NovoEight® - a new rFVIII

Mirella Ezban

Senior Principle Scientist (VP)

Haemophilia biology

Novo Nordisk A/S, Måløv, Denmark

Victor Da Silva Melcunas Severe haemophilia A; Brazil

Four aspects of the molecule make a difference

Well defined molecule

Intact molecules

Full Tyr-1680 sulphation

Low MHC affinity of B-domain

NovoEight® is a well-defined truncated rFVIII molecule

HC = Heavy chain, LC = Light chain, SC = Single chain

Modified from Thim et al. Haemophilia 2010; 16:349-59

LC

NovoEight® Advate®

HC LC

SC

HC with various cleaved B domains

NovoEight®

A1

372 740

HC A2 A1

LC C2 C1 A3

1689

B

1689

A2 A1 HC

LC

B

FVIII with B domains of various lengths

C2 C1 A3

372 740

SDS-PAGE analysis of rFVIII molecules with different B domain sequences

Activated NovoEight® is identical to activated FVIII derived from the full-length sequence

Activation by thrombin

LC

A1

A2

SDS-PAGE analysis of rFVIIIa

NovoEight® Advate®

1689

A2 A1

C2 C1 A3

372 740

Purification process for NovoEight®

Modified from Thim et al. Haemophilia 2010; 16:349-59, Ezban et al. Eur J Haematol 2014. DOI: 10.1111/ejh.12366.

Detergent inactivation and concentration*

FVIII immunoaffinity chromoto-graphy

Anion exchange chromato-graphy

Nanofiltration 20 nm pore-size filter

Size exclusion

*) Hydrophobic interaction/ion exchange, virus inactivation with Triton X-100 detergent

FVIII immunoaffinity chromato-graphy

– recombinant monoclonal antibody

Intact molecules

in final product

Selective immuno purification removes incomplete molecules

Adapted from Thim L et al. Haemophilia 2010;16:349–59

A2

A1

B

A3

C1

C2

720 740

A2

A1

B

A3

C1

C2

A2

A1

B

A3

C1

C2

A2

A1

A2

A2

A2

A3

C1

C2

A2

A1

A2

A2

A2

A3

C1

C2

A2

A1

A2

A2

A2

A3

C1

C2

Intact HC including B-domain

Approx 20% of molecules cleaved at fragile motif at 720

A2

740

A2 A1 B

A3 C1 C2

A2 A1

A3 C1 C2

720

+ + + _ _ _

116

66.3

55.4

36.5

31.0

97.4

thrombin:

200.0

116.3 97.4

66.3

55.4

36.5

31.0

thrombin:

A1

A2

thrombin

LC HC

_ _ + + + _ _ +

SC

NovoEight® is homogenous and well-defined

HC = Heavy chain, LC = Light chain, SC = Single chain

Kristensen AK et al. ISTH 2013, abstract no. PO 058

Intact molecules

in final product

B A2 A1

Blue: NovoEight®

Grey: published FVIII structure

Purity of NovoEight® allowed crystallisation and confirms molecular integrity

Svensson et al, Biol Chem 2013; 394, 761

Ngo et al, Structure 2008; 16, 597

Methionine

Sucrose

NovoEight® formulation technology enables long-term, room-temperature stability

NovoEight® summary of product characteristics

Storage conditions & shelf life • 2-8 °C for up to 24 months • Up to 30 °C for up to 6

months Stability after reconstitution • Max 4 hours at ≤ 30 °C • Max 24 hours at 2-8 °C

B-domain sequence

I E P R S F S Q S R H N P N P P L K R V H Q R E I T S Q

P R S F N P P L V Q S

ReFacto AF® NovoEight®

Q S R H N P N P P L V Q S

B-domain of NovoEight® predicted to have low affinity to few MHC-II variants

Kimchi-Sarfaty et al., Trends Pharmacol Sci 2013; 34(10):534-48

FVIII is sulphated at six tyrosines

A2 A1 a2

C2 C1 A3 a3

B

1 372

1639

a1

740 761

1648 2020 2173 2332

SulfoTyr718

SulfoTyr719

SulfoTyr723

SulfoTyr1664 SulfoTyr1680

SulfoTyr346

HC

LC

In the absence of tyrosine sulphation at Tyr-1680 the affinity for VWF is reduced 5-fold

Sulphation of Tyr-1680 important for VWF binding

Leyte A et al. J Biol Chem 1991; 266: 740-746, Michnick DA et al., J Biol Chem 1994; 269: 20095-20102

NovoEight® has full sulphation of Tyr-1680

CHO = Chinese Hamster Ovary; BHK = baby hamster kidney cells; HEK = human embryonic kidney

Nielsen PF et al. Haemophilia 2012;18:e397–8 Kannicht C et al. Throm Res 2013;131:78–88

Product Origin Non-sulphated Tyr-1680 (%)

ReFacto AF® CHO 3.7–5.3

NovoEight® CHO Below detection limit (0.5%)

Advate® CHO 5.3–9.6

Human-cl rhFVIII HEK Below detection limit

Kogenate FS® BHK 1.5–1.6

Octanate® plasma-derived Below detection limit (0.5%)

In vitro VWF binding

Kd for VWF binding (nM)

NovoEight® Method

nM Kd values result in essentially all FVIII bound to VWF in vivo

Advate®

Christiansen MLS et al. Haemophilia 2010;16:878-87

ELISA 0.24 ± 0.04 0.48 ± 0.13

Surface plasmon resonance 0.23 ± 0.13 0.45 ± 0.07

Field study: laboratory monitoring of NovoEight®

• Laboratories - 36 clinical laboratories representing 19 countries participated

• 33 laboratories used one-stage clotting assay

• 5 laboratories used chromogenic (2 labs both)

• Wide variability in reagents used - 15 different APTT reagents

- 11 buffers for dilution

- 15 FVIII deficient plasmas

- 4 different chromogenic kits

- 29 different instruments used for measurement

Viuff D et al. Haemophilia 2011; 17:695–702

Reliable monitoring of NovoEight® with standard assays and reagents1

• Comparable and consistent estimates of target value were observed for NovoEight® and Advate®

• No need for separate standard • Assay variability was similar for both compounds

*Octocog alfa = Advate®

Turoctocog alfa = NovoEight®

1. Adapted from Viuff D et al. Haemophilia 17:695–702

NovoEight® effective in haemophilia A blood

• NovoEight® and Advate® added to haemophilia A blood

• Whole blood clot formation measured by kaolin-initiated thromboelastography (TEG®)

Christiansen MLS et al. Haemophilia 2010;16:878-87

MTG

Sulphation at Tyr-346 and Tyr-1664 important for optimal activation by thrombin

Michnick DA et al., J Biol Chem 1994; 269: 20095-20102

A2 A1 a2

C2 C1 A3 a3

B

1 372

1639

a1

740 761

1648 2020 2173 2332

SulfoTyr718

SulfoTyr719

SulfoTyr723

SulfoTyr1664 SulfoTyr1680

SulfoTyr346

HC

LC

NovoEight® effective in F8-KO mice bleeding model

• Tail cut 5 min after dosing

Elm T et al. Haemophilia 2012; 18: 139-145

mean and SEM, n = 8

NovoEight® is a high quality rFVIII

• Well defined and intact molecule

• Long-term, room-temperature stability

• State-of-the-art 20 nm filtration

• Low MHC-II affinity of B-domain

• Full Tyr-1680 sulphation

Victor Da Silva Melcunas, Severe haemophilia A; Brazil

Summary