Extended Half Life and Conventional Clotting Factors for Hemophilia Glenn Pierce MD PhD WFH Board of Directors La Jolla, California USA World Federation of Hemophilia (WFH) Indonesian Hemophilia Society (IHS) Symposium on Hemophilia Care Sanur, Bali, Indonesia 23 April 2017

Evolution of modern hemophilia treatment Investigational therapies (2015–) ► Gene therapy ► Novel agents Therapeutic Value Protein engineering era EHL clotting factors (2014–) ► Prolonged half-life (FVIII/FIX) Recombinant clotting factors FVIII, FIX, FVIIa (1990s) Recombinant era Plasma-derived clotting factors (1969) ► Widespread viral contamination Evolution of Products Pierce/WFH

Why Were Extended Half Life Products Developed? Conventional plasma-derived and recombinant clotting factors have short half lives Most prophylaxis regimens don’t prevent crippling arthritis, they delay it Frequent intravenous infusions required to control bleeding Extended half life FVIII and FIX decrease the frequency and burden of treatment Mazepa et al Blood. 2016 Mar 16. pii: blood-2015-10-675140. [Epub ahead of print]; Dunn AL The long and short of it: using the new factor products ASH Education 2015; Pierce/WFH

Extended Half-Life Products Extend the Time Between Infusions Half life: amount of time it takes for 50% of drug to be eliminated Half-lives are averages of a population Individuals can have half-lives that are average, or at the extremes of the population Half life dictates how frequently clotting factor must be administered to protect from bleeding Pierce/WFH

How to Create Extended Half-Life Products? 2 Primary Methods Fusion- Protein Engineering FIX-albumin (Idelvion) FVIII-Fc (Eloctate) and FIX-Fc (Alprolix), a portion of IgG Both albumin and IgG remain in the circulation for weeks Neonatal Fc receptor (FcRn) recycles Fc/Alb fusion proteins 8+ Fc proteins have been FDA approved with a good safety profile IgG-Fc is responsible for tolerance induction and placental transfer Pegylation- Manufacturing Process PEG, a petroleum derivative, slows degradation when attached to protein Site specific- BAY 94-9027 via Cysteine mutation Sugars attached to clotting factor- N8-GP, N9-GP Random-BAX855 (Adynovate) Multiple Pegylated proteins have been FDA approved Chamow S and Ashkenazi A Trends Biotechnol 1996; Dumont JA et al. BioDrugs. 2006;20:151-160; Rath T et al Crit Rev Biotechnol, Early Online: 1–20 Pierce/WFH

Neonatal Fc Receptor Recycling Pathway Figure 3. FcRn recycling pathway.29 IgG and Fc fusion proteins are taken up from circulation into cells by nonspecific pinocytosis and/or endocytosis mechanisms.49 As the endosomes become acidic, the Fc domain of IgG or Fc fusion proteins binds to FcRn. Once the endosome fuses back at the cell surface, Fc dissociates from FcRn at neutral pH and IgG and Fc fusion proteins are released back into the circulation.49 In contrast, circulating proteins that do not interact with FcRn are trafficked to endosomal and lysosomal degradation pathways.29,50 Ultimately, Fc degrades naturally and does not accumulate in the body.51 Despite its name, the expression of human FcRn is stable throughout life.52 Reprinted with permission from Roopenian and Akilesh.29 Roopenian DC, Akilesh S. FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol. 2007;7:715-725 Pierce/WFH

Eloctate (rFVIIIFc) and Alprolix (rFIXFc) How are they similar to conventional recombinant and plasma-derived Factor VIIII and IX concentrates? How are they different? Half life Dosing frequency Dose (rFIXFc) Optimal usage? Pierce/WFH

PIVOTAL STUDY RESULTS Blood. 2014;123(3):317-325 Regulatory approvals in multiple geographies: US, Canada, Japan, Australia, EU Pierce/WFH

Extended Half Life clotting factors Prolongation of the half-life Reduction of the number of injections required for treatment and prophylaxis Pierce/WFH

Dosage and Administration: Eloctate (rFVIIIFc) Dosing Guidelines ELOCTATE, Antihemophilic Factor (Recombinant), Fc Fusion Protein, is a recombinant DNA derived, antihemophilic factor indicated in adults and children with Hemophilia A Similar to other FVIIIs Calculation of the required dose of Factor VIII based on empirical finding- 1 IU Factor VIII/kg raises plasma Factor VIII level 2 IU/dL. In vivo peak increase in Factor VIII level expressed as IU/dL (or % of normal) estimated using : Estimated Increment of Factor VIII (IU/dL or % of normal) = [Total Dose (IU)/kg] x 2 (IU/dL per IU/kg) The dose to achieve a desired in vivo peak increase in Factor VIII level may be calculated using the following formula: Dose (IU) = body weight (kg) x Desired Factor VIII Rise (IU/dL or % of normal) x 0.5 (IU/kg per IU/dL) The label notes that one IU of ELOCTATE per kg body weight increases the circulating level of Factor 8 by 2 IU/dL and provides two formulas: One calculating the expected in vivo peak given a certain dose, and another to identify the dose required to reach a desired factor 8 rise Pierce/WFH

Dosage and Administration: Alprolix (rFIXFc) Dosing Guidelines ALPROLIX, Coagulation Factor IX (Recombinant), Fc Fusion Protein, is a recombinant DNA derived, coagulation Factor IX concentrate indicated in adults and children with hemophilia B 1 IU ALPROLIX/kg increases circulating Factor IX 1% [IU/dL]. Estimate required dose or expected in vivo peak increase in Factor IX level expressed as IU/dL (or % of normal) using the following formulas: IU/dL (or % of normal) = [Total Dose (IU)/Body Weight (kg)] x Recovery (IU/dL per IU/kg) OR  Dose (IU) = Body Weight (kg) x Desired Factor IX Rise (IU/dL or %) x Reciprocal of Recovery (IU/kg per IU/dL) Dose adjustment may be necessary in pediatric patients under 12 years of For patients 12 years of age or older, dose adjustment is not usually required. Similar Pierce/WFH

How do extended half-life products change treatment in hemophilia? Acute bleeding Surgery Prophylaxis Pierce/WFH

Dosing regimens for Acute Bleeding events Efficacy comparable to standard coagulation factors: impact of EHL rFVIII is less remarkable than EHL rFIX products Type of bleeding Dosage Frequency of dosing (hours)   rFVIII Standard rFVIII EHL Minor/Moderate 20-40 IU/kg 20-30 IU/kg 12 to 24 24 to 48 Major (Life threatening hemorrhages) 40-50 IU/kg 8 to 24 Doses similar, frequency differs Type of bleeding Dosage Frequency of dosing (hours)   rFIX Standard rFIX EHL Minor/Moderate 30-60 IU/kg 12 to 24 48 Major (Life threatening hemorrhages) 60-80 IU/kg 80-100 IU/kg 24 for the first 3 days, then every 48 hours US Product inserts, Eloctate and Alprolix Mahlangu et al Blood 2014 Powell et al N Engl J Med 2013 Pierce/WFH

Routine prophylaxis rFVIII extended half-life   Standard EHL Low dose prophylaxis with EHL# Recommended dose 20-40 IU/kg 25-65 IU/kg 10-20 IU/kg Frequency 2 days 3-5 days 4-7 days Doses similar, frequency differs #No data available for the EHL yet Eloctate product insert Mahlangu et al Blood 2014 Pierce/WFH

Routine prophylaxis rFIX extended half-life   Standard EHL Low dose prophylaxis with EHL# Recommended dose 50 IU/kg 100 IU/kg 10-20 IU/kg Frequency 3-4 days 7 days 10-14 days 7-10 days Doses similar, frequency differs, overall dose less #No data available for the EHL yet Alprolix product insert Powell et al NEJM 2013 Pierce/WFH

Efficacy - rFIX extended half-life These protein engineered drugs simplify prophylactic regimens Conventional products  2 infusions/week rFIX Extended half-life  1 infusion/1-2 weeks Reduction in injection frequency  50-75% Monday Thursday Sunday Critical level Conventional products  3-4 infusions/week rFIX Extended half-life  1-2 infusion/week Reduction in injection frequency  33-66% Extended protection from bleeding Improving adherence and burden of therapy Pierce/WFH

Surgery rFVIII extended half-life The rFVIII extended half-life products may offer small impact on dosing frequency during the first 48 hours The daily dose adopted with rFVIII-Fc is similar to that of standard rFVIII rFVIII requires median total dose of 62.50 IU/kg (day of surgery) rFVIII-Fc requires median total dose of 58.31 IU/kg (day of surgery) As with conventional FVIII use peri-surgery, dose and frequency should be based upon measured FVIII activity levels (Mahlangu JN et al. Thromb Haemost 2016; 116: http://dx.doi.org/10.1160/TH15-10-0780) Pierce/WFH

Surgery rFVIII extended half-life Initial dose similar, frequency differs (Highlights of prescribing information to use standard rFVIII) (Highlights of prescribing information to use Eloctate, rFVIII-Fc) Pierce/WFH

Surgery rFIX extended half-life rFIX extended half-life products enable marked reduction in dosing frequency for major surgical procedures Daily dose rFIX-Fc less than standard rFIX rFIX median dose 154 IU/kg (day of surgery) rFIX-Fc median dose 84.16 IU/kg (day of surgery) After 3 days, dosing intervals may be extended Dose and frequency should be based upon measured FIX activity levels (Powell JS et al. Br J Haematol. 2015;168:124–34.; Ragni MV et al. J Haemophilia 2002; 8: 91–7) Pierce/WFH

Surgery rFIX extended half-life Initial dose similar, frequency differs (Highlights of prescribing information to use standard rFIX) (Highlights of prescribing information to use Alprolix, rFIX-Fc) Pierce/WFH

Warnings and Precautions Hypersensitivity Reactions Hypersensitivity reactions, including anaphylaxis, possible with ELOCTATE and ALPROLIX. Early signs of hypersensitivity reactions that can progress to anaphylaxis may include angioedema, chest tightness, dyspnea, wheezing, urticaria, and pruritus. Immediately discontinue administration and initiate appropriate treatment if hypersensitivity reactions occur. Neutralizing Antibodies Formation of neutralizing antibodies (inhibitors) to Factor VIII or Factor IX can occur following administration of ELOCTATE or ALPROLIX. If the plasma Factor level fails to increase as expected or if bleeding is not controlled after Factor administration, suspect the presence of an inhibitor (neutralizing antibody). Monitoring Laboratory Tests: Factor VIII results can be obtained with all instruments and reagents. Factor IX results can be affected by the type of aPTT reagent used. Measurement with a one-stage clotting assay using a kaolin-based aPTT reagent will likely result in an underestimation of activity level. Ellagic acid and silica reagents are ok. Class Effects The Warnings and Precautions section of the label states that hypersensitivity reactions, including anaphylaxis, are possible with ELOCTATE. Early signs of hypersensitivity reactions that can progress to anaphylaxis may include angioedema, chest tightness, dyspnea, wheezing, urticaria, and pruritus. The label states to immediately discontinue administration and initiate appropriate treatment if hypersensitivity reactions occur. Furthermore, formation of neutralizing antibodies – inhibitors - to Factor 8 can occur following administration of ELOCTATE. The label states to monitor all patients for the development of Factor 8 inhibitors by appropriate clinical observations and laboratory tests. If the plasma Factor 8 level fails to increase as expected or if bleeding is not controlled after ELOCTATE administration, the label indicates that one should suspect the presence of an inhibitor. Pierce/WFH

Use in Specific Populations Pediatric Use Pharmacokinetic studies in children show shorter half-life and lower recovery of Factor VIII and Factor IX compared to adults. Because clearance has been shown to be significantly higher in the younger, pediatric population (2 to 5 years of age), higher and/or more frequent dosing based on body weight may be needed. Dosing needs similar, frequency differs The label states that pharmacokinetic studies in children have demonstrated a shorter half-life and lower recovery of Factor 8 compared to adults. Because clearance has been shown to be significantly higher in the younger, pediatric population, specifically those 2 to 5 years of age, higher and/or more frequent dosing based on body weight may be needed. Safety and efficacy studies have been performed in 56 previously treated, pediatric patients less than 18 years of age who received at least one dose of ELOCTATE as part of routine prophylaxis, on-demand treatment of bleeding episodes, or perioperative management. Pierce/WFH

In Summary Extended half life FVIII and FIX products have been approved for treatment and prevention of bleeding in Hemophilia A and B, respectively. These products contain an active Factor VIII or Factor IX molecule EHL are similar to conventional FVIII and FIX products Doses required to treat major and minor bleeding episodes and surgery Assays to measure FVIII and FIX activity (with exceptions)* EHL are different from conventional FVIII and FIX products Prolonged dosing intervals for prophylaxis, acute treatment, and surgery Second dose to treat bleeding may not be required Next generation research: better protection and greater decrease in treatment burden, including gene therapy *Eloctate- no assay issues Alprolix- do not use kaolin to initiate One Stage assay Pierce/WFH

Immune Tolerance Induction Not in Product Label Eloctate Anecdotal case reports of rapid induction of tolerance using Eloctate Groomes et a., Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein, Pediatr Blood Cancer 2016 DOI 10.1002/pbc Malec, Ragni, et al., ASH 2015. 3 patients who received 100-200 IU/kg of rFVIIIFc for ITI therapy every other day or thrice weekly and achieved inhibitor level less than 0.6 B.U in 12 weeks Alprolix Extreme caution is warranted due to risk of anaphylaxis and/or nephrotic syndrome- class effect Pierce/WFH

Thank You Glenn Pierce MD PhD glennfpierce@gmail.com

Alprolix and Eloctate Bibliography-1 Peters RT, Low SC, Kamphaus GD, Dumont JA, Amari JV, Lu Q, Zarbis-Papastoitsis G, Reidy TJ, Merricks EP, Nichols TC, Bitonti AJ. Prolonged activity of factor IX as a monomeric Fc fusion protein. Blood, 2010, 11;115:2057-64. Shapiro AD, MV Ragni, LA Valentino, NS. Key, NC Josephson, JS. Powell, G Cheng, AR Thompson, J Goyal, KL Tubridy, RT. Peters, JA. Dumont, D Euwart, L Li, B Hallen, P Gozzi,1 AJ Bitonti, H Jiang, A Luk, GF Pierce, Recombinant factor IX-Fc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients Blood, 2012, 119: 666-672. Dumont JA, T Liu, SC Low, X Zhang, G Kamphaus, P Sakorafas, C Fraley, D Drager, T Reidy, J McCue, HG Franck, EP Merricks, T Nichols, AJ Bitonti, GF Pierce, H Jiang. Prolonged Activity of a Recombinant Factor VIII-Fc Fusion Protein In Hemophilia A Mice And Dogs Blood, 2012, 119: 3024-3030. Powell JS, NC Josephson, DQuon3, MV Ragni, G Cheng5, E Li, H Jiang, L Li, JA Dumont, J Goyal, X Zhang, J Sommer, J McCue, M Barbetti, A Luk, GF Pierce Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients Blood, 2012, 119: 3031-3037. Peters RT, G Toby, Q Lu, T Liu, JD Kulman, SC Low, AJ Bitonti, GF Pierce. Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein. Thromb Haemost, 2013, 11:132-141. Powell JS, J Pasi, MV Ragni, MC Ozelo, L Valentino, JN Mahlangu, NC Josephson, D Perry, MJ Manco-Johnson, S Apte, RI Baker, GC Chan, N Novitzky, RS Wong, S Krassova, G Allen, H Jiang, A Innes, S Li, LM Cristiano, J Goyal, JM Sommer, JA Dumont, K Nugent, G Vigliani, A Brennan, A Luk, GF Pierce, on behalf of the B-LONG investigators. Phase 3 study of recombinant factor IX Fc in hemophilia B. N Engl J Med, 2013, 369:2313-2323. Mahlangu J, J Powell, M Ragni, P Chowdary, N Josephson, I Pabinger, H Hanabusa, N Gupta, R Kulkarni, P Fogarty, D Perry, A Shapiro, J Pasi, S Apte, I Nesterov, H Jiang, S Li, S Neelakantan, LM Cristiano, J Goyal, J Sommer, JA Dumont, N Dodd, K Nugent, G Vigliani, A Luk, A Brennan, GF Pierce, on behalf of the A-LONG investigators. Phase 3 study of recombinant factor VIII Fc in hemophilia A. Blood, 2014, 123:317-325. Sommer JM, N Moore, B McGuffie-Valentine, S Bardan, Y Buyue, GD Kamphaus, BA Konkle, GF Pierce. Comparative field study evaluating the activity of recombinant Factor VIII-Fc fusion protein (rFVIIIFc) in plasma samples at clinical haemostasis laboratories. Haemophilia, 2014, 20:294-300. McCue J, D Osborne, J Dumont, R Peters1, B Mei1, GF Pierce, K Kobayashi, D Euwart. Validation of the manufacturing process used to produce long- lasting recombinant factor IX Fc fusion protein. Haemophilia 2014; 20:e327-e335. Sommer JM, Y Buyue* S Bardan, RT Peters, H Jiang, GD Kamphaus, E Gray, GF Pierce. Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity. Thromb Haemost 2014; 112: 932-40. Shapiro A, Ragni M, Kulkarni R, Oldenburg J, Srivastava A, Quon D, Pasi J, Hanabusa H, Pabinger I, Mahlangu J, Fogarty P, Lillicrap D, Kulke S, Potts J, Neelakantan S, Nestorov I, Li S, Dumont J, Jiang H, Brennan A, Pierce GF. Dosing Recombinant Factor VIII Fc Fusion Protein: Relationship of Extended Dosing Interval to Prior Treatment Regimen and Baseline von Willebrand Factor Level. J Thromb Haemost, 2014; 12:1788-1800. Powell J, A Shapiro, M Ragni, C Negrier, J Windyga, M Ozelo, J Pasi, R Baker, J Potts, S Li, B Mei, GF Pierce, B Robinson. Switching to recombinant factor IX Fc fusion protein prophylaxis results in fewer infusions, decreased FIX consumption, and lower bleeding rates. Brit J Haematol, 2015; 168:113-123. Pierce/WFH

Alprolix and Eloctate Bibliography-2 Powell J, S Apte, H Chambost, C Hermans, S Jackson, N Josephson, J Mahlangu, M Ozelo, K Peerlinck, J Pasi, D Perry, M Ragni, X Wang, H Jiang, S Li, LM Christiano, A Innes, K Nugent, A Brennan, A Luk, G Allen, GF Pierce, B Robinson. Long-Lasting Recombinant Factor IX Fc Fusion (rFIXFc) for Perioperative Management of Subjects with Haemophilia B in the Phase 3 B-LONG Study. Brit J Haematol, 2015; 168:124-134. G Young, J Mahlangu, R Kulkarni, B Nolan, R Liesner, J Pasi, C Barnes, S Neelakantan‡, G Gambino, LM Cristiano, GF. Pierce, G Allen, for the Kids A-LONG Investigators. Recombinant factor VIII Fc fusion protein for the prevention and treatment of bleeding in children with severe hemophilia A. J Thromb Haemost, 2015; 13:967-977. Nolan B, J Mahlangu, D Perry, G Young, R Liesner, B Konkle, S Rangarajan, S Brown, H Hanabusa, KJ Pasi, I Pabinger, S Jackson, LM Cristiano, X Li, GF Pierce, G Allen. Long-term safety and efficacy of recombinant factor VIII Fc fusion protein (rFVIIIFc) in subjects with haemophilia A. 2015, Haemophilia doi: 10.1111/hae.12766 McCue J, Ri Kshirsagar, K Selvitelli, B Mei, R Peters, GF Pierce, J Dumont, S Raso, H Reichert. Manufacturing process used to produce longer lasting recombinant factor VIII Fc fusion protein, Biologicals 2015; 43:213-219. Dumont J, KS Loveday, DR Light, GF Pierce, H Jiang. Evaluation of the Toxicology, Pharmacokinetics, and Local Tolerance of Recombinant Factor IX Fc Fusion Protein in Animals. Thromb Res 2015; 136:371-378. Hanabusa H, M Shima, K Nogami, T Matsushita, K Fukutake, M Taki, M Sekai, Y Dong, S Neelakantan, LM. Cristiano, GF. Pierce, B Mei. Evaluation of the Safety, Pharmacokinetics, and Efficacy of Recombinant Factor VIII Fc Fusion Protein in Japanese Subjects With Severe Haemophilia A: Analysis from the A-LONG Study. Haemophilia. 2015. In press. M Shima, K Fukutake, H Hanabusa, T Matsushita, M Taki, M Sakai, T Hirakata, Y Dong, S Li, LM Cristiano, GF Pierce, B Mei. Japanese Subject Subpopulation Analysis of B-LONG: A Phase 3 Study of Long-Acting Recombinant Factor IX Fc Fusion Protein. Japanese J Thromb Haemost. Toby GG, T Liu, Buyue Y, X Zhang, AJ Bitonti, GF Pierce, J Sommer, H Jiang, RT Peters. Recombinant factor IX-Fc maintains full procoagulant properties and exhibits prolonged efficacy in hemophilia B mice. 2015, PLOS One, Submitted. Krishnamoorthy S, T Liu, D Drager, S Patarroyo-White, E Seth-Chhabra, R Peters, N Josephson, D Lillicrap, R Blumberg, GF Pierce, H Jiang. Tolerance Induced by Recombinant Factor VIII Fc (rFVIIIFc) Fusion Protein in Hemophilia A Mice. Submitted Wyrwich KW, S Krishnan, P Auguste, J-LL Poon, R von Maltzahn, R Yu, GF Pierce, B Mei, J Mahlangu, S von Mackensen. Health-Related Quality of Life Changes with Treatment of Longer Acting Clotting Factors: Haem-A-QoL Scores in the A­LONG and B-LONG Clinical Studies of Recombinant Factor VIII and IX Fc Fusion Proteins. Submitted. Dumont, J, KS Loveday, DR Light, GF Pierce, H Jiang. Evaluation of the Toxicology and Pharmacokinetics of Recombinant Factor VIII Fc Fusion Protein in Animals. 2015. Submitted Thromb Res. AD Shapiro, JN Mahlangu, D Perry, J Pasi, DV Quon, P Chowdary, E Tsao, S Li, A Innes, GF Pierce, GA Allen, B Robinson. Treatment of Bleeding Episodes With Recombinant Factor VIII Fc Fusion Protein in Subjects With Severe Hemophilia A in the Phase 3 A-LONG Study. Submitted K Fischer, R Kulkarni, B Nolan, J Mahlangu, S Rangarajan, G Gambino, L Diao, A Ramirez-Santiago, GF Pierce, G Allen. Recombinant Factor IX Fc Fusion Protein in Children with Hemophilia B. Submitted. J Pasi, M Ozelo, J Mahlangu, M Ragni, D Perry, E Tsao, S Li, A Innes, G Pierce, B Mei, B Robinson. Treatment of Bleeding Episodes in Subjects With Hemophilia B Using Recombinant Factor IX Fc Fusion Protein in the Phase 3 B-LONG Study. Submitted Rath T, K Baker, JA Dumont, RT Peters, H Jiang, S-W Qiao, WI Lencer, GF Pierce, RS Blumberg. Fc-fusion proteins and FcRn: structural insights for longer lasting and more effective therapeutics. Crit Rev Biotechnol. 2015 Jun;35(2):235-54. Pierce/WFH

Structure of Fc Fusions 1 FVIII/FIX:2 Fc S S S S S S A1 A2 A3 C1 C2 Fc Eloctate Fc metal ion-mediated non-covalent bond glycosylated asparagine disulfide bridge free cysteine S sulfated tyrosine g g g g g g PRO GLA H EGF-B EGF-A ACT PEP CATALYTIC Fc PRE g g g g g g b S P Fc N-glycosylation Interchain disulfide bond g Gamma carboxylated glutamic acid S Tyrosine sulfation P Serine phosphorylation O-glycosylation b b-hydroxylation Alprolix Pierce/WFH

12: Clinical Pharmacology 12.1: Mechanism of Action ELOCTATE is a recombinant fusion protein that temporarily replaces the missing Coagulation Factor VIII needed for effective hemostasis. ELOCTATE contains the Fc region of human immunoglobulin G1 (IgG1), which binds to the neonatal Fc receptor (FcRn). FcRn is part of a naturally occurring pathway that delays lysosomal degradation of immunoglobulins by cycling them back into circulation and prolonging their plasma half-life. The Mechanism of Action section states that ELOCTATE is a recombinant fusion protein that temporarily replaces the missing Coagulation Factor 8 needed for effective hemostasis. ELOCTATE contains the Fc region of human immunoglobulin G1, which binds to the neonatal Fc receptor (FcRn). FcRn is part of a naturally occurring pathway that delays lysosomal degradation of immunoglobulins by cycling them back into circulation and prolonging their plasma half-life. Pierce/WFH

Global study including 17 countries Enrolling centers: 50 A-LONG B-LONG Global study including 17 countries Enrolling centers: 50 Overall 93.5% of subjects completed study 55 subjects had ≥50 EDs Global study including 19 countries Enrolling centers: 60 Overall, 92.7% of subjects completed study At end of study, 111 subjects completed ≥50 EDs Pierce/WFH

How is rFVIIIFc Produced? Different B-domain deleted (BDD)-rFVIIIFc produced by recombinant DNA technology from a human embryonic kidney (HEK) cell line, which has been extensively characterized. HEK cell line expresses BDD-rFVIIIFc into defined, cell culture medium containing no materials derived from animal or human sources. BDD-rFVIIIFc purified using a series of chromatography steps, including affinity capture with a recombinant, single chain antibody fragment produced in a yeast expression system. No human or animal derived proteins are used in the purification or formulation processes. The production process also incorporates two dedicated viral clearance steps - detergent treatment step for inactivation and 15 nm filtration step for removal of viruses. Similar BDD-rFVIIIFc is produced by recombinant DNA technology from a human embryonic kidney (HEK) cell line, which has been extensively characterized. The HEK cell line expresses BDD-rFVIIIFc into a defined, cell culture medium that does not contain any proteins derived from animal or human sources. → BDD-rFVIIIFc is purified using a series of chromatography steps, including affinity capture with a recombinant, single chain antibody fragment produced in a yeast expression system. → No human or animal derived proteins are used in the purification or formulation processes. → The production process also incorporates two dedicated viral clearance steps - a detergent treatment step for inactivation and a 15 nm filtration step for removal of viruses. Pierce/WFH

Dosage and Administration: Routine Prophylaxis Different Recommended starting regimen: 50 IU/kg of ELOCTATE administered every 4 days Regimen may be adjusted based on patient response: 25-65 IU/kg at 3-5 day intervals More frequent or higher doses up to 80 IU/kg may be required in children less than 6 years of age. For routine prophylaxis the recommended starting regimen is 50 IU/kg of ELOCTATE administered every 4 days. →The regimen may be adjusted based on patient response with dosing in the range of 25 to 65 IU/kg at 3 to 5 day intervals. More frequent or higher doses up to 80 IU/kg may be required in children less than 6 years of age. Pierce/WFH

Clinical Pharmacology Pharmacokinetics PK of ELOCTATE (rFVIIIFc) was evaluated in 28 subjects following 10 minute intravenous infusion of single 50 IU/kg dose. PK profile obtained at week 14, after repeated dosing, was comparable with the PK profile obtained after the first dose. PK data demonstrate that ELOCTATE has a prolonged circulating half-life. Conventional FVIII half life is ~12 hr in adults Table 4: Pharmacokinetic Parameters* (arithmetic mean, 95% CI) PK Parameters rFVIIIFc (95% CI) N = 28 Cmax (IU/dL) 109 (102, 116) Terminal half-life (h) 19.7 (17.4, 22.0) CL (mL/h/kg) 2.06 (1.78, 2.34) Vss (mL/kg) 49.5 (46.9, 52.2) Incremental Recovery (IU/dL per IU/kg) 2.26 (2.13, 2.40) Time to 1% (days) 5.10 (4.54, 5.66) Different The pharmacokinetics of ELOCTATE were evaluated in 28 subjects following a 10 minute intravenous infusion of a single dose of 50 IU/kg. The PK parameters in table 4 were based on plasma 8 activity measured by the one-stage clotting assay. The PK profile obtained at week 14, after repeated dosing, was comparable with the PK profile obtained after the first dose. The PK data demonstrate that ELOCTATE™ has a prolonged circulating half-life Section 12.3 goes on to detail the pharmacokinetic parameters—expressed as the arithmetic mean—in Table 4 of Section 12. For those of you familiar with the primary manuscript of the A-LONG paper, you may recall that the half-life was listed as 19.0 hours; whereas Table 4 gives the half-life as 19.7 hours. It is important to understand the difference between these two values. The half-life listed in the label was calculated based on the arithmetic mean, whereas the half-life in the primary manuscript was expressed as a geometric mean. Geometric means are more conservative than arithmetic means. Industry standard for labeling uses arithmetic means.   (note to programmer: add in time for the viewer to read the table). *Based on a 50 IU/kg dose; Abbreviations: CI = confidence interval; Cmax = maximum observed activity; CL = clearance; Vss = body weight adjusted volume of distribution at steady-state; Time to 1% = time after dose when FVIII activity has declined to 1 IU/dL above baseline. Pierce/WFH

Clinical Pharmacology Pharmacokinetics Table 5: Comparison of PK Parameters of ELOCTATE by Age Pediatric Study Phase 3 Study 2 to 5 years N = 10 6 to 11 years N = 27 12 to 17 years N = 11 IR (IU/dL per IU/kg) 1.89 (1.75, 2.03) 2.44 (2.02, 2.85) 1.85 (1.58, 2.12) t½ (h) 12.0 (9.55, 14.4) 14.6 (11.5, 17.7) 16.4 (14.1, 18.6) CL (mL/h/kg) 3.88 (2.91, 4.49) 2.70 (2.30, 3.09) 2.66 (2.34, 2.98) Vss (mL/kg) 58.7 (54.7, 62.6) 49.9 (44.5, 55.3) 60.3 (53.3, 67.3) Table 5 of Section 12.3 outlines the PK parameters for patients 2 to 5 years, 6 to 11 years and 12 to 17 years of age. Again, note that these PK parameters are expressed as the arithmetic mean.   (note to programmer: add in time for the viewer to read the table). Table 5 presents the PK parameters calculated from the pediatric data of 48 subjects, less than 18 years of age after receiving a single 50 IU/kg dose. 1PK parameters are presented in arithmetic mean (95% CI); Abbreviations: CI = confidence interval IR=incremental recovery;1/2 = elimination half-life; CL = clearance; Vss = volume of distribution at steady-state Pierce/WFH

Bleeding Episode Etiology Individualized Prophylaxis Clinical Studies Table 7: Median (IQR)1 Annualized Bleed Rate by ELOCTATE Treatment Arm Bleeding Episode Etiology Individualized Prophylaxis (N=117) Weekly Prophylaxis (N=23) Episodic (On-Demand) Overall ABR 1.60 (0.00, 4.69) 3.59 (1.86, 8.36) 33.57 (21.14, 48.69) Spontaneous ABR 0.00 (0.00, 2.03) 1.93 (0.00, 4.78) 20.24 (12.21, 36.81) Joint ABR (0.00, 3.11) (0.00, 7.62) 22.76 (15.07, 39.02) Table 7 of the clinical studies section of the label lists the median annualized bleeding rate by treatment arm. 117 patients had an overall median ABR of 1.6 in the individualized prophylaxis arm. 23 subjects had an overall median ABR of 3.59 in the fixed-weekly prophylaxis arm and 23 subjects had an overall median ABR of 33.57 in the episodic arm.   Spontaneous annualized bleeding rates were 0 in the individualized arm, 1.93 in the fixed-weekly arm and 20.24 in the episodic arm. 1Median (interquartile range, 25th and 75th percentiles). Pierce/WFH

6: Adverse Reactions 6.2: Immunogenicity Clinical trial subjects were monitored for neutralizing antibodies to Factor VIII. No subjects developed confirmed, neutralizing antibodies to Factor VIII. One 25 year old subject had a transient, positive, neutralizing antibody of 0.73 BU at week 14, which was not confirmed upon repeat testing 18 days later and thereafter. The detection of antibodies that are reactive to Factor VIII is highly dependent on many factors, including: the sensitivity and specificity of the assay, sample handling, timing of sample collection, concomitant medications and underlying disease. Immunogenicity is described in section 6.2. Subjects in the phase 3 clinical trials were monitored for neutralizing antibodies to Factor VIII. No subjects developed confirmed, neutralizing antibodies to Factor VIII. One 25 year old subject had a transient, positive, neutralizing antibody of 0.73 BU at week 14, which was not confirmed upon repeat testing 18 days later and thereafter. The label notes that the detection of antibodies that are reactive to Factor 8 is highly dependent on many factors, including: the sensitivity and specificity of the assay, sample handling, timing of sample collection, concomitant medications and underlying disease Pierce/WFH

Dosage and Administration Routine Prophylaxis Alprolix The recommended starting regimens are either: 50 IU/kg once weekly OR: 100 IU/kg every 10 days Adjust dosing regimen based on individual response.   Different Pierce/WFH

Pharmacokinetics: ALPROLIX Adults PK data demonstrate that ALPROLIX has prolonged circulating half-life. Table 4: Pharmacokinetic Parameters (Arithmetic Mean, CV) PK Parameters rFIXFc (50 IU/kg) (N = 22) Cmax (IU/dL) 46.04 (68.3%) CL (mL/kg/h) 3.304 (28.4%) Vss (mL/kg) 327.0 (28.2%) Terminal T1/2 (h) 86.52 (37.2%) IR (IU/dL per IU/kg) 1.0154 (58.7%) Time to 1% FIX activity (d) 11.489 (23.8%) Different PK parameters are presented in arithmetic mean (CV%) Abbreviations: IR = incremental recovery; T1/2 = elimination half-life; CL = body weight adjusted clearance; Vss = body weight adjusted volume of distribution at steady-state, Time to 1% FIX activity = estimated time in days after dose when FIX activity has declined to approximately 1 IU/dL above baseline Pierce/WFH

Pharmacokinetics Table 5: Comparison of Pharmacokinetic Parameters of rFIXFc by Age Category PK parameter1 2 to 5 years (N = 5) 6 to 11 years (N = 13) 12 to 17 years (N = 11) IR (IU/dL per IU/kg) 0.5980 (15.7%) 0.7422 (29.2%) 0.8929 (36.4%) T½ (h) 66.40 (32.1%) 72.23 (23.1%) 83.59 (19.1%) CL (mL/h/kg) 4.406 (16.8%) 3.613 (25.1%) 3.483 (25.6%) Vss (mL/kg) 349.0 (19.2%) 303.0 (28.5%) 326.0 (24.9%) Different 1PK parameters are presented in arithmetic mean (CV%) Abbreviations: IR = incremental recovery; T ½= elimination half-life; CL = body weight adjusted clearance; Vss = body weight adjusted volume of distribution at steady-state Pierce/WFH

Pharmacokinetics: ALPROLIX Adults 100 IU/kg dose ALPROLIX™ Pharmacokinetics Cmax, IU/dL 99.89 (20.1%) Clearance, mL/kg/h 2.65 (21.7%) Time to 1% FIX activity, days 15.8 (21.3%) n=27 Different Pierce/WFH

Similar Clinical Studies 2.95 1.38 17.69 Table 7: Median Annualized Bleeding Rate by Treatment Arm Bleeding Episode Etiology Prophylaxis Fixed Weekly Interval (N=61) Prophylaxis Individualized Interval (N=26) Episodic (On Demand) (N=27) Overall ABR (IQR)* 2.95 (1.01, 4.35) 1.38 (0.00, 3.43) 17.69 (10.77, 23.24) Spontaneous ABR (IQR)* 1.04 (0.00, 2.19) 0.88 (0.00, 2.30) 11.78 (2.62, 19.78) * IQR=interquartile range Five subjects (2 in prophylaxis fixed weekly interval arm, 3 in prophylaxis individualized interval arm) did not have sufficient data to be included in the efficacy analysis. Pierce/WFH

Pediatric Use Safety, efficacy and pharmacokinetics of rFIXFc in children with haemophilia B: Results of the Kids B-LONG study International Society on Thrombosis and Haemostasis - 25th Congress June 20-15, 2015 Toronto Kathelijn Fischer1, Roshni Kulkarni2, Beatrice Nolan3, Johnny Mahlangu4, Savita Rangarajan5, Giulia Gambino6, Lei Diao6, Alejandra Ramirez-Santiago6, Glenn F. Pierce6, Geoffrey Allen6 Pierce/WFH

rFVIIIFc (Eloctate) and rFIXFc (Alprolix) Dosing Regimens Pierce/WFH

Long Term Safety Pierce/WFH

Surgery Using Eloctate and Alprolix Brit J Haematol, 2015; 168:124-134 Surgery Using Eloctate and Alprolix Pierce/WFH

Pierce/WFH

Thromb Haemost 2014; 112: 932–940 Pierce/WFH