS,6S

Epox

2ANAc,6X

Porcine Mucosa HP

ANX,6S

ANX,6OH

2ANS,3S,6X

2ANAc,6X 2ANH2

2ANS,6S

Bovine Lung HP

ANS,6X red

I2Sred

ANAc,6X-(I)

ANS,6X-(I ) ANS,3S,6X

ANS,6X-(I2S)+ANAc-(G)

ANS,6X-(G) I2S-(ANS,6X)

G2S

I-(A6S)

I-(A6OH)

Xyl-(Serox)

G-(ANS,6X)

Bovine Lung HP

G-ANAc

Gal2

G-(ANS,3S,6X)

G + Gal1

1H/13CRing Region

A6S

ANS,6X-(I2S)

ANS,6X-(I)

ANS,6X-(G)

ANS,3S,6X

ANAc-(G)

ANAc,6X-(I)

ANH2

ANSɑred

A-epox

ANAca red

A-(GalA)+

Unk 0

10

20

30

40

50

60

70

80

90

100

0

2

4

6

8

10

12

14

16

18

20

Amine Residues%

mola

r

% m

ola

r

A6S

ANS,6X

-

(I2S)

Characterization: HSQC Compositional Analysis BLH vs. PMH

• Higher degree of sulfation at position 6-O of glucosamine.

• Lower content of N-acetylated glucosamine residues.

• Higher content of trisulfated disaccharide.

• Similar content of 3-O-sulfated glucosamine.

BLH (n=5)PMH (n=10)

BLH

I2S-

(ANS,

6X)

I-(A6

S)

I-(A)

G-(A

NS,3S

,6X)

G-(A

NS,6X

)

G-ANAc

Epox

GalA

G2S

0

10

20

30

40

50

60

70

80

90

100

0

1

2

3

4

5

6

7

8

9

Uronic Acid Residues%

mola

r%

mola

r

I2S-

(ANS,

6X)


• Higher degree of sulfation at position 2-O of iduronic acid.

• Lower content of glucuronic acid residues.

• Lower content of pentasaccharide.

BLH (n=5)PMH (n=10)

BLH

G+Gal1 Gal2 Xyl -ser ox Xyl-ser 0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Linkage Region

% m

ola

r


• BLH: Lower content of linkage region.

BLH (n=5)PMH (n=10)

Composition of Amine Residues (%molar)

ANS,6X-(I2S) ANS,6X-(I) ANS,6X-(G) ANS,3S,6X ANAc-(G) ANAc,6X-(I) ANH2 ANSɑred A-epoxANAc

ɑred

A-(GalA)+Unk

A6S

Average BLH 2015

(n=5)74.8 3.9 8.0 4.6 3.4 0.2 1.1 2.4 0.0 0.0 1.5 87.7

Range 74.0-763.5-4.4

6.8-8.7 3.9-5.3 2.5-4.5 0-0.9 0.8-1.2 1.5-2.7 -- --1.3-2.0

86.1-88.5

Average BLH 1980

(n=3)77.4 3.2 7.6 3.6 2.7 0.0 0.9 2.2 0.0 0.0 2.4 89.6

Range 76.7-78.7

3.0-3.4

7.1-8.2 3.5-3.7 2.4-3.4 -- 0-1.5 1.3-3.0 -- --1.4-3.9

88.8-90.4

Composition of Uronic Acid Residues (% molar) Linkage Region (% molar monosaccharides)

I2S-(ANS,6X) I-(A6S) I-(A6OH) G-(ANS,3S,6X) G-(ANS,6X) G-ANAc Epox GalA G2S G+Gal1

Gal2

Xyl -ser ox Xyl-ser

Average BLH 2015 (n=5) 89.5 2.8 0.9 1.0 3.0 2.1 0.0 0.0 0.6 3.0 0.9 0.9 0.0

Range 87.8-90.52.5-3.7

0.8-1.1

0.9-1.22.3-3.8

1.8-2.7

-- -- 0-0.72.5-3.4

0.8-1.1 0.7-1.0 --

Average BLH 1980 (n=3) 91.5 2.6 0.3 1.2 2.6 1.4 0.0 0.0 0.5 1.1 0.0 0.0 0.0

Range 90.1-92.82.4-2.7

0-0.9 1.1-1.22.2-2.8

0.9-1.9

-- -- 0-0.90.8-1.5

-- -- --

Characterization: HSQC Compositional Analysis BLH 2015 vs. 1980

• New BLH samples are slightly less sulfated and slightly more heterogeneous.

G8015-dig2_6_01_870.d: BPC 200.00000-1500.00000 -All MS, Smoothed (2.32,1,GA)




0

2

4

7x10Intens.

0

2

4

7x10Intens.

0

1

2

3

4

7x10Intens.

0

2

4

7x10Intens.

0 10 20 30 40 50 Time [min]





0

2

4

7x10Intens.

0

2

4

7x10Intens.

0

1

2

3

4

7x10Intens.

0

2

4

7x10Intens.

0 10 20 30 40 50 Time [min]





0.0

0.5

1.0

1.5

7x10Intens.

0.5

1.0

1.5

7x10Intens.

1

2

3

47x10

Intens.

0.5

1.0

1.5

7x10Intens.

36 38 40 42 44 46 48 50 52 Time [min]

∆U4,3,1

∆U4,5,0





0.0

0.5

1.0

1.5

7x10Intens.

0.5

1.0

1.5

7x10Intens.

1

2

3

47x10

Intens.

0.5

1.0

1.5

7x10Intens.

36 38 40 42 44 46 48 50 52 Time [min]

∆U4,4,1 ∆U3,4,0 ∆U4,5,0

∆U4,5,1

∆U4,6,0∆U4,3,1 – H2O

∆U2,4,0

635

1185

System peak

1428

418

System peak

∆U4,3,1

∆U4,7,0

∆U6,8,0-H2O

∆U5,7,0-H2O

∆U-ANAc

∆U-Gal-Gal-Xyl-CH2COOH+ ∆U-Gal-Gal-CH(CH2OH)COOH

ANS,6S +

∆UGal-ANS

∆U-ANAc,6S

∆U-ANS

∆U2S-ANAc

U2,2,0 + ∆U3,1,1

∆U-ANS,6S

∆U2S-ANS

ANS,3S,6S

∆U2S-ANAc,6S

∆U4,2,1 – H2O

∆U2S-ANS,6S

∆U-ANSLR-ox

635

System peak

System peak

∆U4,5,0

Characterization: Oligosaccharide Mapping by IPRP-HPLC-MS

∆U4,6,0 (3 isomers)∆U3,4,0

PM

HB

LH

1

∆U-ANAc

ANS,3S,6S

System peak

ΔU-A

NAc

ΔU-A

NS

ΔU2S

-ANAc

ΔU-A

NAc,6

S

ΔU-A

NS,6S

ΔU2S

-ANS

ΔU2S

-ANAc

,6S

ΔU2S

-ANS,

3S,6

S0.0

1.0

2.0

3.0

4.0

5.0

6.0

Disaccharides PMH

BLH 1

BLH 2

BLH 3

BLH 4

Are

a %

ΔU 2S-A NAc

ΔU-A NAc

,6S

ΔU-A NS,6S

ΔU 2S-A NS

ΔU 2S-A NAc

,6S

ΔU2S-A NS,

3S,6

S

ΔU-A NAc

ΔU-A NS

PMH BLH 1 BLH 2 BLH 3 BLH 4

ΔU-ANS,6S/ΔU2S-ANS 1.9 0.9 0.9 1.0 1.0


Empty Bars: < LOQ (0.1 %)

• BLH: Higher content of ΔU2S-ANS,3S,6S

• BLH: Lower content of disaccharides containing N-Acetylglucosamine

ΔU 4

,2,1

-H2O

ΔU 4

,3,1

-H2O

ΔU 4

,3,1

ΔU 4

,4,1

ΔU 4

,4,0

-H2O

ΔU 4

,5,0

ΔU 4

,5,1

(+ 4

18)

ΔU 4

,6,0

(iso

mer

1)

ΔU 4

,6,0

(iso

mer

2)

ΔU 4

,6,0

(iso

mer

3)

ΔU 4

,7,0

Δ 5,

7,0-

H2O

ΔU 6

,8,0

-H2O

0.0

0.5

1.0

1.5

2.0

2.5

Resistant Oligosaccharides PMH

BLH 1

BLH 2

BLH 3

BLH 4

Are

a %

ΔU # monosaccharide, # sulfates, # N-acetyl


Empty Bars: <LOQ (0.1 %)

+ (ΔU2S-ANS,3S,6S)ΔU2S-ANS,6S-G-ANS,3S,6S

ΔU-ANAc,6S-G-ANS,3S,6S

ΔU-ANS,6S-G-ANS,3S,6S

PF4/Heparin (molar ratio)

Characterization: In vitro interaction with human PF4

Sample Ratio PF4/HPSEC-TDA

Mw (Da)

PMH G5460

5.0 19,000

BLH 3 9.0 16,000

Photon Correlation Spectroscopy

Sample Ratio PF4/HPSEC-TDA

Mw (Da)PMH

G5460~5.0 19,000

BLH 3 ~9.0 16,000

Zeta Potential

PF4/Heparin (molar ratio)

• BLH has a stronger interaction with PF4 than PMH.

Summary

• The results of this study were obtained using a limited number of samples prepared by the same process/producer. Therefore, they do not reflect the possible structural variability of a commercial product.

•Research samples of BLH were obtained using the steps and conditions of a current heparin manufacturing process for unfractionated heparin.• Only slight structural differences were found between these samples and BLH

samples manufactured in the 80s.

• Comparative characterization of the new BLH samples and PMH, using state-of-the art analytical methods, showed that both heparins are different in terms of molecular weight, anticoagulant activity, sulfation pattern, disaccharide composition of the chain, structure and amount of the AT-binding domain and interaction with PF4.

• These results are in agreement with previous studies comparing bovine lung and porcine mucosa heparins.

• Work is in progress to increase the number of samples.

Acknowledgments

Bioiberica, Barcelona, Spain.

Andreu Franco

Ronzoni Institute, Milano, Italy. Giangiacomo Torri

Annamaria NaggiMarco Guerrini

NIBSC, South Mimms, UK. John Hogwood


https://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=https://twitter.com/bioiberica&ei=_8e2VLDcBImsUcS-gtAC&bvm=bv.83640239,d.d24&psig=AFQjCNEaBbFlsWnsxQiVI7b3cwWeQGvP-g&ust=1421351221437578

Thank you!


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Updating the comparison between bovine lung and porcine mucosal heparins. USP & NIBSC 6th Workshop...

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