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 Torri 2, Annamaria Naggi 2, Marco Guerrini 2, Andreu Franco 1, John Hogwood 3. 3 National Institute for Biological Standards and Control South Mimms, UK. 1 Bioiberica, Barcelona, Spain. 2 Ronzoni Institute, Milano, Italy.

Outline Background and Objective. Sample Preparation Sample Characterization Summary

Background 2014 Proposal to reintroduce bovine heparin in the USA market Discovery of heparin st International Standard Lungs Mid-to late 50s Beginning of a gradual shift to porcine mucosa st commercialization in USA (Roche-Organon) nd International Standard 1933 Scott &Charles bovine lungs as raw material ~ 20 YEARS st clinical trials in humans 90s Outbreak of BSE Bovine lung heparin withdrawn from the market. Evolution of bovine lung heparin (BLH) rd 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? Concerns Reintroduction BLH Differences BLH vs PMH Available Information: How BLH looked in the past.

Aim of the Pres entation 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 H 2 O 2 BLH Fractionation with Organic Solvent /Dry Heparin Number of Samples 150 Kg of SM600 Kg of SM Overall Yield (‰) BLH PMH Pilot Plant Laboratory

USP ParametersBLH 1BLH 2BLH 3BLH 4BLH 5 Nucleotidic Impurities NMT 0.1% (w/w) 0.03 Protein Impurities NMT 0.1% (w/w) Galactosamine in Total Hexosamine NMT 1.0% <LOQ 1.01<LOQ0.82 All the BLH samples comply the acceptance criteria of the USP monograph. Characterization: Impurity Profile

USP Parameters Mw (15,000-19,000 Da) M 24,000 (NMT 20%) M 8,000-16,000 / M 16,000-24,000 (NLT 1.0) PMH 215, PMH 315, PMH 415, Mean PMH (n=3) 15, SD BLH 113, BLH 214, BLH 313, BLH 413, BLH 514, Mean BLH (n=5) 13, SD 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 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 BLH ( ) 116 ( ) ( ) 124 ( ) 202 ( ) BLH ( ) 119 ( ) ( ) 130 ( ) 206 ( ) BLH ( ) 131 ( ) ( ) 119 ( ) 212 ( ) BLH ( ) 145 ( ) ( ) 136 ( ) 250 ( ) BLH (123 – 135) 141 (133 – 151) (141 – 148) 110 (101 – 119) 279 (255 – 306) Potencies (IU/mg) were estimated against 07/328, 6 th 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 1 H/ 13 C Anomeric Region G+Gal1 G-A NAc G-(A NS,6X ) I-(A 6OH ) A NS,6X -(G) A NS,6X -(I 2S )+A NAc - (G) A NS,6X -(I) Epox A NAc,6X -(I) A NS,6X  red A NAc  red I-(A 6S ) A NS,3S,6X I 2S -(A NS,6X ) Gal 2 G-(A NS,3S,6X ) Xyl-(Ser,ox) Porcine Mucosa HP A NX,6S A NX,6OH 2 A NS,3S,6X 2 A NH2 2 A NS,6S Epox 2 A NAc,6X Porcine Mucosa HP A NX,6S A NX,6OH 2 A NS,3S,6X 2 A NAc,6X 2 A NH2 2 A NS,6S Bovine Lung HP A NS,6X  red I 2S  red A NAc,6X -(I) A NS,6X -(I ) A NS,3S,6X A NS,6X -(I 2S )+A NAc -(G) A NS,6X -(G) I 2S - (A NS,6X ) G 2S I-(A 6S ) I-(A 6OH ) Xyl-(Ser ox ) G-(A NS,6X ) Bovine Lung HP G-A NAc Gal 2 G- (A NS,3S,6X ) G + Gal 1 1 H/ 13 C Ring Region

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) Characterization: HSQC Compositional Analysis BLH vs. PMH 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

Characterization: HSQC Compositional Analysis BLH vs. PMH BLH: Lower content of linkage region. BLH (n=5) PMH (n=10)

Composition of Amine Residues (%molar) A NS,6X -(I 2S )A NS,6X -(I)A NS,6X -(G)A NS,3S,6X A NAc -(G)A NAc,6X -(I)A NH2 A NSɑred A-epox A NAc ɑred A- (GalA)+U nk A 6S Average BLH 2015 (n=5) Range Average BLH 1980 (n=3) Range Composition of Uronic Acid Residues (% molar) Linkage Region (% molar monosaccharides) I 2S -(A NS,6X )I-(A 6S )I-(A 6OH )G-(A NS,3S,6X )G-(A NS,6X )G-A NAc EpoxGalAG 2S G+Gal 1 Gal 2 Xyl -ser ox Xyl-ser Average BLH 2015 (n=5) Range Average BLH 1980 (n=3) Range Characterization: HSQC Compositional Analysis BLH 2015 vs New BLH samples are slightly less sulfated and slightly more heterogeneous.

∆U4,3,1 ∆U4,5,0 ∆U4,4,1 ∆U3,4,0 ∆U4,5,0 ∆U4,5,1 ∆U4,6,0 ∆U4,3,1 – H 2 O ∆U2,4, System peak System peak ∆U4,3,1 ∆U4,7,0 ∆U6,8,0-H 2 O ∆U5,7,0-H 2 O ∆U-A NAc ∆U-Gal-Gal-Xyl-CH 2 COOH + ∆U-Gal-Gal-CH(CH 2 OH)COOH A NS,6S + ∆UGal-A NS ∆U-A NAc,6S ∆U-A NS ∆U 2S -A NAc U2,2,0 + ∆U3,1,1 ∆U-A NS,6S ∆U 2S -A NS A NS,3S,6S ∆U 2S -A NAc,6S ∆U4,2,1 – H 2 O ∆U 2S -A NS,6S ∆U-A NS LR-ox 635 System peak ∆U4,5,0 Characterization: Oligosaccharide Mapping by IPRP-HPLC-MS ∆U4,6,0 (3 isomers)∆U3,4,0 PMH BLH 1 ∆U-A NAc A NS,3S,6S System peak

ΔU-A NAc ΔU-A NS PMHBLH 1BLH 2BLH 3BLH 4 ΔU-A NS,6S /ΔU 2S -A NS Characterization: Oligosaccharide Mapping by IPRP-HPLC-MS Empty Bars: < LOQ (0.1 %) BLH: Higher content of ΔU 2S -A NS,3S,6S BLH: Lower content of disaccharides containing N-Acetylglucosamine

ΔU # monosaccharide, # sulfates, # N-acetyl Characterization: Oligosaccharide Mapping by IPRP-HPLC-MS Empty Bars: <LOQ (0.1 %) + (ΔU 2S -A NS,3S,6S ) ΔU 2S -A NS,6S -G-A NS,3S,6S ΔU-A NAc,6S -G-A NS,3S,6S ΔU-A NS,6S -G-A NS,3S,6S

PF4/Heparin (molar ratio) Characterization: In vitro interaction with human PF4 SampleRatio PF4/HP SEC-TDA Mw (Da) PMH G ,000 BLH ,000 Photon Correlation Spectroscopy SampleRatio PF4/HP SEC-TDA Mw (Da) PMH G5460~5.019,000 BLH 3~9.016,000 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 Naggi Marco Guerrini NIBSC, South Mimms, UK. John Hogwood USP & NIBSC 6th Workshop on the Characterization of Heparin Products, São Paulo, Brazil August 6-7, 2015

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