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
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