Facts On Quality And Safety Characteristics Of Fresh Frozen Plasma Single DonationsAnd octaplasLG®

Jürgen Römisch, Ph.D.

Senior Vice President R&D Plasma

Octapharma PPGmbH

Vienna, Austria

BPAC Meeting, Rockville, MDSeptember 20, 2012

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Table of Contents

Quality and Safety Aspects of Fresh Frozen Plasma Single Donations

Plasma Transfusion Related Adverse Events (TRALI): Biochemical / cellular background

octaplasLG® - Manufacturing Process

octaplasLG® - Pathogen Safety

octaplasLG® - Plasma Quality / Biochemical Profile

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Quality and Safety Aspects of FFP Single DonationsDonor screening requirements in USA for fresh frozen plasma

Unique donor identification and registration in a validated computer system

Deferral check (history of donor)

Donor questionnaire (preferably electronic); completed prior to each donation[1]

Education on blood borne diseases and transmission risks

Physical donor assessment by a health professional

Exclusion of donors who do not meet the established acceptance criteria (defined in 21 CFR 640.3 and other guidance documents)[2, 3]

Individual donations are tested on several parameters such as on the absence of HBsAg and antibodies against HIV, HCV and Syphilis

[1] Guidance for Industry: Implementation of acceptable full-length donor history questionnaire and accompanying materials for use in screening donors of blood and blood components.FDA, October 2006[2] http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr=640.63[3] http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr=640.3

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Transfusion Related Acute Lung Injury has emerged as the leading cause of fatalities after blood/plasma transfusion

Incidence (TRALI Study Group)[4]: 2.57-0.81/10,000 transfused units (2006-2009)

Typical presentation of TRALI is development of dyspnea, hypoxemia, hypotension and fever 6 hrs after transfusion (bilateral pulmonary infiltrates)[5]

– Immune-mediated TRALI is usually caused by antibodies of the blood/plasma donor transferred to the recipient

Reactive antibodies to Human Leukocyte Antigens (HLA) or Human Neutrophil Antigens (HNA) cause complement and cell activation and aggregation

Release of cytokines and induction of pro-inflammatory reactions

Damage of vascular endothelium and exudation of fluid across the pulmonary basement membrane

Edema and associated/subsequent events

TRALI

Plasma Transfusion Related Adverse Events (TRALI) Biochemical/cellular background

[4] Transfusion Related Acute Lung Injury Letter; October 19, 2001http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ucm105850.htm[5] Toy P et al. For the TRALI Study Group. Transfusion-related lung injury: incidence and risk factors. Blood 2012; 119:1757-1767

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octaplasLG® Manufacturing ProcessOverview: FFP pooling

Pooling of individual donations (630-1,520 FFP units) facilitates

Dilution and immune neutralization of - allergens - pathogenic antibodies - infectious agents

Leveling out individual heterogeneity, thus reducing variability of contents of plasma componentsresulting in a consistent product quality

YY

Y

Y Y

Y

Individual antibody specificities of different donations

Individual antigen structures contained in single FFP units

YYYYY

Cell fragments

Antibodies to WBCs were detected in a certain number of FFPs, but not in Octaplas® batches tested [6a,6b,7]

[6a] Sachs U. et al. Transfusion 2005; 45:1628-1631[6b] Barz D. et al. Anaesthesiol. Reanimate 1994; 156: 155-158[7] Sinnott P. et al. Eur. J. Immunogen. 2004; 31:271-274

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octaplasLG® Manufacturing ProcessOverview: safety measures: pathogen safety

Antibodies to WBCs (TRALI)Non-Enveloped Viruses ADRs related to cells and fragments

Lipid-Enveloped Viruses

Bacteria & Parasites

Safety against

Infectious Prions

Fast thawing and pooling of single FFP units

Removal of cells, fragments and aggregates by 1.0 µm filtration

Full quality control and release of final product

Castor oil extraction of TNBP, clear filtration and solid phase extraction of Octoxynol

Addition of glycine

Affinity chromatography for PrPsc capture

Sterile filtration (0.45 µm and 0.2 µm)

Aseptic filling, sealing of bags and fast freezing (≤ -60°C) and storage (≤ -30°C)

S/D treatment (1% TNBP & 1% Octoxynol for 1-1.5 hrs at +30°C ± 1°C)

Pooling of individual donations (630-1,520 FFP units) facilitates

Dilution and immune neutralization of - allergens - pathogenic antibodies - infectious agents

Leveling out of individual heterogeneity and reducing variability of contents of plasma components, resulting in a consistent product quality

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Pathogen SafetySafety measures

Beyond the basis precautionary safety measures ensured for FFP, such as donor screening and look-back procedures, the following octaplasLG® process steps add to the pathogen safety profile

– Plasma pool testing (HIV, HBsAG, HAV, HBV, HCV, HEV and Parvovirus B19)

– Solvent / Detergent (S/D) treatment for the inactivation of the most hazardous transfusion related enveloped viruses such as HIV, HBV, HCV and WNV– and other potentially emerging lipid-enveloped viruses

– Immune neutralization by specific antibodies in the plasma pool and the final product such as HAV, Parvovirus B19 and HEV– Pooling of on average 1,000 plasma donations ensures the presence of those antibodies– Respective minimum antibody titers are specified and must be met for product release

– Micro-filtration minimizes the risk of bacteria and parasites presence

– Specific affinity chromatography with a high binding capacity can remove potentially present infectious prion protein, which is not inactivated or sufficiently removed by other manufacturing steps

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Plasma pool testing

Pathogen Safety

Parameter* Limit Plasma Pool*

anti-HIV 1/2 negative

HBsAg negative

anti-HAV** ≥ 1 IU/ml

HAV-PCR negative

HBV-PCR negative

HCV-PCR negative

HEV-PCR# negative

HIV-PCR negative

Parvo B19 PCR ≤ 10.0 IU / µl

The plasma pool for octaplasLG® (630-1,520 FFP units) is tested for:

* under FDA review ** tested in IP sample 1 # Implementation Nov. 2012

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Pathogen SafetyS/D treatment

Solvent / Detergent (S/D) treatment causes disruption of the lipid envelopes of LEV resulting in complete, fast and robust virus inactivation[8,9]

PRV: model virus for HBV; BVDV: model virus for HCV

[8] Dichtelmüller H.O. et al. Transfusion 2009; 49:1931-1943[9] Hellstern P. et al. Transfus. Med. Hemother. 2011: 38:65-70

Red line — = kinetics – Blue square = below limit of detection (LOD)

0 10 20 30 40 50 600

1

2

3

4

5

6

7

8

9

WNV

Time [Minutes]

Mea

n V

iru

s L

oad

[lo

g10

TC

ID50

]

0 10 20 30 40 50 600

1

2

3

4

5

6

7

8

9BVDV

Time [Minutes]

Mea

n V

iru

s L

oad

[lo

g10

TC

ID50

]

0 10 20 30 40 50 600

1

2

3

4

5

6

7

8

9PRV

Time [Minutes]

Mea

n V

iru

s L

oad

[lo

g10

TC

ID50

]

≥ 5.09 log10

≥ 5.24 log10 ≥ 5.99 log10

At 85% S/D

concentration

(robustness)

0 10 20 30 40 50 600

1

2

3

4

5

6

7

8

9

HIV

Time [Minutes]

Mea

n V

iru

s L

oad

[lo

g10

TC

ID50

]

≥ 5.47 log10

At 85% S/D

concentration

(robustness)

Total inactivation 4 log10 to LOD within 1 minute, i.e. 98.9% time-safety-margin for 1 hours S/D

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Pathogen SafetyImmune neutralization

Immune neutralization depends on the virus load and the specific antibody content of the plasma pool and the final container

For non-enveloped viruses, which are not inactivated by S/D treatment, minimum titers of neutralising antibodies were specified, thus must be present

Virus Plasma pool octaplasLG®

HAV PCRanti-HAV IgG

negative≥ 1 IU/ml ≥ 1 IU/ml

B19 PCRanti-B19 IgG

≤ 10.0 IU/µl≥ 11 IU/ml

HEV PCRanti-HEV IgG

Implementation Nov. 2012≥ 0.2 IU/ml

HAV: Hepatitis A VirusB19: Parvovirus B19HEV: Hepatitis E Virus

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Pathogen SafetyMicro-Filtration

Filtration steps at different stages of the manufacturing process minimize the risk of a bacterial or parasite transfection

– 1.0 µm filtration subsequent to FFP pooling– 0.45 µm and 0.2 µm sterile filtrations prior to aseptic filling

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Pathogen SafetyGlobal virus reduction factors during octaplasLG® manufacturing

OctaplasLG. Biological Licence Application (STN 125416/0). 3.2.A.2.4.3 Summary Tables of Virus Validation Factors.

PCR of HAV, B19, HBV, HCV, and HIV genomes are performed on plasma pool level; HEV PCR is being implemented

Anti- HAV (pool and product), HBsAg (pool), anti HIV 1/2 (pool), anti-B19 (product) and anti-HEV (product) antibody titers are tested and must comply with the defined specifications

Step HIV-1 PRV SBV BVDV WNV VACV HSV-1 HAV COX-B6 POL-1

Immune neutralisation (log10)

n.a. n.a n.a n.a n.a n.a≥ 11.1(pool)

≥ 10.0(pool)

≥ 8.6(pool)

≥ 10.9(pool)

S/D treatment (log10)

≥ 6.18 ≥ 5.03 ≥ 5.31 ≥ 5.12 ≥ 5.63 ≥ 5.00 n.a n.a n.a n.a

Global reduction factor (log10)

≥ 6.18 ≥ 5.03 ≥ 5.31 ≥ 5.12 ≥ 5.63 ≥ 5.00≥ 11.1(pool)

≥ 10.0(pool)

≥ 8.6(pool)

≥ 10.9(pool)

Lipid-enveloped viruses Non-enveloped viruses

HIV: Human Immunodeficiency Virus Type 1 PRV: Pseudorabies Virus; model for Herpes VirusSBV: Sindbis Virus; model for Hepatitis C Virus BVDV: Bovine Viral Diarrhea Virus; model for Hepatitis C VirusWNV: West Nile Virus VACV: Vaccinia VirusHSV-1. Herpes Simplex Virus Type 1 HAV: Hepatitis A VirusCOX-B6: Coxsackie Virus B6 POL-1: Poliovirus type 1

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Pathogen SafetyPrPSc Affinity Chromatography

A sensitive and robust assay to detect eventually present infectious prion protein in blood and plasma donations is not yet available

The affinity chromatography with a specific immobilized ligand has a high capacity to adsorb infectious prion protein, thus reduces the risk of transfusion related Creutzfeldt-Jakob Disease

– Prion removal capacity (PrPSc) of octaplasLG® was confirmed in animal bioassay studies[10]

– 5.64 log10 ID50 / ml gel

[10] Heger A. et al. Vox Sang 2012; 104:294-301.

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Plasma Quality & Biochemical ProfileQC release parameters

For product release each octaplasLG® batch is tested on a number of parameters (following slides) in the Quality Control department

Specifications for these parameters have been set and some of them were revised for US for octaplasLG®, also taking into consideration the history in US of a first generation S/D treated plasma product from another manufacturer[11]

These specifications must be met for release of each batch to safeguard the presence of physiologically relevant concentrations of the tested plasma components, in particular

– Balance of coagulation factors and their haemostasis regulating proteins (inhibitors)

[11] Coignard P. et al. Hepatology 2002; 36 (4): Pt.2BPAC 102nd Meeting May, 2012: Evaluation of potential new plasma products manufacturedfollowing storage at room temparature for up to 24 hours

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Plasma Quality & Biochemical ProfileProtein S and Plasmin Inhibitor

In the course of the implementation of the Ligand Gel chromatography the S/D treatment exposure time was shortened

– maintaining a sufficient safety margin for complete inactivation of lipid-enveloped viruses

– but facilitating an improved plasmin inhibitor in-vitro recovery[12,13]

[13]

[12] Heger A. et al. Vox Sang 2012; 103:30[13] Heger A. et al. Vox. Sang. 2009; 96:225

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Plasma Quality & Biochemical ProfileProtease inhibitors and cofactors

ParametersFFP

(n=12)[14]

Final Product Specification

octaplasLG® USA #

octaplasLG®

(n=33)[12]

Antithrombin [IU/ml] 1.06 (0.77-1.23) n.s. 0.92 (0.74-1.00)

Heparin cofactor II [IU/ml] 0.95 (0.76-1.19) n.s. 1.06 (0.82-1.44)

Protein C [IU/ml] 0.89 (0.79-1.05) ≥ 0.7 0.97 (0.79-1.10)

Protein S [IU/ml] 1.03 (0.71-1.39) ≥ 0.4 0.61 (0.50-0.72)

Plasmin inhibitor [IU/ml] 1.04 (0.95-1.18) ≥ 0.4 0.52 (0.30-0.64)

α1-Antitrypsin [mg/ml]* 1.57 (0.98-2.22) n.s. 1.29 (0.88-1.76)

C1-inhibitor [IU/ml] 0.87 (0.72-0.97) n.s. 0.89 (0.67-1.23)

n.s., not specified

*different test methods used for FFP (kinetic nephelometry) and octaplasLG® (ELISA)

# under review by FDA

[12] Heger A. et al. Vox. Sang. 2012; 103:130[14] Beeck H. et al. Vox. Sang. 1998; 74:219-223

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Plasma Quality & Biochemical ProfileCoagulation factors and proteins of the haemostatic system

ParametersFFP

(n=12) [14]

Final Product Specification

octaplasLG® USA

octaplasLG®

(n=33) [12]

Fibrinogen [mg/ml] 2.6 (1.9-3.6) 2.0-4.0 2.7 (2.5-3.1)

Factor II [IU/ml] 0.88 (0.77-1.18) ≥ 0.7 0.92 (0.79-1.08)

Factor V [IU/ml] 0.90 (0.73-1.50) ≥ 0.6 0.81 (0.70-1.00)

Factor VII [IU/ml] 0.95 (0.67-1.38) ≥ 0.6 1.03 (0.70-1.20)

Factor VIII [IU/ml] 0.76 (0.52-1.13) ≥ 0.5 0.85 (0.60-1.30)

Factor IX [IU/ml] 1.02 (0.82-1.28) n.s. 0.94 (0.74-1.30)

Factor X [IU/ml] 0.79 (0.62-0.99) ≥ 0.8 0.93 (0.85-1.04)

Factor XI [IU/ml] 1.13 (0.96-1.80) ≥ 0.7 0.88 (0.80-1.00)

Factor XII [IU/ml] 0.82 (0.45-1.12) n.s. 1.00 (0.81-1.35)

Factor XIII [IU/ml] 1.06 (0.68-1.69) n.s. 1.01 (0.85-1.20)

VWF:RCo [IU/ml] 0.89 (0.77-0.98) n.s. 0.88 (0.72-1.01)

ADAMTS13 [IU/ml][15] 1.24 (1.07-1.44) ≥ 0.7 0.91 (0.75-1.30)

Plasminogen [IU/ml] 0.98 (0.82-1.39) n.s. 0.91 (0.75-1.03)

n.s.: not specified[12] Heger A. et al. Vox. Sang. 2012; 103:130[14] Beeck H. et al. Vox. Sang.1998; 74:219-223[15] Heger A. et al. Vox. Sang. 2007; 92:206-212 (Octaplas, n=18)

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Plasma Quality & Biochemical ProfileRanges of plasma protein concentrations in octaplasLG® batches and FFPs are equivalent

Facto

r II

Facto

r VII

Facto

r IX

Facto

r XI

Facto

r XIII

ADAMTS13

HCII

Prote

in S

A1-AT

Facto

r V

Facto

r VIII

Facto

r X

Facto

r XII

VWF:R

co

ATIII

Prote

in C

Plasm

in in

h

C1-IN

H

0

20

40

60

80

100

120

140

160

180

octaplasLG® batches (n=33) Single-donor FFP bags (n=12)

Po

ten

cy [

%]

[12] Heger A. et al. Vox. Sang. 2012; 103:130[14] Beeck H. et al. Vox. Sang. 1998; 74:219-223

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The Thrombin Generation Assay demonstrated the overall coagulation potential of octaplasLG®, comparable to the range measured in FFPs

Plasma QualityGlobal coagulation assays and specific assays

[12] Heger A. et al. Vox Sang. 2012; 103:130[16] Pock K. et al..Transfusion Apher. Sci, 2007; 37:223-231

OctaplasLG® (US plasma, n=12)

[12,16]

ParametersFFP

(n=18) [16]

octaplasLG®

(n=33) [12]

Peak thrombin [nM] 331 (195-437) 396 (286-507)

Lag phase [min] 14.6 (5.3-20.5) 12.2 (10.4-16.0)

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ADAMTS13 levels and von Willebrand Factor multimeric structures are equivalent in octaplas® batches and in FFP [15,17]

Plasma QualityGlobal coagulation assays and specific assays

1 2 3 4 5 6 7 8 9 10 11

HighVWF-MM

IntermediateVWF-MM

LowVWF-MM

HighVWF-MM

IntermediateVWF-MM

LowVWF-MM

octaplas®

Normal Plasma Ref. Standard

ADAMTS13

VWF

VWF multimer structure: densitometry FFP octaplas®

[15] Heger A. et al. Vox Sang. 2007; 92:206-212 [17] Heger A. et al. Haemophilia. 2006; 12 (2):05PO125

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Summary

In addition to the basic safeguarding measures for FFP with respect to a potential transfusion related infection, the octaplasLG® manufacturing process adds S/D treatment, micro-filtrations and a dedicated PrPSc adsorption step (LG); immune neutralization of non-enveloped viruses and PCR testing in the plasma pool are ensured for each octaplasLG® batch

FFP pooling of (on average 1,000 donations) results in neutralization of pathogenic antibodies and substances (minimization TRALI risk)

The overall biochemical profiles of FFPs and octaplasLG® are comparable

– Reduction of S/D exposure period increased the plasmin inhibitor in-vitro recovery in octaplasLG®

– Balanced coagulation factor and inhibitor contents are ensured by product release specifications (several inhibitor activities) for each octaplasLG® batch

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Acknowledgements

Andrea Heger

Simone Meindl

Andrea Neisser-Svae

Barbara Rangetiner

Torben Schmidt

Tor-Einar Svae

Michael Szkutta