Clinical trial NCT00323856
Conditions: Severe Hemophilia A
Keywords: Hemophilia A Plasma-derived treatment Factor VIII Inhibitor
Sponsor: Grifols Biologicals, LLC
Start date: 2003-04-08; Completion date: 2018-12-14
Enrollment: 51 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to determine the immunologic and overall safety associated with long-term use of Alphanate in subjects diagnosed with severe hemophilia A (Factor VIII:C less than 0.01 IU/ml), who have been previously treated with plasma-derived Factor VIII products other than Alphanate and who have no history of developing either antibody inhibitors to Factor VIII or nonspecific inhibitors of coagulation.

Detailed description:
This is a Phase IV, non-randomized, multicenter study of at least 50 evaluable subjects diagnosed with severe hemophilia A. Enrolled subjects will be treated at home and with in-clinic therapy exclusively with Alphanate as their sole source of Factor VIII concentrate for prophylaxis and treatment of all bleeding episodes and surgical procedures. Subjects will be treated for at least 2 years and a minimum of 50 exposure days, or if 50 exposure days are not reached, for a maximum of 30 months and in accordance with the subject's usual pre-study treatment regimen. Subjects will continue treatment as above or until they develop inhibitors to Factor VIII at a titer greater than or equal to 5 Bethesda units (BU/ml); Factor VIII becomes ineffective at providing hemostasis, or the subject exhibits severe or serious adverse events that prevent completion of the study.

Arms: arm 1: Anti-Hemophilic coagulation factor VIII (Human) Alphanate SD/HT
Interventions: intervention 1: Alphanate SD/HT
Intervention descriptions: intervention 1: Plasma-derived preparation of Factor VIII

Protocol documents:
Prot_000.pdf: 
 
Official Title: 
 
A STUDY OF IMMUNOLOGIC SAFETY FOR ALPHANATE® IN PREVIOUSLY 
TREATED PATIENTS DIAGNOSED WITH SEVERE HEMOPHILIA A 
 
NCT Number: 
NCT00323856 
 
Document Date: 
 
Protocol Amendment 6: 10 January 2007 
 
 
 
 
 
 
 
 


 
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ANTIHEMOPHILIC FACTOR (HUMAN) ALPHANATE® 
SOLVENT-DETERGENT, HEAT-TREATED 
 
 
PHASE IV PROTOCOL: GBI 04-01 
(formerly ATC 02-01 of Alpha Therapeutic Corporation) 
 
 
A STUDY OF IMMUNOLOGIC SAFETY FOR ALPHANATE® IN 
PREVIOUSLY TREATED PATIENTS DIAGNOSED WITH SEVERE 
HEMOPHILIA A 
 
 
 
 
 
 
 
 
GRIFOLS INC. 
2410 Lillyvale Avenue 
Los Angeles, CA 90032 
 
 
 
10 January 2007 
(Incorporating Protocol Amendment Number Six) 
 
 
 
 
Confidential 
The information submitted herein constitutes trade secrets and/or is confidential information, 
which is the property of Grifols Inc., as provided by 21 CFR § 20.61.   Do not disclose without 
the written permission of Grifols Inc. 


 
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Contains Confidential and Proprietary Information of Grifols Inc. 
 
 
PROTOCOL NUMBER: GBI 04-01 (formerly ATC 02-01) 
 
Amendment Tracking 
 
Please indicate any amendments to this protocol by writing the Amendment Number and the 
Date of the Amendment in the space below.  This page will be used to track original 
protocols and their amendments. This is the original protocol if no amendment number is 
listed below. 
 
Original Protocol: 
Title: 
A STUDY OF IMMUNOLOGIC SAFETY FOR ALPHANATE IN 
PREVIOUSLY TREATED PATIENTS DIAGNOSED WITH SEVERE 
HEMOPHILIA A (OF ALPHA THERAPEUTIC CORPORATION) 
 
 
 
DATE: 
26 MARCH 2002 
 
 
 
NO.: 
ATC 02-01  
 
Amendment Number Six 
 
Date of Amendment: 10 January 2007 
 
Section and/or Item  
Amendment 6  
 
Section 4 Study Design 
 
 
 
 
 
 
Delete: All subjects will have CD4 counts greater than or equal to 
400 cells/µL at baseline. 
 
Add: All subjects will have adequate immune system function (CD4 
count must be greater than or equal to 400 cells/µL and subject 
must not have an immune system impairment or damage by 
disease or treatment). 
 
 
Section 5.2 Exclusion 
Criteria 
 
Add: The subject is immunocompromised (including HIV+ status or 
has an impaired immune system due to disease or treatment). 
 
Section 7.1 Study 
Procedures 
Delete: CD4 count will be determined. 
 
Add: Immune system function will be assessed, including a CD4 
count.. Any patient with suspicion of a compromised immune 
system must be approved by the sponsor prior to enrollment. 
 
Section 10.2 Serious 
Adverse Events 
Add: … 
, who can be contacted during working hours 
(8:00 a.m. to 5:00 p.m. Pacific Time) at 
 or 
 during non-working hours. 
 


 
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PROTOCOL NUMBER: GBI 04-01  
(formerly ATC 02-01 of Alpha Therapeutic Corporation) 
 
A STUDY OF IMMUNOLOGIC SAFETY FOR ALPHANATE® IN PREVIOUSLY 
TREATED PATIENTS DIAGNOSED WITH SEVERE HEMOPHILIA A  
 
Study Sponsor: 
Grifols Inc. 
2410 Lillyvale Avenue 
Los Angeles, CA 90032 
Sponsor Contact(s) 
 
and 
 
Person(s) Authorized to 
Sign Protocol and 
Protocol Amendment(s) 
for the Sponsor: 
, PhD 
 
Telephone: 
   Facsimile: 
 
 
, PhD 
 
Telephone: 
   Facsimile: 
 
 
 
 
Tel: 
   Facsimile: 
 
 
Project Manager: 
, PhD 
 
Telephone: 
   Facsimile: 
 
 
Statistician: 
 
 
Medical Safety Officer 
, R.N. 
 
Telephone: 
   Cellular: 
 
 
Date of Protocol: 
26 March 2002 (as ATC 02-01) 
Amendments:  
Amendment Number One: 11 July 2002 
Amendment Number Two: 14 October 2002 
Amendment Number Three: 17 April 2003 
Amendment Number Four: 16 December 2003 (as GBI 04-01) 
Amendment Number Five 1 May 2006 
Amendment Number Six: 10 January 2007 


 
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Proprietary Notice: 
The concepts and information contained herein are considered 
proprietary and shall not be disclosed in whole or in part without 
the expressed consent of Grifols Inc.  


 
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TABLE OF CONTENTS 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.................................... 5 
1 
INTRODUCTION.................................................................................................. 8 
2 
RATIONALE ........................................................................................................ 9 
3 
OBJECTIVE....................................................................................................... 10 
4 
STUDY DESIGN ................................................................................................ 10 
5 
SELECTION OF SUBJECTS............................................................................. 12 
5.1 
Inclusion Criteria ....................................................................................... 12 
5.2 
Exclusion Criteria...................................................................................... 13 
6. 
TEST MATERIAL............................................................................................... 13 
7 
STUDY PROCEDURES (SEE APPENDIX 2).................................................... 14 
7.1 
Screening.................................................................................................. 14 
7.2 
Enrollment and Assignment of Subject Numbers...................................... 16 
7.3 
At seven to fourteen days after the first infusion of Alphanate® ................ 17 
7.4 
Every 3 Months until Month 30 and/or Off-Study ...................................... 17 
7.5 
Unscheduled Visits ................................................................................... 18 
7.6 
In Hospital and In Clinic Treatments ......................................................... 19 
7.7 
Home Treatment....................................................................................... 19 
7.8 
Miscellaneous ........................................................................................... 19 
7.9 
Administration of Alphanate® .................................................................... 20 
7.9.1 Product usage and subject diaries ................................................. 20 
7.9.2 Alphanate® dosing guidelines......................................................... 20 
7.10 Quarterly Clinic/Office Visits ..................................................................... 22 
7.11 Subjects Who Develop Inhibitors .............................................................. 22 
7.12 Treatment with Other Blood Products....................................................... 23 
7.13 Concurrent Medication.............................................................................. 23 
7.14 Discontinuation of Subjects....................................................................... 23 
8 
CLINICAL ENDPOINTS..................................................................................... 24 
8.1 
Primary Endpoint ...................................................................................... 24 
8.2 
Secondary Endpoints................................................................................ 25 
9 
STATISTICAL PLAN ......................................................................................... 25 
9.1 
Sample Size.............................................................................................. 25 
9.2 
Planned Method of Analysis ..................................................................... 25 
9.2.1 Primary Endpoint............................................................................ 25 


 
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9.2.2 Secondary Endpoints..................................................................... 26 
Viral safety will be determined by a lack of seroconversion to 
reactivity against the specific blood-borne viruses for which 
subjects will have been tested and found to be nonreactive at 
the time of enrollment.............................................................. 26 
10 ADVERSE EVENTS........................................................................................... 27 
10.1 Adverse Event Monitoring......................................................................... 27 
10.2 Serious Adverse Events............................................................................ 27 
10.3 Grading Scale ........................................................................................... 28 
10.4 Causality................................................................................................... 28 
11 SUBJECT DEATHS........................................................................................... 29 
12 INFORMED CONSENT FORM AND PATIENT AUTHORIZATION FORM....... 29 
13 ADMINISTRATIVE PROCEDURES AND DOCUMENTS.................................. 31 
13.1 Institutional Review Board and Privacy Board .......................................... 31 
13.2 Documents Required Before a Study Is Initiated ...................................... 32 
13.3 Documents Required Within 30 Days of Study Initiation........................... 32 
14 REPORTING AND MONITORING OBLIGATIONS OF INVESTIGATORS ....... 32 
14.1 Case Reports............................................................................................ 32 
14.2 Clinical Monitoring..................................................................................... 33 
14.3 Investigator's Final Report ........................................................................ 33 
14.4 Regulatory Correspondence ..................................................................... 33 
15 RECORDS ......................................................................................................... 34 
16 HANDLING OF INVESTIGATIONAL DRUG ..................................................... 34 
16.1 Investigational Drug Utilization Record Instructions.................................. 34 
16.2 Disposition of Clinical Supplies ................................................................. 36 
17 MISCELLANEOUS PROVISIONS..................................................................... 36 
17.1 Use of Information and Publication Rights................................................ 36 
18 REFERENCES................................................................................................... 38 
19 INVESTIGATOR’S AGREEMENT ..................................................................... 40 
20 APPENDICES.................................................................................................... 41 
APPENDIX 1: KARNOFSKY PERFORMANCE SCORE.................................... 41 
APPENDIX 2: SCHEDULE OF EVENTS ........................................................... 42 


 
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
AHF 
ALT 
AP 
AST 
ATC 
BU 
BUN 
CBC 
CEDs 
CFR 
CPMP 
 
CRF 
FDA 
FVIII 
FVIII:C 
GI 
HAV 
HBsAg 
HBcAb 
HBsAb 
HBV 
HCV 
HIV-1 
HIV-2 
IDUR 
IRB 
LDH 
PCR 
PEG 
PTP 
PUP 
SD/HT 
seronegative 
seropositive 
SWI  
USP 
VWF 
Antihemophilic Factor 
Alanine aminotransferase 
Alkaline phosphatase 
Aspartic aminotransferase 
Alpha Therapeutic Corporation 
Bethesda unit 
Blood urea nitrogen 
Complete blood count 
Cumulative exposure days 
Code of Federal Regulations (U.S.) 
European Commission Committee for Propriety Medicinal 
Products 
Case report form (s) 
United States Food and Drug Administration 
Factor VIII 
Factor VIII coagulant activity 
Grifols Inc. 
Hepatitis A virus 
Hepatitis B surface antigen 
Hepatitis B core antibody 
Hepatitis B surface antibody 
Hepatitis B virus 
Hepatitis C virus 
Human immunodeficiency virus type 1 
Human immunodeficiency virus type 2 
Investigational drug utilization record 
Institutional review board 
Lactic acid dehydrogenase 
Polymerase chain reaction 
Polyethylene glycol 
Previously-treated patients 
Previously-untreated patients 
Solvent Detergent/Heat Treated 
non-reactive in an ELISA test for antibody to the virus in question 
reactive in an ELISA test for antibody to the virus in question 
sterile water for injection  
United States Pharmacopeia 
von Willebrand Factor  
 


 
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PROTOCOL SUMMARY 
 
Protocol Number: 
 
GBI 04-01 (formerly ATC 02-01 of Alpha Therapeutic 
Corporation) 
Title: 
A Study of Immunologic Safety for Alphanate in Previously 
Treated Patients Diagnosed with Severe Hemophilia A 
Study Phase: 
Phase IV 
Study Duration: 
At least 2 years 
Name of Drug: 
Antihemophilic Factor (Human), Alphanate® Solvent Detergent/ 
Heat Treated 
Drug Dosage: 
An appropriate dosage for the prevention and/or control of 
bleeding 
Concurrent Control: 
None 
Route and Form:  
Intravenous 
Objectives: 
To determine the immunologic and general safety of long-term 
use of Alphanate® in individuals with severe hemophilia A.  
Subject Population:  
Males diagnosed with severe hemophilia A who have been 
previously treated with Factor VIII concentrates, cryoprecipitate, 
or whole blood for a total of 150 cumulative exposure days; at 
least six years and not more than 65 years of age, who have no 
history of exposure to Alphanate® and who have never been 
diagnosed with antibody inhibitors to Factor VIII or to nonspecific 
inhibitors of coagulation.  
Structure: 
Multi-center study 
Blinding: 
Unblinded 
Method of Subject 
Assignment: 
Non-randomized 
Total Sample Size:  
At least fifty evaluable subjects 
Pharmacokinetics:  
Not Applicable 
Efficacy Endpoints 
Not Applicable 


 
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Primary Endpoint 
  
Secondary Endpoints: 
 
1. The incidence of Factor VIII inhibitor development. 
 
1. The frequency, nature, causality and severity of adverse      
events. 
2. The occurrence of changes in biochemical parameters which 
would indicate renal or hepatic impairment. 
3. The incidence of seroconversion to human immunodeficiency 
virus type 1 and 2 (HIV-1/HIV-2), hepatitis A virus (HAV), 
hepatitis B virus (HBV), hepatitis C virus (HCV) or parvovirus 
B19 in subjects seronegative for these viruses at the time of 
enrollment. 
4. The amount of product used per year as part of at-home 
prophylaxis and therapy for bleeding episodes. 
5. A physician’s qualitative assessment of hemostasis 
(evaluated as “None”, “Moderate”, “Good”, or “Excellent” in 
response to therapy with Alphanate® received by the subject 
in a hospital or another location under the physician’s direct 
supervision. 
 
 
 


 
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1 
INTRODUCTION 
The development of an immune response to Factor VIII (i.e., the generation of 
antibody inhibitors of Factor VIII) by patients diagnosed with hemophilia A and 
treated with Factor VIII concentrates is a serious complication in the treatment 
of hemophilia A.1,2  Estimates of inhibitor formation from retrospective and 
prospective clinical trials vary from as low as four percent to as high as 28 
percent.  All studies agree that prevalence of antibody inhibitors is higher 
among individuals with severe hemophilia A (Factor VIII:C less than 0.01 
IU/mL) than among individuals with mild or moderate disease, with estimates 
ranging from eight to 28 percent.3,4 
In clinical trials in which previously-untreated or minimally-treated patients 
(PUPs) were treated with recombinant Factor VIII concentrates, the incidence 
of inhibitors to Factor VIII was measured as a function of cumulative exposure 
days (CEDs).  In a clinical trial conducted by Bray et al.5, inhibitors developed 
in 17 of 71 (24%) PUPs treated with Recombinate® (Baxter Bioscience); 5 of 
the 71 (7%) patients developed high-responder inhibitors. The median number 
of CEDs was 11 for all patients and nine for patients who developed inhibitors.   
Similarly, in a clinical trial conducted by Lusher et al. of subjects receiving 
Kogenate® (Bayer), inhibitor formation occurred in 16 of 62 (27%) subjects.6  
Seven of the 62 (11%) patients  developed high-responder inhibitors. The 
median number of CEDs was nine for patients who developed inhibitors and 
28 for subjects who exhibited no immune response to Kogenate®. 
Several clinical trials have been conducted in which PUPs were enrolled and 
then treated with plasma-derived Factor VIII concentrates of varying purity and 
produced using differing virucidal treatments (e.g., terminal dry heat, 
pasteurization, solvent-detergent).7-9  The rates of incidence for antibody 
inhibitors ranged from 21 to 28 percent, a range similar to that observed with 
recombinant Factor VIII products.  Consequently, the investigators for these 
trials concluded that recombinant DNA products do not pose any greater risk 
for inhibitor formation in PUPs than do plasma-derived products. 
However, a substantially increased incidence of Factor VIII inhibitors (a 4.5-
fold increase) was reported by Peerlinck et al.10 and Rosendaal et al11 in 
clinical trials of previously treated individuals (PTPs) treated with pasteurized 
factor VIII concentrate of an intermediate purity that is available in Europe.  
The increase in inhibitor formation was observed even in individuals who had 
been previously treated with multiple Factor VIII products (“multitransfused” 
patients). The authors suggested that pasteurization might have compromised 
the integrity or conformation of Factor VIII, which in turn could have resulted in 
increased immunogenicity.  


 
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Guérois et al.12 conducted a clinical trial involving 56 PUPs treated with 
HPSD-VIII® 
(a 
plasma-derived, 
high-purity, 
solvent-detergent 
treated 
concentrate).  The incidence of immune response was monitored over a 
period of 76 months (median = 29 months) and for patients experiencing 100 
CEDs (median = 26 CEDs).  The median number of CEDs prior to detection of 
inhibitors was 16 days.  Inhibitors developed in five patients (9 percent), only 
one of whom was categorized as a high responder (2 percent).  Therefore, it 
appeared that the use of HPSD-VIII® was associated with a lower incidence of 
antibody inhibitor than was observed with the administration of recombinant 
DNA products or a combination of plasma-derived concentrates.  Additionally, 
inhibitors appeared later (16 CEDs) in individuals receiving HPSD-VIII® than in 
those treated with the recombinant DNA products (nine CEDs). 
Addiego et al.13 investigated the use of Hemofil M®, a plasma-derived Factor 
VIII concentrate treated with a solvent-detergent process, in 23 PUPs.  Only 
two of 23 (9%) patients developed inhibitors after 14 to 26 CEDs.  Although 
the number of subjects was limited in this trial, the median number of CEDs 
experienced by this population, 32 days, exceeded those in the populations 
studied by Guérois et al.12, Bray et al.5 and Lusher et al.6  A more limited study 
by Lusher and Salzman with the pasteurized Factor VIII concentrate  
Monoclate P® showed an incidence of inhibitor formation of 18 percent.14 
A study of the incidence of inhibitors in previously-treated patients (PTPs) 
carried out by McMillan et al. included 1306 hemophilia patients whose mean 
age was 18, and who were followed for up to four years. Thirty-one of these 
individuals (2.4 percent) developed inhibitors to Factor VIII; of these, 29 had 
been diagnosed with severe hemophilia A. The incidence of new inhibitors 
was eight per 1,000 patient-years of observation. 
In a study of PTPs treated with recombinant Factor VIII (Kogenate®), by 
Schwartz et al, inhibitor formation was observed in only 1 of 86 (1.2%) patients 
following repeated exposure to the product.16  The mean annual per capita 
consumption of Factor VIII in this subgroup was estimated to be approximately 
78,000 units annually. 
2 
RATIONALE 
Alphanate® is a high-purity plasma-derived preparation of Factor VIII 
(Antihemophilic Factor, AHF) in which Factor VIII:C is present in complexes of 
high-molecular weight multimers of von Willebrand Factor (VWF). 
Alphanate® is prepared from plasma found to be nonreactive for hepatitis B 
surface 
antigen 
(HBsAg) 
and 
negative 
for 
antibodies 
to 
human 
immunodeficiency virus HIV-1 and HIV-2 and hepatitis C virus (HCV) using 
FDA approved assays.  All units of plasma used in the manufacture of 


 
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Alphanate® are also tested for HIV and HCV genomic material using 
polymerase chain reaction (PCR) assays and using a three-stage sample 
pooling method approved by the US Food and Drug Administration (FDA).  In 
addition, all units of plasma used in the preparation of Alphanate® have an 
alanine aminotransferase (ALT) level less than twice the upper limit of normal 
for the assay method used. 
Two specific viral reduction processes are used in the manufacture of 
Alphanate®: 
(1) treatment 
with 
an 
organic 
solvent/detergent 
mixture 
[tri(n-butyl)phosphate (TNBP)/polysorbate 80] to inactivate lipid-
enveloped viruses such as HIV-1, HIV-2, hepatitis B (HBV) and 
HCV; and  
(2) exposure of the lyophilized product in the final container to heating 
for 72 hours at 80ºC. 
These processes have the theoretical potential to expose new antigenic sites 
in the Factor VIII molecule.  If new antigenic entities are created, individuals 
with hemophilia A who have not developed antibody inhibitors in response to 
treatment with other plasma-derived Factor VIII concentrates could develop 
inhibitors to these new antigenic sites.  The generation of antibody inhibitors to 
Factor VIII could result in treatment complications.  
3 
OBJECTIVE 
The objective of this study is to determine the immunologic and overall safety 
associated with long-term use of Alphanate® in individuals diagnosed with 
severe hemophilia A (Factor VIII:C less than 0.01 IU/mL), who have been 
previously treated with plasma-derived Factor VIII products other than 
Alphanate® and who have no history of developing either antibody inhibitors to 
Factor VIII or nonspecific inhibitors of coagulation. 
4 
STUDY DESIGN 
This is a phase IV, non-randomized, multicenter study involving at least 50 
evaluable subjects diagnosed with severe hemophilia A (levels of Factor VIII:C 
in plasma less than 0.01 IU/mL), who are male, at least six years of age and 
not more than 65 years of age, and with a Karnofsky Performance Score of at 
least 50.  All subjects will have adequate immune system function (CD4 count 
must be greater than or equal to 400 cells/µL and subject must not have an 
immune system impairment or damage by disease or treatment). 


 
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Enrolled subjects will be treated at home and with in-clinic therapy exclusively 
with Alphanate® as their sole source of FVIII concentrate for prophylaxis and 
treatment of all bleeding episodes and surgical procedures for a period of at 
least two years and a minimum of 50 exposure days, or, if 50 exposure days 
are not reached, for a maximum of 30 months. Alphanate® will be 
administered in accordance with the subject’s usual pre-study treatment 
regimen.  An exposure day is defined as any day on which a subject receives 
one or more infusions of any Factor VIII containing product.  However, only 
one exposure day will be accrued for each surgical episode, regardless of the 
number of days of treatment or the number of infusions of Factor VIII 
associated with that surgical procedure. 
A subject will continue treatment, as specified above, unless  
• The subject develops inhibitors to Factor VIII at a titer greater than or equal 
to 5 Bethesda units (BU/mL);  
• treatment with Factor VIII becomes ineffective at providing hemostasis 
even for a subject who has developed inhibitors with a titer of less than 5 
BU/mL; or 
• the subject exhibits severe or serious adverse events that prevent 
completion of participation in the study. 
In all instances, only the inhibitor titer obtained by the central reference laboratory will 
be used to determine the subject’s eligibility to continue his participation in the trial. 
The primary endpoint of this study is: 
 
 
the incidence of Factor VIII inhibitor after exposure to Alphanate®. 
 
 
The secondary endpoints are: 
 
 
1. The frequency, nature, causality and severity of adverse events.  
2. The occurrence of changes in biochemical parameters which would 
indicate alterations in renal or hepatic function. 
3. Evidence for seroconversion to HIV-1/HIV-2, HAV, HBV, HCV and 
parvovirus B19 for subjects who are seronegative for these viruses 
upon enrollment. 
4. The amount of product used per year as part of at-home prophylaxis 
and therapy for bleeding episodes. 


 
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5. A physician’s qualitative assessment of hemostasis (evaluated as 
“None,” “Moderate,” “Good,” or “Excellent”) in response to therapy with 
Alphanate® received by the subject in a hospital or at another location 
under the physician’s direct supervision. 
 
The design of this study satisfies the recommendations of the European 
Commission Committee for Propriety Medicinal Products (CPMP) regarding 
eligibility criteria and end points for studies, in previously-treated patients, of 
concentrates with marketing authorization in which a manufacturing process 
has been added.  In addition, this study conforms to the CPMP 
recommendation that all enrolled subjects be vaccinated against hepatitis A 
and B.  Any candidate for this trial who has not yet been immunized against 
hepatitis A will be offered a course of hepatitis A vaccination. 
5 
SELECTION OF SUBJECTS 
The following entrance criteria must be satisfied for a patient to be enrolled 
into this study: 
5.1 Inclusion Criteria 
1. The subject must be male, at least six years of age and not more than 65 
years of age. 
2. The subject must have signed and dated an Informed Consent Form and 
Patient Authorization for Release of Information approved by the 
appropriate Institutional Review Board (IRB) prior to screening and 
enrollment.  If the subject is a minor (i.e., less than 18 years of age) both 
he and his parent or legal guardian must sign and date the informed 
consent. 
3. The subject must have been diagnosed with severe hemophilia A and must 
have levels of Factor VIII less than 0.01 IU/mL. 
4. The subject must have received treatment with cryoprecipitate, Factor VIII 
concentrates, and/or whole blood, for at least 150 cumulative exposure 
days (CEDs) prior to enrollment. 
5. The subject must not have received treatment with cryoprecipitate, Factor 
VIII concentrate, or any other blood product, for at least 72 hours prior to 
screening. 
6. The subject must never have been diagnosed with inhibitors to Factor VIII 
at any detectable titer. 


 
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7. The subject must never have been diagnosed with nonspecific inhibitors of 
coagulation. 
8. The subject must test negative for the presence of Factor VIII inhibitors at 
screening and enrollment. 
9. The subject must have CD4 counts greater than or equal to 400 cells/ µL. 
10. The subject must have been vaccinated against hepatitis A and hepatitis B, 
or have evidence of antibodies against hepatitis A and hepatitis B.  
However, a subject who has no prior immunity against hepatitis A will be 
offered a course of vaccination for hepatitis A. 
11. The subject must have a Kamofsky Perfomrance Score of at least 50 
(Appendix 1). 
 
5.2 Exclusion Criteria 
1. The subject is receiving any immunosuppressive medications including 
intravenous immunoglobulins at the time of enrollment. 
2. The subject has clinical signs or symptoms of an infection, such as fever, 
chills or nausea during screening or enrollment. 
3. The subject has a history of frequent reactions to Factor VIII concentrates 
(e.g., chills or headaches). 
4. The subject has received treatment with Alphanate® (Solvent-Detergent/ 
Heat-Treated). 
5. The subject is immunocompromised (including HIV+ status or has an 
impaired immune system due to disease or treatment). 
 
6. 
TEST MATERIAL 
Alphanate® is prepared from plasma found to be nonreactive for hepatitis B 
surface antigen (HBsAg) and negative for antibodies of HIV-1/HIV-2 and HCV 
using FDA approved assays.  All units of plasma used in the manufacture of 
Alphanate® are also tested for HIV and HCV genomic material using 
polymerase chain reaction (PCR) assays and using a three-stage sample 
pooling method approved by the US Food and Drug Administration (FDA).  In 
addition, all units of plasma used in the preparation of Alphanate® have an 


 
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alanine aminotransferase (ALT) level less than twice the upper limit of normal 
for the assay method used. 
Alphanate® is manufactured by cryoprecipitation, polyethylene glycol (PEG) 
precipitation, heparin-agarose chromatography and glycine precipitation.  Two 
specific viral reduction procedures are used in the manufacturing process: 
• At an intermediate step in the manufacture of Alphanate®, the product is 
treated with an organic solvent/detergent mixture [tri(n-butyl)phosphate 
(TNBP)/polysorbate 80] to inactivate lipid-enveloped viruses such as 
HIV-1/HIV-2, hepatitis B and C (HBV, HCV); and 
• The lyophilized product is heated at 80ºC for 72 hours in the final container 
and meets all internal specifications of Grifols Inc. 
However, virus reduction methodologies and testing techniques cannot fully 
eliminate the risk of viral transmission.  
Alphanate® is provided as a lyophilized powder in a vial containing at least 40 
International Units Factor VIII:C/mL (200 IU/vial) (IU; all subsequent 
references to IU will be expressed as units) after reconstitution.  A label 
indicating the manufacturer, lot number, number of Factor VIII:C units, and 
reconstitution volume is affixed to each vial.  A vial containing the appropriate 
volume of sterile water for injection (SWI), USP, is provided with each vial of 
Alphanate®. 
Alphanate® used in this clinical trial has met all internal product release 
specifications of Grifols Inc. 
7 
STUDY PROCEDURES (SEE APPENDIX 2)  
7.1 Screening 
All of the following procedures and laboratory procedures will be performed: a) 
at least 72 hours after the most recent administration of cryoprecipitate, 
Factor VIII concentrate or any other blood product, and b) before the first 
administration of Alphanate®. 
 
See the table in Appendix 2 for the listing of laboratory tests to be performed. 
1. Before any invasive screening tests are performed, the subject will be 
presented with an IRB-approved informed consent form and patient 
authorization form, which will be explained to him.  The subject will not 
participate in any part of the clinical trial, including the initial screening until 
he has signed and dated the forms to indicate that he understands and 


 
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agrees to its contents (if he is less than 18 years of age, his parent or legal 
guardian must agree and sign). 
2. The subject’s medical and past treatment history will be reviewed to 
ascertain whether he meets all the entry requirements for the study. 
3. Each subject will receive a physical examination that will include: vital 
signs (pulse, respiratory rate, oral temperature, blood pressure), height, 
weight, and a description of any physically observable abnormal 
conditions. 
4. Hematologic status will be assessed by: complete blood count (CBC) with 
white blood cell (WBC) differential and platelet count. 
5. Immune system function will be assessed, including a CD4 count.. Any 
patient with suspicion of a compromised immune system must be 
approved by the sponsor prior to enrollment. 
6. Factor VIII coagulant activity (Factor VIII:C) will be assessed both by the 
local laboratory and the central reference laboratory  The value obtained 
by the central reference laboratory will determine the subject’s 
eligibility.  
7. The Factor VIII inhibitor level (BU/mL) will be determined. Two sets of 
samples will be drawn: one for analysis at the local laboratory of the 
clinical site, and the other sample for analysis at the central reference 
laboratory.  The value obtained by the central reference laboratory 
will determine the subject’s eligibility. 
8. Serum chemistry tests will be performed: alanine aminotransferase (ALT), 
aspartic aminotransferase (AST), alkaline phosphatase (AP), lactic acid 
dehydrogenase (LDH), blood urea nitrogen (BUN), total bilirubin and  
creatinine.  
9. Viral and serological tests will be conducted: including testing for HBsAg, 
and for antibodies to HAV, HBcAb, HBsAb, HCV, HIV-1/HIV-2 and 
parvovirus B19 (by routine immunoassay). 
10. A sample of venous blood will be drawn from which approximately 5 mL of 
serum will be prepared and aliquoted into three vials of at least 1.5 mL of 
sample each.  The samples will be frozen and retained at -20°C in the 
event that they are required for later serological or virology tests. 


 
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7.2 Enrollment and Assignment of Subject Numbers 
After the investigator has assured that the subject meets all of the enrollment 
criteria, the screening and enrollment case report forms will be completed.  
Note that with respect to Factor VIII coagulant activity (Factor VIII:C) and the 
Factor VIII inhibitor levels, the values obtained by the central reference 
laboratory will determine the subject’s eligibility.  The investigator may use the 
results from the local laboratory of the site to support clinical decisions in 
patient management. 
After assuring that the subject meets all of the enrollment criteria, the following 
clinical and laboratory assessments will be performed: 
1. Abbreviated physical examination including: interim medical history, vital 
signs (pulse, respiratory rate, oral temperature, blood pressure) and 
weight. 
2. If the subject has been treated with any of the products mentioned 
above in Section 7.1 (cryoprecipitate, Factor VIII concentrate or any 
other blood product) after the screening tests are performed, blood 
will again be drawn no more than three days prior to the first treatment 
with Alphanate® for baseline testing of the following parameters: 
(a)  Factor VIII coagulant activity (Factor VIII:C) for central lab testing, 
(b) Factor VIII inhibitor levels (BU/mL) for central testing, 
(c) 
Repeat viral and serological tests including anti-HIV-1/HIV-2, HBsAg, 
anti-HAV, anti-HBV, and anti-HCV, and anti-parvovirus B1. 
3. A sample of venous blood will be drawn from which approximately 5 mL of 
serum will be prepared and aliquoted into three vials of at least 1.5 mL of 
sample each.  The samples will be frozen and retained at -20°C in the 
event that they are required for later serological or virology tests. 
In order to complete subject enrollment and receive the Subject Number, the 
participating study site will provide to the clinical monitor at Grifols Inc. a copy 
of the completed screening and enrollment CRFs, screening and enrollment 
tests and medical history.  The results of the screening and enrollment tests 
and procedures will be reviewed and the clinical monitor will confirm the 
individual's eligibility and assign a subject number.  Grifols Personnel assigned 
to the study can be contacted by telephone during working hours (8:00 a.m. to 
5:00 p.m. Central Time) at 
 or fax 
. 


 
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7.3 At seven to fourteen days after the first infusion of Alphanate® 
1. Parvovirus B19 PCR assay - only in subjects seronegative for parvovirus 
B19 at enrollment.  
2. Collect a 2 mL sample of citrated plasma for inhibitor testing at the central 
reference laboratory. 
 
7.4 Every 3 Months until Month 30 and/or Off-Study 
 
Samples for quarterly testing will be drawn ± 10 days of the scheduled visit date.  At 
each of these visits, the following procedures will be performed: 
 
1. Abbreviated physical examination including: interim medical history, vital signs 
(pulse, respiratory rate, oral temperature, blood pressure) and weight; 
2. Factor VIII coagulant activity (Factor VIII:C); 
3. Factor VIII inhibitor levels (BU/mL) in two sets of samples;  
4. Hematologic status will be assessed by: complete blood count (CBC) with 
white blood cell (WBC) differential and platelet count. 
5. Serum chemistry tests including: alanine aminotransferase (ALT), aspartic 
aminotransferase 
(AST), 
alkaline 
phosphatase 
(AP), 
lactic 
acid 
dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine and total 
bilirubin; 
6. Viral and serological tests including: testing for HBsAg, and for antibodies to 
HAV, HBcAb, HBsAb, HCV, HIV-1/HIV-2 (only if seronegative at baseline) and 
parvovirus B19 by routine immunoassay (only in subjects who are 
seronegative at baseline); 
7. A sample of venous blood will be drawn from which approximately 5 mL of 
serum will be prepared and aliquoted into three vials of at least 1.5 mL of 
sample each.  The samples will be frozen and retained at -20°C in the event 
that they are required for later serological or virology tests. 
If at any quarterly visit, the subject tests positive for the presence of Factor VIII 
inhibitors (inhibitor titer of greater than 0.6 modified BU/mL), based upon the 
value determined by the central reference laboratory, the procedures specified in 
section 7.12 (Subjects Who Develop Inhibitors) will also be performed. 
 


 
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7.5 Unscheduled Visits 
 
For each unscheduled visit, the following tests will be performed: 
 
1. Abbreviated physical examination including: interim medical history, vital 
signs (pulse, respiratory rate, oral temperature, blood pressure) and 
weight; 
2. Factor VIII coagulant activity (Factor VIII:C); 
3. Factor VIII inhibitor levels (BU/mL) in two sets of samples (to be 
performed only if: a) there is clinical indication that the normal 
dosage of Alphanate® is not providing adequate hemostasis, or b) 
surgery; 
4. Hematologic status will be assessed by: complete blood count (CBC) with 
white blood cell (WBC) differential and platelet count; 
5. Serum chemistry tests including: alanine aminotransferase (ALT), aspartic 
aminotransferase 
(AST), 
alkaline 
phosphatase 
(AP), 
lactic 
acid 
dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine and total 
bilirubin; 
6. Viral and serological tests (to be performed only if subject exhibits 
clinical or subjective symptoms indicating possible viral infection) 
including: testing for HBsAg, and for antibodies to HAV, HBcAb, HBsAb, 
HCV, HIV-1/HIV-2 (only if seronegative at baseline) and parvovirus B19 
by routine immunoassay (only in subjects who are seronegative for 
parvovirus B19 at baseline); 
7. A sample of venous blood will be drawn from which approximately 5 mL of 
serum will be prepared and aliquoted into three vials of at least 1.5 mL of 
sample each.  The samples will be frozen and retained at -20°C in the 
event that they are required for later serological or virology tests. 
If at an unscheduled visit, a test for Factor VIII is positive for the presence of 
Factor VIII inhibitors (inhibitor titer of greater than 0.6 modified BU/mL), 
based upon the value determined by the central reference laboratory, the 
procedures specified in section 7.12 (Subjects Who Develop Inhibitors) will be 
performed. 
 


 
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7.6 In Hospital and In Clinic Treatments 
A physician’s qualitative assessment of hemostasis (evaluated as “None,” 
“Moderate,” “Good,” or “Excellent”) in response to therapy with Alphanate® 
received by the subject will be recorded for all treatments administered in the 
hospital, clinic, or at another location under the physician’s direct supervision. 
7.7 Home Treatment 
Following enrollment, the subject will have the choice of obtaining Alphanate® 
treatment at the participating study site in response to bleeding episodes, or to 
receive treatment at home (See Section 7.9.1). 
 
7.8 Miscellaneous 
 
All enrollment tests should be performed prior to the first administration of 
Alphanate®.  Samples drawn for quarterly testing will be drawn ± 10 days of 
the scheduled visit date.  The presence of inhibitors will be determined by the 
Nijmegen modification of the Bethesda assay (see below); the Factor VIII 
source for this assay will be normal pooled plasma.  Inhibitor titers will be 
considered valid if the subject’s Factor VIII level is at or below baseline 
(less than 0.01 IU/mL), therefore, the subject should be scheduled for the 
quarterly visit at least three days after an infusion with Alphanate®. 
If the subject’s Factor VIII levels are higher than baseline at the time the 
plasma sample is drawn, then the subject will be rescheduled for testing within 
one week of the notification of the invalid result. 
Assays for inhibitor to Factor VIII will be performed locally (i.e., at the 
coagulation testing laboratory for each clinical site) to support decisions 
regarding treatment.  Duplicate plasma samples will be collected at each time 
point for confirmatory testing at a central reference laboratory.  The central 
laboratory will utilize the Nijmegen modification of the Bethesda assay to 
improve reliability.17 The modification involves the following changes in 
reagents: (1) buffering of the normal pooled plasma used in the assay and 
control with 0.1 M imidazole to pH 7.4 and (2) replacing the imidazole buffer in 
the control mixture with immunodepleted Factor VIII-deficient plasma.  In 
addition, to further improve the accuracy of the assays, a standardized 
immunodepleted plasma will be used for the performance of the assay at all 
clinical sites, as well as at the central reference laboratory. 
The local laboratories will not be required to test for Factor VIII inhibitors using 
the Nijmegen modification of the Bethesda assay, but are required to provide 
the sponsor with a detailed description of the procedure used. 


 
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Only the results of the assays from the central reference laboratory will be 
used in the determination and analysis of the primary endpoint for the clinical 
trial. 
To determine the risk of transmission of parvovirus B19, subjects who are 
seronegative at enrollment will be tested approximately one week after 
receiving the first infusion with Alphanate® for the presence of parvovirus B19 
by Polymerase Chain Reaction (PCR) testing. 
7.9 Administration of Alphanate® 
7.9.1 Product usage and subject diaries 
Alphanate® will be administered as needed during the study.  Indications for 
infusion include: 
• bleeding episode or hemorrhage; 
• routine prophylaxis; 
• prophylactic treatment prior to physiotherapy or surgery. 
Following enrollment, the subject will have the choice of obtaining Alphanate® 
treatment at the participating study site in response to bleeding episodes, or to 
receive treatment at home.  If the Alphanate® is to be self-administered, then 
the subject must maintain an infusion diary, which will contain the following 
information: 
 
• date of infusion; 
• number of units infused; 
• number of vials used; 
• lot number(s); 
• reason for infusion; 
• any adverse events 
The site should provide the subject with adequate drug supply for three 
months based on the subject’s anticipated needs. 
 
7.9.2 Alphanate® dosing guidelines 
All subjects will be infused with the appropriate dose of Alphanate® (as 
determined by the investigator) for the prevention and/or control of bleeding, 
as needed.  The dose and duration of therapy with Alphanate® will depend on 
the severity of the Factor VIII deficiency, the location and extent of the 
bleeding and the individual’s physical condition.  The amount of coagulation 
factor administered and the frequency of dosing will always take into account 
the clinical effectiveness of the product in each individual.  However, the 


 
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following regimen should be used as a guide for dosage calculation in the 
treatment of adults with hemophilia A. 
Antihemophilic factor (Factor VIII:C activity) potency is expressed in 
International Units (IU) on the product label, where one unit approximates the 
activity in one milliliter of normal plasma.  Replacement therapy studies have 
shown a linear dose-response relationship, with a 2.0-2.5 % increase (0.020-
0.025 IU/mL) in Factor VIII activity for each unit of Factor VIII:C per kg of body 
weight transfused, from which an approximate factor of 0.5 IU/kg can be 
calculated.18, 19   
The following formula provides a guide for dosage calculation (the plasma 
Factor VIII may vary depending upon the age, weight, severity of hemorrhage, 
or surgical procedure of the subject): 
 
 
 
Number of Factor VIII:C Required =  
 
 
 Body Weight (kg) x 0.50 (IU/kg) x Factor VIII:C Desired Increase (percent) 
 
For example, in order to increase the Factor VIII:C concentration to 30 percent 
in a subject with a body weight of 50 kg, the formula is applied as follows: 
50 kg x 0.50 IU/kg x 0.30 IU/mL × 100 = 750 IU Factor VIII:C 
 
Mild to moderate hemorrhages may usually be treated with a single 
administration sufficient to raise the plasma Factor VIII level to 0.20 to 
0.30 IU/mL.  In more serious hemorrhages, the subject's plasma Factor VIII 
level should be raised to 0.3 to 0.5 IU/mL.  Infusions are generally required at 
twice daily intervals over several days. 19  
Surgery requires that the Factor VIII level be raised to 0.50 to 0.80 IU/mL with 
the level maintained at or above 0.30 IU/mL for approximately two weeks 
postoperatively.  For dental extractions, the Factor VIII level should be raised 
to 0.50 IU/mL immediately prior to the procedure; further Factor VIII may be 
given if bleeding recurs. 20 
In subjects who experience frequent hemorrhages, Antihemophilic Factor 
(Human), Alphanate®, may be administered prophylactically on a daily or 
every other day schedule to raise the Factor VIII level to approximately 0.15 
IU/mL. 21 
In pediatric subjects, no clinical trials for safety and effectiveness have been 
conducted in 16 years of age or younger.  Across well-controlled half-life and 
recovery clinical trials in patients previously treated with Factor VIII 
concentrates for Hemophilia A, the one pediatric subject receiving Alphanate® 
(Solvent/Detergent) responded similarly when compared with 12 adult 
subjects. 22  


 
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Factor VIII levels should be monitored periodically to evaluate the response of 
individual patients to a given dosage regime. 
 
7.10 Quarterly Clinic/Office Visits 
 
Each participating subject will return to the study site at least quarterly for 
examination and collection of his infusion diary and for his quarterly drug 
supply.  At each visit, the subject will bring all used and unused containers of 
Alphanate®, in addition to the diary.  During the quarterly visits an abbreviated 
physical examination will be performed by the investigator or a staff member 
designated by the investigator.  Blood samples will be collected for required 
testing, and the usage of the study drug, prescription medication and blood 
products will be determined.  At each visit, the study nurse will discuss the 
information in the diary with the subject and will reconcile drug use.  A new 
diary and a new supply of Alphanate® will be given to the subject.   
 
 
Participating sites will: 
• maintain these diaries in the subject files as part of the case report forms; 
• verify, to the extent possible, their accuracy by comparing Alphanate® 
usage in these diaries against their own prescription information; and 
• discuss with the subject any adverse events and their severity. 
 
7.11 Subjects Who Develop Inhibitors 
If at any quarterly visit, the subject tests positive for the presence of Factor VIII 
inhibitors, (inhibitor titer of 0.6 modified BU/mL or greater) based on the local 
lab test and confirmed by the central reference laboratory test result, the 
following steps should be taken: 
• For an inhibitor titer of greater than or equal to 10 BU/mL,  
 
 
- Collect a 10 mL sample of citrated plasma and send to the central  
 
 
  reference laboratory. 
- Withdraw the subject only after performing all required testing (see 
Section 7.4) and only after receiving confirmation of the inhibitor titer 
from the central reference laboratory. 
• For an inhibitor titer less than 10 BU/mL,  


 
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- Collect a 50 mL sample of citrated plasma. This sample can be 
obtained in multiple draws within a two-week period and sent to the 
central reference laboratory. 
 
 
- Repeat inhibitor titer assay at two-week intervals until: 
1. the inhibitor titer decreases to < 0.6 modified BU/mL or 
2. the subject’s participation in the study is ended or 
3. it becomes apparent that, after repeated assays at two-week intervals, the 
inhibitor titer will not decrease during the course of the study (the 
investigator should first consult with the Project Manager prior to making 
this determination). 
• If the subject’s inhibitor level is less than 5 BU/mL and treatment with 
Alphanate® at higher doses remains effective in achieving hemostasis, 
then the subject will continue the current treatment regimen and testing 
will be performed as described in Section 7.4 above. 
• If Alphanate® treatment either becomes ineffective in achieving 
hemostasis, even if the inhibitor titer remains below 5 BU/mL, or if the 
inhibitor level rises to greater than or equal to 5 BU/mL, as confirmed by 
test results from the central reference laboratory, then the subject will 
be withdrawn from the study. 
Subjects withdrawn from the study may either be treated with alternate 
concentrates or commence a treatment program with Alphanate® for the 
induction of immune tolerance.  The treating physician, in consultation with 
Grifols Office of Medical Affairs, will determine the alternate treatment or 
product and the immune tolerance protocol. 
7.12 Treatment with Other Blood Products 
 
Subjects will be treated for hemophilia A exclusively with Alphanate® during 
the course of the study.  If whole blood or other blood products are used 
during surgery or during the control of hemorrhagic episodes, the exact dose, 
date and duration of treatment and prescription etiology will be entered in the 
appropriate case report forms. 
7.13 Concurrent Medication 
 
All prescription and non-prescription medication administered to the subjects, 
including the name of the drug, dose, duration of treatment as well as the 
indication for use will be entered on the appropriate case report forms.  
7.14 Discontinuation of Subjects 
A subject will be discontinued from the study if any of the following occurs: 


 
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• A severe or serious adverse event, which, based on medical judgment, 
prevents completion of participation in the study (See Section 10 for 
classification of adverse events). 
• Development of a Factor VIII inhibitor either  
- 
at a titer at or above 5 BU/mL or  
- 
at a titer lower than 5 BU/mL but that renders Alphanate® treatment 
ineffective in providing hemostasis. 
• Treatment of the subject with another Factor VIII concentrate while in the 
study. 
• Treatment of the subject with immunosuppressive drugs. 
• Unwillingness and non-compliance of the subject with the provisions of the 
protocol. 
• Withdrawal of the subject from the study on his/her own volition. 
• Death of the subject during participation in the study. 
If a subject withdraws after treatment, the clinical site will attempt to obtain all 
possible follow-up safety data from that subject. 
8 
CLINICAL ENDPOINTS 
The safety of Alphanate® will be evaluated based on the following criteria: 
8.1 Primary Endpoint 
Incidence of antibody inhibitors to Factor VIII in at least 50 evaluable subjects 
will be determined following a period of at least two years and a minimum of 
50 exposure days or if 50 exposure days are not reached, a maximum of 30 
months. Only one exposure-day will be accrued for each surgical episode 
independent of the number of days of treatment or the number of infusions.  
For the purpose of this endpoint only, a subject is determined to be evaluable 
when his clinical experience after the date of enrollment includes at least 50 
CEDs of treatment with Alphanate® or when he develops inhibitors with a titer 
greater than or equal to 5 BU/mL as determined by the central reference 
laboratory. 


 
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8.2 Secondary Endpoints 
All subjects will be considered to be evaluable for the secondary endpoints. 
1. The frequency, nature, causality and severity of adverse events.  
2. The occurrence of variations in biochemical parameters which would 
indicate renal or hepatic impairment. 
3. Evidence for seroconversion to HIV-1/HIV-2, HAV, HBV, HCV and 
parvovirus B19 for subjects who are seronegative for these viruses upon 
enrollment. 
4. The amount of product used per year as part of at-home prophylaxis and 
therapy for bleeding episodes. 
5. A physician’s qualitative assessment of hemostasis (evaluated as “None,” 
“Moderate,” “Good,” or “Excellent”) in response to therapy with Alphanate® 
received by the subject in a hospital or at another location under the 
physician’s direct supervision. 
With this section, the design of this clinical trial satisfies the recommendations 
of the CPMP for trials in previously-treated patients of concentrates with 
marketing authorization in which a manufacturing process has been added. 
9 
STATISTICAL PLAN 
9.1 Sample Size 
 
The sample size of 50 to 80 evaluable patients followed for at least two years 
for renal or hepatic function and evidence of seroconversion to HIV-1/HIV-2, 
HAV, HBV, HCV and parvovirus B19 is in accordance with the 
recommendations of the CPMP regarding post-authorization surveillance of 
previously-treated patients infused with a coagulation factor concentrate: 
• for which a marketing authorization has already been granted and 
• in the production of which a manufacturing process has been added. 
9.2 Planned Method of Analysis 
9.2.1 Primary Endpoint  
Incidence of Antibody Inhibitors to Factor VIII in Individuals Treated with 
Alphanate® 
Assuming a maximum biannual rate of inhibitor incidence of three percent in 
multi-transfused hemophilia patients, the 95% one-sided confidence interval 
for this incidence rate in a sample of 50 subjects is up to two percent.  Thus, 


 
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with a sample of 50 evaluable subjects monitored for two years, if one subject 
develops an antibody inhibitor to Factor VIII with a titer higher than 0.6 
modified BU/mL, that persists for at least one month, the incidence of 
inhibitors to Factor VIII will be judged to be higher than that for patients with 
hemophilia A who are treated with currently marketed Factor VIII concentrates.  
If the sample size is increased to 80 subjects, no more than 1 subject may 
develop an antibody inhibitor to Factor VIII. 
9.2.2 Secondary Endpoints 
1. Adverse Events 
The percentage of subjects experiencing one or more adverse events will be 
calculated.  The frequency of adverse events will be reported as a proportion 
of the total number of infusions with Alphanate.  Causality [the proportions of 
adverse events judged to be definitely, probably, possibly, probably not and 
definitely not related to treatments with Alphanate] and severity will be 
reported as percentages of the total number of adverse events occurring 
during the course of the clinical trial. 
2. Assessment of Biochemical Tests on Serum 
Descriptive statistics will be presented, with a focus on parameters which 
when elevated may indicate alterations in hepatic or renal function. 
3. Viral Safety 
Viral safety will be determined by a lack of seroconversion to reactivity against 
the specific blood-borne viruses for which subjects will have been tested and 
found to be nonreactive at the time of enrollment. 
4. Consumption Analysis 
Descriptive statistics will be employed to evaluate the use of Alphanate by 
subjects, both for self-administration and for treatment by the investigator at 
the clinical site or other places of treatment. 
5. Hemostasis 
Descriptive statistics will be employed to evaluate the physicians’ qualitative 
assessments of the ability of Alphanate to control bleeding episodes when 
administered at the clinical site. 


 
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10 ADVERSE EVENTS  
 
Any adverse events observed by the investigator or study staff or reported by 
the subject and which occur during the study will be recorded on the 
appropriate case report form.  These events will be reported regardless of the 
perceived relation of the event to study treatment. 
10.1 Adverse Event Monitoring 
At each visit of the subject to his treatment center an assessment will be 
performed of any adverse events occurring since the previous visit.  Any 
adverse event, which is observed by the investigator or study staff or reported 
by the subject and which occurs during the study, will be reported on the 
appropriate CRF.  Any substantive or unexpected new abnormality will be 
recorded and monitored until the abnormal finding returns to normal for that 
subject. 
Each new event that appears to be independent of any prior event will be 
reported separately.  The description of the event will include: 
• its nature, 
• the date of onset, 
• the duration and severity of each sign or symptom, 
• the investigator's opinion regarding the likelihood of causal relationship to 
the study drug, 
• the course of action taken and 
• the resolution of the event. 
10.2 Serious Adverse Events 
Any serious adverse event, including death, that occurs while the subject is 
receiving an investigational drug, irrespective of the investigator's opinion 
regarding drug relationship, will be reported by telephone within 24 hours of 
its occurrence to the Grifols’ Personnel assigned to the study, 
, 
who can be contacted during working hours (8:00 a.m. to 5:00 p.m. Pacific 
Time) at 
 or 
 during non-working hours. 
 
A serious adverse event is defined by FDA regulations in the Code of Federal 
Regulations (21 CFR 312.32) as any adverse event that suggests a significant 
hazard, contraindication, side effect or precaution.  With respect to human 
clinical events, a serious event includes any event that results in the following 
outcomes: 
Death, 
a 
life-threatening 
adverse 
drug 
event, 
inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or 
significant disability/incapacity, or a congenital anomaly/birth defect.  Important 
medical events that may not result in death, be life-threatening, or require 


 
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hospitalization may be considered serious adverse drug events when, based 
upon appropriate medical judgment, they may jeopardize the patient or subject 
and may require medical or surgical intervention to prevent one of the 
outcomes listed in the definition. 
If a serious adverse event occurs, based on the investigator's judgment, 
appropriate therapy should be administered. Subjects will then be monitored 
closely as appropriate. 
Oral reports of serious adverse events must be confirmed in writing within five 
(5) calendar days of the occurrence to Grifols Inc. and to the Institutional 
Review Board within their reporting guidelines. 
10.3 Grading Scale 
An adverse event may occur during or following the administration of the 
investigational compound or control agent.  Each event will be graded 
according to severity using the following criteria as appropriate: 
• Mild: the event was noted but the administration of the compound was not 
interrupted; the event resolved spontaneously or no treatment was required 
beyond administration of nonprescription analgesics. 
• Moderate: the administration of the compound was not necessarily 
interrupted; the event required momentary treatment with prescription 
drugs and produced no sequelae. 
• Severe: the event caused interruption of the administration of the 
compound, produced sequelae and required prolonged treatment. Infusion 
of the compound will not be resumed until all symptoms are resolved. 
10.4 Causality 
The investigator must attempt to explain each adverse event and its 
relationship to drug treatment (definite, probable, possible, probably not, 
definitely not).  Criteria for determining the relationship of clinical adverse 
reactions to test drug administration are as follows: 
• Definite: an event that follows a reasonable temporal sequence from 
administration of the drug or in which the drug level has been established 
in body fluids or tissues; that follows a known response pattern to the 
suspected drug; and that is confirmed by improvement on stopping the 
drug (dechallenge), and reappearance of the reaction on repeated 
exposure (rechallenge). 
• Probable: an event that follows a reasonable temporal sequence from 
administration of the drug; that follows a known response pattern to the 
suspected drug; that is confirmed by dechallenge; and that could not be 


 
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reasonably explained by the known characteristics of the subject's clinical 
state. 
• Possible: an event that follows a reasonable temporal sequence from 
administration of the drug; that follows a known response pattern to the 
suspected drug; but that could have been produced by the subject's clinical 
state or other modes of therapy administered to the subject. 
• Probably not: an event that follows a reasonable temporal sequence from 
administration of the drug; that does not follow a known response pattern 
to the suspected drug; and that could be reasonably explained by the 
known characteristics of the subject's clinical state. 
• Definitely not: an event that occurs prior to the administration of study 
drug, is proven to be caused by the subject’s disease or condition or 
another drug through rechallenge, or does not recur following rechallenge. 
11 SUBJECT DEATHS 
All deaths of subjects, regardless of cause, occurring within 24 months of the 
subject's entry into the protocol and which are known to the investigator will be 
reported in the case report on the appropriate CRF.  Documentation of the 
subject's cause of death and a copy of the autopsy report, if any, will also be 
provided. 
Grifols Inc. must be notified within 24 hours by telephone of all subject deaths; 
written follow-up must be received within 5 calendar days of initial notification. 
12 INFORMED CONSENT FORM AND PATIENT AUTHORIZATION FORM  
The investigator will be responsible for obtaining from every subject prior to his 
participation in the study a written Informed Consent signed and dated by the 
subject in accordance with US Federal regulations.  Any study enrolling 
subjects less than 18 years of age must allow for the signing of informed 
consents by the subject's legally authorized representative.  A written Informed 
Consent will be obtained from the subject after the investigator has provided a 
full explanation, both verbally and in writing, of the purpose, risks and 
discomforts involved, and potential benefits of the study.  The original signed 
copy of the Informed Consent must be maintained in the institution's records, 
and is subject to inspection by a representative of Grifols Inc. The Informed 
Consent document must be approved by the Institutional Review Board and 
Grifols Inc. prior to study initiation and must conform to federal regulations.  
The document must include the following: 
• A statement that the study involves research, an explanation of purposes 
of the research and the expected duration of the subject's participation, a 


 
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description of the procedures to be followed, and identification of any 
procedures that are experimental. 
• A description of any reasonably foreseeable risks or discomforts to the 
subject. 
• A description of any benefits to the subject or to others which may 
reasonably be expected from the research. 
• A disclosure of appropriate alternative procedures or courses of treatment, 
if any, that might be advantageous to the subject. 
• A statement describing the extent, if any, to which confidentiality of records 
identifying the subject will be maintained and that notes the possibility that 
representatives from the Food and Drug Administration and/or other 
regulatory agencies and Grifols Inc. may inspect and obtain copies of the 
records, including medical records. 
• For research involving more than minimal risk, an explanation as to 
whether any compensation is provided and an explanation as to whether 
any medical treatments are available if injury occurs and if so, what they 
consist of, or where further information may be obtained. 
• An explanation of whom to contact for answers to pertinent questions 
about the research and subject's rights, and whom to contact in the event 
of a research-related injury to the subject. 
• A statement that participation is voluntary, that refusal to participate will 
involve no penalty or loss of benefits to which the subject is otherwise 
entitled, and that the subject may discontinue participation at any time 
without penalty or loss of benefits to which the subject is otherwise entitled.  
The subject must be assured that refusal to participate in the study or 
withdrawal from the study will not affect the quality of the medical care 
which he/she will receive. 
• A statement that the particular treatment or procedure may involve risks to 
the subject (or to the embryo or fetus, if the subject is or may become 
pregnant) which are currently unforeseeable. 
• Anticipated circumstances under which the subject's participation may be 
terminated by the investigator without regard to the subject's consent. 
• Any additional costs to the subject that may result from participation in the 
research. 
• The consequences of a subject's decision to withdraw from the research 
and procedures for orderly termination of participation by the subject.  The 
subject must be assured that withdrawal will not affect the level of medical 
care which he/she will receive. 


 
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• A statement that significant new findings developed during the course of 
the research which may relate to the subject's willingness to continue 
participation will be provided to the subject. 
• The approximate number of subjects involved in the study. 
 
In conjunction with the Informed Consent, the investigator will also be responsible 
for obtaining the Patient Authorization Form for Release of Information, in 
accordance with the Health Insurance Portability and Accountability Act (HIPAA), 
effective 14 April 2003, prior to the subject’s participation in the study.   
 
The Patient Authorization Form will allow the investigator and his support staff to 
use or disclose identifiable health information to Grifols Inc., its employees and 
agents, members of, and auditors hired by, the independent board that approved 
this clinical trial; and members of government agencies in the US and other 
countries that grant approvals for drugs to be sold and used to treat patients.  The 
authorization will apply to subjects enrolled in the clinical sites in the United States 
and other participating countries. 
If the subject chooses not to sign the authorization form, he cannot participate in 
the trial.  However, treatment not related to the study will not be withheld from 
him. 
 
13 ADMINISTRATIVE PROCEDURES AND DOCUMENTS 
13.1 Institutional Review Board and Privacy Board 
US Federal regulations require that an Institutional Review Board (IRB) 
oversee all investigational drug studies (21 CFR 56).  This board or 
committee, the makeup of which must conform to federal regulations, and any 
state and local guidelines regarding such, will approve all aspects of the study, 
including said protocol and written Informed Consent to be used, prior to 
initiation of the study.  The investigator will provide Grifols Inc. with a copy of 
the communication from the committee to the investigator indicating approval 
of the protocol and consent form.  All amendments to the protocol must be 
reviewed and approved prior to implementation, except where necessary to 
eliminate apparent immediate hazards to human subjects. 
THE INVESTIGATOR WILL BE RESPONSIBLE FOR OBTAINING ANNUAL 
IRB RENEWAL FOR THE DURATION OF THE STUDY.  COPIES OF THE 
INVESTIGATOR'S REPORT AND/OR COPIES OF THE IRB EXTENSION 
APPROVAL MUST BE SENT TO GRIFOLS INC. 
The IRB or a duly established Privacy Board will review and approve the 
Patient Authorization Form to allow the release of the subject’s protected 
health information to Grifols Inc. and other entities as required to fulfill the 


 
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obligations of this protocol.  Any requested changes to the Authorization Form 
by the Board must be submitted to Grifols Inc. 
13.2 Documents Required Before a Study Is Initiated 
Prior to study implementation and drug shipment, the following documents 
must be submitted to Grifols Inc. for the purpose of FDA submission and/or 
procedural compliance: 
• Executed Form FDA 1572, listing all subinvestigators and signed by the 
principal investigator. 
• Investigator's Agreement from the final protocol signed by the principal 
investigator. 
• Current Curricula Vitae for the principal investigator and all subinvestigators. 
• Copy of the correspondence from the IRB indicating approval of the protocol 
and written Informed Consent, signed by the IRB chairperson or designee 
and containing the name and address of the IRB. 
• A sample of the written Informed Consent that was reviewed and approved 
by the IRB, or a revised document if changes were requested by the 
committee. 
• A sample Patient Authorization Form that was reviewed and approved by 
the IRB or Privacy Board, or a revised document if changes were 
requested by the Board. 
 
13.3 Documents Required Within 30 Days of Study Initiation 
• A membership roster of the IRB, listing names and occupations.  In order to 
avoid a conflict of interest, investigators, subinvestigators and others directly 
participating in the study, should indicate in writing their abstention from voting 
on the protocol if they are also IRB members. 
• Clinical laboratory ranges for all tests required for the protocol and documenta-
tion of laboratory licensure. 
14 REPORTING AND MONITORING OBLIGATIONS OF INVESTIGATORS 
14.1 Case Reports 
A case report, comprised of individual case report forms, will be completed for 
every subject who signed a written Informed Consent form for participation in 
the study. 


 
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• Case report pages will be filled out in indelible dark ink or will be 
typewritten. 
• Any corrections will be made by a single line stroke through the entry to be 
corrected, and the correct entry will be made above or near the deleted 
entry, initial and dated.  Correction fluid must not be used. 
• The Investigator must sign and date the certification of the case report form 
completion and return the originals, not copies, to Grifols, Inc.  This 
signature will indicate that thorough inspection of the data has been made 
and will thereby certify the contents of the case report form. 
All original source documentation (laboratory results, treatment records, audit 
queries, etc.) will be retained by the investigator or institution unless specified 
otherwise by the protocol.  The results as they become available will be 
entered on the appropriate case report forms.  Legible reproductions of the 
original laboratory reports for selected tests or variables may be included with 
the completed case report forms.  The research manager will indicate which 
reports, if any, need to be reproduced.  The investigator will retain a copy of 
the completed case report forms and necessary supporting laboratory data, 
and documentation before entrusting the original(s) to the mail. 
Case report forms will be reviewed at the study site by a clinical monitor of 
Grifols Inc. who will make a decision as to their acceptability in regard to 
completeness and accuracy of the data.  Query letters will be generated for 
omissions, corrections and clarifications.  Data may also be reviewed in-house 
by a clinical assistant or data management personnel.  
14.2 Clinical Monitoring 
 
A representative of Grifols Inc. will visit the institution prior to initiating the 
study and periodically thereafter (at least once a year) to monitor acceptability 
of facilities, the agreement between CRF entries and original source 
documentation, adherence to the protocol and to applicable FDA and other 
regulatory agencies regulations and the maintenance of adequate clinical 
records.  The monitor will have access to subject records, medication sheets, 
laboratory data and other source documentation. 
14.3 Investigator's Final Report 
 
A summary report must be submitted to the Institutional Review Board and a 
copy to Grifols Inc. within eight weeks after the study's completion or 
termination. 
14.4 Regulatory Correspondence 
 
The investigator will notify Grifols Inc. within three days following FDA or other 
regulatory agency contact with the investigative site.  The investigator will 
provide Grifols in a timely fashion, with copies of all correspondence with the 


 
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FDA or other regulatory agency which may affect the review of the current 
study (e.g., Form 483, Inspection Observations, compliance responses) or 
their qualification as an investigator in studies conducted by Grifols Inc. (e.g., 
warning letters). 
15 RECORDS 
The Food and Drug Administration requires that an investigator will retain 
records for a period of two years following the date of the New Drug 
Application or Product License Application is approved for the drug for the 
indication for which is being investigated; or, if no application or license is to 
be filed or, if the application or license is not approved for such indication, until 
two years after the investigation is discontinued (21 CFR 312.62).  In 
compliance with CPMP requirements, however, all records must be retained 
for a period of 15 years following the date of approval for the New Drug 
Application or Product License Application. 
Investigator should ensure that the following records are maintained for the 
appropriate time period: 
• Patient files containing copies of completed case reports and supporting 
documentation and the signed Informed Consent form. 
• Investigator files containing copies of the documents required for the 
initiation of the study (executed Form FDA 1572, signed Investigator's 
Agreement, 
current 
Curricula 
Vitae 
for 
the 
principal 
and 
all 
subinvestigators, copy of the IRB approval of the protocol and Informed 
Consent form), copies of correspondence received from and sent to Grifols 
Inc..  In addition to these records required by regulations, Grifols Inc. 
requests that the investigator keep a copy of the Financial Agreement 
between Grifols Inc. and the investigator. 
• Pharmacy files containing copies of the Investigational Drug Utilization 
Records (IDUR) or an equivalent form approved by Grifols Inc., instructions 
for the use of the IDUR and package inserts and/or the Investigator's 
Brochure. 
16 HANDLING OF INVESTIGATIONAL DRUG 
16.1 Investigational Drug Utilization Record Instructions 
In accordance with federal regulations (21 CFR 312.62), all investigators are 
required to keep accurate records showing final disposition of all 
investigational drugs.  The pharmacist in charge of the investigational drug 
may keep the records and sign the IDUR or the equivalent form approved by 
Grifols Inc.  These records must be transmitted to Grifols Inc. on an ongoing 


 
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basis, showing accurate reconciliation of each and every shipment of 
investigational drug or control agent.  A separate/new IDUR (or the equivalent) 
must be made for each new lot of drug received. 
THE INVESTIGATOR MUST NOT USE MATERIAL PROVIDED FOR THIS 
PARTICULAR STUDY IN ANOTHER STUDY WITHOUT PRIOR WRITTEN 
APPROVAL FROM GRIFOLS INC. 
Investigator or his/her designee will record: 
• Person Responsible:  The investigator or designee should sign the first 
report in full.  On the Distribution Log, if the same individual signs the forms 
from day to day, his/her title need not be recorded after the first time. 
• Lot Number:  The lot number is indicated on the label applied to each 
container of the product. 
• Expiration Date:  The expiration date will be listed on the label of the 
product. 
• Date Used:  Date administered or dispensed to the subject. 
• Disposition of Material:  Indicate if administered, destroyed, damaged in 
transit and destroyed, or other final disposition of material.  Material cannot 
be transferred for preclinical or other use without prior written approval 
from Grifols Inc. 
• Unused Containers:  Indicate any unopened units returned, as well as 
opened and partially used containers. 
• Date Returned to Grifols Inc. or Destroyed:  At the termination of the study, 
unused and opened and partially used containers will be returned to the 
clinical project manager or destroyed.  However, the investigator will not 
destroy the supplies without prior consultation with the 
 
.  The 
, 
, PhD, 
may be contacted at 
. 
• Disposition of Investigational Drug Utilization Records:  When an IDUR (or 
the approved equivalent form) is complete for any given lot, it is to be 
signed by the principal investigator or pharmacist in charge of the 
investigational drug.  The white original (if Grifols’ form) is to be mailed to 
Grifols Inc., the yellow copy is to remain in the pharmacy file and  the pink 
copy is to be filed in the on-site investigator's file.  Appropriate copies of an 
equivalent form will be filed at the pharmacy, the investigator's file and 
Grifols Inc. 


 
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16.2 Disposition of Clinical Supplies 
Ultimate accountability for the receiving, dispensing and inventory of the test 
material lies with the investigator.  US federal regulation requires that storage 
of the substance be in a secure enclosure, access to which is limited, to 
prevent theft or diversion.  Neither the pharmacist nor investigator may supply 
the test material to any person outside of this protocol. (See previous section 
for details of the record-keeping for the investigational material.) 
The investigational drug provided by Grifols Inc. for use in this study is 
intended for use only in the clinical trial outlined in this protocol and will be 
administered only to patients appropriately enrolled in this trial.  The use of the 
drug for other clinical or preclinical situations is strictly prohibited.  The use of 
the investigational drug by the investigator, subinvestigators or any third party, 
outside of the provisions stated in this protocol, without the express written 
permission of Grifols Inc., is strictly prohibited. 
Alphanate® is an investigational drug for use in Clinical Trial GBI 04-01.  The 
possession and use of Alphanate® by investigators must be closely controlled 
and monitored. All Alphanate® that is not used in the course of this clinical trial 
will be returned to Grifols Inc. or disposed of according to the written direction 
of the Clinical Research Manager. 
On the written approval of the Project Manager, outdated Alphanate® will be 
destroyed and the containers and/or labels returned to Grifols Inc. 
Material that is not outdated at the completion of the study will be returned by 
the investigator to an address provided by Grifols Inc. or will be handled 
otherwise according to written instructions from Grifols Inc. 
17 MISCELLANEOUS PROVISIONS 
17.1 Use of Information and Publication Rights 
All information and data, including the terms of this protocol, and all data, 
clinical results and research conducted hereunder concerning Grifols Inc.'s 
products and operations including Grifols’ patent applications, formulas, 
manufacturing processes, basic scientific data and formulation information that 
has been supplied by Grifols Inc. and not previously published are considered 
confidential by Grifols and will remain the sole property of Grifols Inc.  The 
investigator understands and agrees that said proprietary and/or confidential 
information disclosed to or produced by him/her thereunder is highly valuable 
to Grifols Inc. and will be used EXCLUSIVELY by the investigator in 
accomplishing this study and will not use it for any other purposes without 
Grifols’ prior written consent.  The investigator agrees that he/she will not use 


 
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any such proprietary and/or confidential information for any other purpose.  
The investigator also understands and agrees that such disclosure will not be 
deemed to grant to the investigator a license for use of said proprietary and/or 
confidential information, except as expressly provided herein. 
It is understood by the investigator that the information developed in the 
clinical study will be used by Grifols Inc. in connection with the development of 
Alphanate®, therefore, may be disclosed and used solely by Grifols as 
required to such third parties and agencies as Grifols, in its sole discretion, 
warrants.  In order to allow for the use of the information derived from the 
clinical studies, it is understood that there is an obligation to provide to Grifols 
complete test results and all data developed in this study. The investigator 
agrees to promptly answer all inquires from the clinical research manager 
regarding completion, legibility or accuracy of trial data in the case report. 
The investigator may publish the results of this study.  In this regard, however, 
at least 30 days before submitting a manuscript to a publisher and 7 days 
before submitting an abstract for presentation at a scientific meeting, a copy 
will be provided by the investigator to Grifols for review. Grifols will have the 
right to modify or amend such a manuscript to ensure that no confidential or 
proprietary information of Grifols is disclosed and to insure that reported data 
are factually correct. Grifols agrees that before it publishes any results of this 
study in a refereed journal, it will provide the investigator, for review, a 
prepublication manuscript at least 30 days prior to the submission of the 
manuscript to the publisher. 


 
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18 REFERENCES 
 
1. Goldsmith JC: Introduction: The challenges of inhibitor patient care. Sem 
Hematol. 30, Suppl 1:1-2, 1993.   
 
2. Aledort L: Inhibitors in hemophilia patients: Current status and management.  
Am. J. Hematol. 47:208-217, 1994. 
 
3. Scharrer I and Neutzling O:  Incidence of inhibitors in hemophiliacs: A review of 
the literature.  Blood Coag. Fibrinol. 4:753-738, 1993. 
 
4.  Aledort L: World registry on factor VIII inhibitor patients: Why? Sem Hematol. 30, 
Suppl 1:7-9, 1993. 
 
 
5. Bray G, Gomperts E, Courter S et al.:  A multicenter study of recombinant factor 
VIII (Recombinate): Safety, efficacy and inhibitor risk in previously untreated 
patients with hemophilia A.  Blood 83: 2428-2435, 1994. 
 
6.  Lusher JM, Arkin S, Abildgaard CF et al.  Recombinant factor VIII for the 
treatment of previously untreated patients with hemophilia A.  N. Engl. J. Med., 
328, 453-459, 1993. 
 
7.  Addiego J, Kasper C, Abildgaard C et al.: Frequency of inhibitor development in 
hemophiliacs treated with low purity factor VIII.  Lancet 342: 462-464, 1993. 
 
8. 
Ehrenforth S, Kreuz W, Scharrer I, et al.: Incidence of development of factor VIII 
and factor IX inhibitors in hemophiliacs. Lancet 339: 594-598, 1992.  
 
9. de Biasi R, Rocino  A, Papa ML et al.: Incidence of factor VIII inhibitor 
development in hemophilia A patients treated with less pure factor VIII 
concentrates.  Thromb. Haemost. 71: 544-547, 1994. 
 
10. Peerlinck  K, Arnout J, Giles JG et al.:  A higher than expected incidence of 
factor VIII inhibitors in multitransfused heamophilia A patients treated with 
intermediate purity pasteurized factor VIII concentrate.  Thromb. Haemost. 69: 
115-118, 1993. 
 
11. Rosendaal FR, Nieuwehuis HK, van der Berg HM et al.:  A sudden increase in 
factor VIII inhibitor development in multitransfused hemophilia A patients in the 
Netherlands.  Blood 81: 2180-2186, 1993. 
 
12. Guérois C, Laurian Y, Rothchild C et al: Incidence of factor VIII inhibitor 
development in severe hemophilia A patients treated with only one brand of 


 
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highly purified plasma-derived concentrate.  Thromb. Haemost. 73: 215-218, 
1995. 
 
13. Addiego JE, Gomperts E, Liu SL et al.:  Treatment of hemophilia with a highly 
purified factor VIII concentrate prepared by anti-FVIIIC immunoaffinity 
chromatography.  Thromb. Haemost. 67: 19-27, 1883. 
 
14. Lusher JM, Salzman PM:  Viral safety and inhibitor development associated with 
factor VIII:C ultra-purified from plasma in hemophiliacs previously unexposed to 
factor VIII:C concentrates.  Sem. Hematol. 27 (Suppl 2): 1-7, 1990. 
 
15. McMillan CW, Shapiro SS, Whitehurst D et al.: The natural history of factor VIII 
inhibitors in patients with hemophilia A: a national cooperative study.  II. 
Observations on the initial development of factor VIII:C inhibitors.  Blood 71: 344-
348, 1988. 
 
17. Schwartz RS, Abildgaard CF, Aledort LM et al.: Human recombinant DNA-
derived  antihemophilic factor (factor VIII) in the treatment of hemophilia A.  N. 
Engl. J. Med. 323:1800-1805, 1990. 
 
16. Verbruggen B, Novakova I, Wessels H et al.: The Nijmegen modification of the 
Bethesda assay for factor VIII: Improved Specificity and Reliability. Thromb. 
Haemost. 73: 247-251, 1995. 
 
17. Shanbrom E., Thelim M: Experimental prophylaxis of severe hemophilia with a 
factor VIII concentrate. JAMA 208(9): 1853-1856, 1969. 
 
18. Levine, P.H., Hemophilia and Allied Conditions, In: Current Therapy in 
Hematology-Oncology: 1983-84, Brain, M.C. (ed) New York: BC Decker, pp.147-
152, 1983. 
 
19. Kasper CK: Hemophilia and hemophiloid disorders.  In "Current Therapy",4th 
Edition, Conn HF, ed., Saunders, Philadelphia, pp 258-263, 1974. 
 
20. Kasper CK, Dietrich SL. Rapaport SI: Hemophilia prophylaxis with Factor VIII 
concentrate.  Arch. Intern. Med, 125: 1004-1009, 1970. 
 
21. Data on file at Grifols Inc. 


Contains Confidential and Proprietary Information of Grifols Inc. 
 
19 INVESTIGATOR’S AGREEMENT 
 
Investigator’s Name: ______________________________________________ 
Institution: ______________________________________________________ 
 
I attest that I have read Clinical Protocol GBI 04-01 (formerly ATC 02-01 of Alpha Therapeutic Corporation) entitled: “A 
Study of Immunologic Safety for Alphanate® in Previously Treated Patients Diagnosed with Hemophilia A”, dated 10 
January 2007 - incorporating protocol amendment six. 
 
I understand the contents of Clinical Protocol GBI 04-01 and agree to adhere to the design, conduct and reporting 
requirements of the study as stated in the clinical protocol and to my obligations to Grifols Inc. as described in the protocol 
and executed contracts between myself, my institution and Grifols Inc. 
 
I understand that if any questions about the design or conduct of the trial or about my responsibilities to Grifols Inc. (or 
those of Grifols Inc. to me) arise before or during the trial, I will promptly contact the clinical project manager at Grifols Inc. 
for clarification. 
 
I understand that only I and the subinvestigators named on Form FDA-1572, completed for participation in Clinical 
Protocol GBI 04-01 are authorized to review and sign the case reports relating to this trial.  I understand that I and my 
subinvestigator(s) are fully responsible for ensuring the completeness, correctness and timely entry of data on all case 
report forms relating to Clinical Protocol GBI 04-01. 
 
I understand that the Alphanate® provided for this study is designated for use in Clinical Protocol GBI 04-01 only.  I will not 
use this material for the performance of any other preclinical or clinical studies unless so requested and approved in 
writing by Grifols Inc. nor will I make this material available to any other person for any reason whatsoever. 
 
I understand that for the purpose of this clinical protocol the Alphanate® consigned to me will become outdated on the 
expiration date listed on the product label or Investigator’s Brochure.  Any outdated material or material remaining at the 
conclusion of the trial will be returned to Grifols Inc. or disposed of according to their directions. 
 
I agree that the data acquired in the performance of Clinical Protocol GBI 04-01 will not be used by me or any of the 
named subinvestigators in any publication except that which reports the results of this study alone and that all 
manuscripts and/or abstracts will be submitted to Grifols Inc. prior to submission for publication as specified in the 
protocol. 
 
I furthermore agree to adhere to applicable Food and Drug Administration regulations regarding the conduct of clinical 
trials and the responsibilities of Clinical Investigators. 
 
_______________________________  ____________________________________  ________ 
Signature of Principal Investigator          Type or Print Name as Signed                         Date 
 
_______________________________   ___________________________________  _________ 
Signature of Project Manager                  Type or Print Name as Signed                        Date 
Grifols Inc. 
 


Contains Confidential and Proprietary Information of Grifols Inc. 
 
 
20 APPENDICES 
APPENDIX 1: KARNOFSKY PERFORMANCE SCORE 
 
100
90
80
70
60
50
40
30
20
10
0
Normal; no complaints, no evidence of disease 
 
Able to carry on normal activity; minor signs of symptoms of disease 
 
Normal activity with effort; some signs of symptoms of disease 
 
Cares for self, unable to carry on normal activity or do active work 
 
Requires occasional assistance, but is able to care for most personal needs 
 
Requires considerable assistance and frequent medical care 
 
Disabled; requires special care and assistance 
 
Severely disabled; hospitalization is indicated, although death is not imminent 
 
Very sick; hospitalization necessary; active support treatment is necessary 
 
Moribund; fatal process progressing rapidly 
 
Dead 
 


Contains Confidential and Proprietary Information of Grifols Inc. 
 
APPENDIX 2: SCHEDULE OF EVENTS 
 
Test 
Screening1 
 
 
Enrollment
2  
Seven to 
14 days 
Post 
Infusion 1 
In Hospital 
and In 
Clinic 
Treatments 
Every Three 
Months until 
Month 30 and/or 
Off-Study3 
 
 
Unsched 
Sign Informed Consent Form 
X 
 
 
 
 
 
Physical Examination 
medical & past treatment history, vital signs, height  & weight 
X 
 
 
 
 
 
Abbreviated Physical Exam 
interim medical history, vital signs, & weight 
 
X 
 
 
X 
X 
Hematology Panel 
CBC with WBC differential and platelet count 
X 
 
 
 
X 
X 
Biochemistry Panel 
ALT, AST, AP, LDH, BUN,  total bilirubin, and creatinine 
X 
 
 
 
X 
X 
Viral Serology 
HBsAg, and antibodies to HAV, HBcAb, HBsAb, HCV, HIV1/HIV-
2, and parvovirus B19 (by routine immunoassay) 
X 
X2 
 
 
X 
X5 
Parvovirus B19 PCR Assay 
performed only if seronegative for parvo B19 at enrollment 
 
 
X 
 
 
 
CD4 Count 
X 
 
 
 
 
 
Factor VIII:C  
X 
X2 
 
 
X 
X 
Factor VIII:C Inhibitor (two sets of samples) 
X 
X2 
 
 
X4 
X 6,7 
Collect 2 mL citrated plasma for inhibitor testing 
 
 
X 
 
 
 
Plasma for inhibitor characterization7 
performed only if subject develops an inhibitor 
 
 
 
 
X 7 
 
Serum for storage Draw approx 5 mL of serum and aliquot 
into three vials of at least 1.5 mL of sample each.  Freeze and 
retain the sample at -20°C for later serological & virological tests. 
X 
X 
 
 
X 
X 
Patient Diary 
if the Alphanate® is to be self-administered 
 
 
X 
X 
X 
X 
Physician's Assessment of Hemostasis 
evaluated as “None,” “Moderate,” “Good,” or “Excellent” 
 
 
 
X 
 
 
1. Perform at least 72 hours after the most recent administration of any F VIII conc. or any other blood products. 
2. These tests are to be performed at enrollment (baseline testing) only if the subject received any Factor VIII, cryoprecipitate, or whole product treatment after 
screening tests were performed. 
3. Quarterly testing will be ± 10 days of scheduled testing date.  Schedule the visit at least three days after an infusion with Alphanate®. 
4. If subject develops an inhibitor, repeat assay at two week intervals until either the inhibitor falls to < 0.6 BU/mL or it is clear that the inhibitor titer will not fall. 
5. Perform only if subject exhibits clinical or subjective symptoms indicating possible viral infection. 
6. Perform only if a) there is a clinical indication that the normal dosage of Alphanate® is not providing adequate hemostasis or b) surgery. 
7. If an inhibitor titer is detected at a titer of greater than or equal to 10 BU/mL, collect a 10 mL sample of citrated plasma. For an inhibitor titer less than 10 
BU/mL, collect a 50 mL sample of citrated plasma. If a 50 mL is required, this sample can be obtained in multiple draws within a two-week period. 

SAP_001.pdf: 
Official Title: 
A Study of Immunologic Safety for Alphanate® in Previously Treated 
Patients Diagnosed With severe Hemophilia A. 
(formerly ATC 02-01 of Alpha Therapeutic Corporation) 
NCT Number:     NCT00323856 
Document Date:  Statistical Analysis Plan Version 1.0: 29 September 2010 


Grifols Biologicals Inc. 
Protocol GBI 04-01 
DOCUMENT: 
PROTOCOL: 
SAP VERSION: 
SAP DATE: 
PROTOCOL DATE: 
SPONSOR: 
PREPARED BY: 
AUTHOR: 
APPROVED: 
Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
CONFIDENTIAL 
GRIFOLS 
Statistical Analysis Plan 
GBI 04-01 
A Study of Immunologic Safety for Alphanate® in Previously 
Treated Patients Diagnosed With severe Hemophilia A. 
(formerly ATC 02-01 of Alpha Therapeutic Corporation) 
Version 1.0 
29 September 2010 
Original: 26 March 2002 
Amendment 1: 11 July 2002 
Amendment 2: 14 October 2002 
Amendment 3: 17 April 2003 
Amendment 4: 16 December 2003 (became GBI 04-01) 
Amendment 5: 1 May 2006 
Amendment 6: 10 January 2007 
Grifols Biologicals Inc. 
Clinipace Worldwide 
-
PhD 
Clinipace Worldwide 
-
MS 
Stat istician 
Clinipace Worldwide 
Date 
Page 1 of 13 


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 2 of 13 
 
TABLE OF CONTENTS 
 
TABLE OF CONTENTS ............................................................................................................................. 2 
1. 
INTRODUCTION ........................................................................................................................... 5 
2. 
STUDY OBJECTIVE(S), TREATMENTS, AND ENDPOINT(S) ............................................................ 5 
2.1 
Study Objectives .......................................................................................................................... 5 
2.2 
Treatment Comparisons .............................................................................................................. 5 
2.3 
Study Endpoints ........................................................................................................................... 5 
2.3.1 
Efficacy .................................................................................................................................... 5 
2.3.2 
Safety ....................................................................................................................................... 5 
3. 
STUDY DESIGN ............................................................................................................................. 6 
3.1 
Overall Study Design .................................................................................................................... 6 
3.2 
Overall Schedule of Time and Events .......................................................................................... 6 
4. 
SAMPLE SIZE CONSIDERATIONS .................................................................................................. 8 
5. 
ANALYSIS POPULATIONS ............................................................................................................. 8 
5.1 
Safety Population ........................................................................................................................ 8 
6. 
CONSIDERATIONS FOR DATA ANALYSIS ...................................................................................... 8 
6.1 
Programming Environment ......................................................................................................... 8 
6.2 
Strata and Covariates .................................................................................................................. 8 
6.3 
Subgroups .................................................................................................................................... 8 
6.4 
Multiple Comparisons and Multiplicity ....................................................................................... 8 
6.5 
Significance Level ......................................................................................................................... 8 
6.6 
Statistical Notation and Methodology ........................................................................................ 8 
7. 
DATA HANDLING METHODS ........................................................................................................ 9 
7.1 
Missing Data ................................................................................................................................ 9 
7.2 
Visit Windows .............................................................................................................................. 9 
7.3 
Data Derivations .......................................................................................................................... 9 
8. 
STUDY POPULATION .................................................................................................................... 9 
8.1 
Subject Disposition ...................................................................................................................... 9 
8.2 
Protocol Deviations ..................................................................................................................... 9 
8.3 
Demographic Characteristics ....................................................................................................... 9 


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 3 of 13 
 
8.4 
Medical History .......................................................................................................................... 10 
8.5 
Concomitant Medications ......................................................................................................... 10 
9. 
STATISTICAL ANALYSIS ............................................................................................................... 10 
9.1 
Primary Analysis ........................................................................................................................ 10 
9.2 
Secondary Analyses ................................................................................................................... 10 
9.2.1 
Adverse Events ...................................................................................................................... 10 
9.2.2 
Serious Adverse Events and Death ....................................................................................... 11 
9.3 
Additional Analyses ................................................................................................................... 11 
9.3.1 
Exposure to Study Drug ......................................................................................................... 11 
9.3.2 
Laboratory Evaluations.......................................................................................................... 11 
9.3.3 
Vital Signs .............................................................................................................................. 11 
10. 
INTERIM AND END-OF-STUDY ANALYSES .................................................................................. 11 
10.1 
Interim Analysis ..................................................................................................................... 12 
10.2 
End-of-Study Analysis ............................................................................................................ 12 
11. 
ATTACHMENTS .......................................................................................................................... 12 
11.1 
Table of Contents for Data Displays ...................................................................................... 12 
 


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
Page 4 of 13 
ABBREVIATIONS: 
AE: 
Adverse event 
BU: 
Bethesda unit 
BUN: 
Blood urea nitrogen 
ALT: 
Alanine aminotransferase 
AP: 
Alkaline phosphatase 
AST: 
Aspartic aminotransferase 
CBC: 
Complete blood count 
CPWW: 
Clinipace Worldwide 
CRF: 
Case report form (s) 
CTM: 
Clinical trial material 
FDA: 
United States Food and Drug Administration 
F VIII: 
Factor VIII 
FVIIIc: 
Factor VIII coagulant activity 
HAV: 
Hepatitis A virus 
HBsAg:  
Hepatitis B surface antigen 
HBcAb: 
Hepatitis B core antibody 
HBsAb: 
Hepatitis B surface antibody 
HBV: 
Hepatitis B virus 
HCV: 
Hepatitis C virus 
HIV-1: 
Human immunodeficiency virus type 1 
HIV-2: 
Human immunodeficiency virus type 2 
LDH: 
Lactic acid dehydrogenase 
MedDRA: 
Medical Dictionary for Regulatory Activities 
mL: 
Milliliter 
SAE: 
Serious adverse event 
SAP: 
Statistical Analysis Plan 
SD: 
Standard deviation 
SOC: 
System organ class 
TEAE: 
Treatment-emergent adverse event 
WBC: 
White blood cell count 
WHO: 
World Health Organization


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 5 of 13 
 
1. 
INTRODUCTION 
This document describes the statistical methods and data presentations to be used in the summary and 
analysis of safety and efficacy data from Protocol GBI 04-01.  Background information is provided for the 
overall study design and objectives.  The reader is referred to the study protocol and case report forms 
(CRFs) for details of study conduct and data collection. 
2. 
STUDY OBJECTIVE(S), TREATMENTS, AND ENDPOINT(S) 
2.1 
Study Objectives 
The objective of this study is to determine the immunologic and overall safety associated with long-term 
use of Alphanate® in individuals diagnosed with severe hemophilia A (Factor VIII:C less than 0.01 IU/mL), 
who have been previously treated with plasma-derived Factor VIII products other than Alphanate® and 
who have no history of developing either antibody inhibitors to Factor VIII or nonspecific inhibitors of 
coagulation. 
2.2 
Treatment Comparisons 
Alphanate® is the only study treatment being administered, so this is an open label, non-comparator 
study. 
2.3 
Study Endpoints 
2.3.1 Efficacy 
There are two secondary efficacy endpoints in this study: 
• 
The amount of product used per year as part of at-home prophylaxis and therapy for bleeding 
episodes, and 
• 
A physician’s qualitative assessment of hemostasis (evaluated as “None”, “Moderate”, “Good”, 
or “Excellent” in response to therapy with Alphanate® received by the subject in a hospital or 
another location under the physician’s direct supervision.  
 
2.3.2 Safety 
Safety will be evaluated on the basis of numerous assessments.  The primary safety assessment will be 
made on the presence/absence of the development of Factor VIII inhibitors.   
Additional secondary safety assessments will be made on: 
• 
The frequency, nature, causality and severity of adverse events, 
• 
The occurrence of changes in biochemical parameters which would indicate renal or hepatic 
impairment, 
• 
The incidence of seroconversion to human immunodeficiency virus type 1 and 2 (HIV-1/HIV-2), 
hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) or parvovirus B19 in 
subjects seronegative for these viruses at the time of enrollment. 
 


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 6 of 13 
 
3. 
STUDY DESIGN 
3.1 
Overall Study Design  
This is a phase IV, non-randomized, multi-center study to assess the safety of Alphanate® as the sole 
source of Factor VIII concentrate for prophylaxis and treatment of all bleeding episodes and surgical 
procedures for a period of at least two years and a minimum of 50 exposure days, or, if 50 exposure 
days are not reached, for a maximum of 30 months.   
3.2 
Overall Schedule of Time and Events 
 
 


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Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 7 of 13 
 
 
Test 
Screening1 
Enrollment2 
Seven to 
14 days 
Post 
Infusion 1 
In Hospital 
and In Clinic 
Treatments 
Every Three 
Months until 
Month 30 and/or 
Off-Study3 
Unsched 
Sign Informed Consent Form 
X 
 
 
 
 
 
Physical Examination 
medical & past treatment history, vital signs, height  & weight 
X 
 
 
 
 
 
Abbreviated Physical Exam 
interim medical history, vital signs, & weight 
 
X 
 
 
X 
X 
Hematology Panel 
CBC with WBC differential and platelet count 
X 
 
 
 
X 
X 
Biochemistry Panel 
ALT, AST, AP, LDH, BUN,  total bilirubin, and creatinine 
X 
 
 
 
X 
X 
Viral Serology 
HBsAg, and antibodies to HAV, HBcAb, HBsAb, HCV, HIV1/HIV-2, and parvovirus 
X 
X2 
 
 
X 
X5 
Parvovirus B19 PCR Assay 
performed only if seronegative for parvo B19 at enrollment 
 
 
X 
 
 
 
CD4 Count 
X 
 
 
 
 
 
Factor VIII:C  
X 
X2 
 
 
X 
X 
Factor VIII:C Inhibitor (two sets of samples) 
X 
X2 
 
 
X4 
X 6,7 
Collect 2 mL citrated plasma for inhibitor testing 
 
 
X 
 
 
 
Plasma for inhibitor characterization7 
performed only if subject develops an inhibitor 
 
 
 
 
X 7 
 
Serum for storage Draw approx 5 mL of serum and aliquot into three 
vials of at least 1.5 mL of sample each.  Freeze and retain the sample at 
X 
X 
 
 
X 
X 
Patient Diary 
if the Alphanate® is to be self-administered 
 
 
X 
X 
X 
X 
Physician's Assessment of Hemostasis 
evaluated as “None,” “Moderate,” “Good,” or “Excellent” 
 
 
 
X 
 
 
1. 
Perform at least 72 hours after the most recent administration of any F VIII conc. or any other blood products. 
2. 
These tests are to be performed at enrollment (baseline testing) only if the subject received any Factor VIII, cryoprecipitate, or whole product treatment after 
screening tests were performed. 
3. 
Quarterly testing will be ± 10 days of scheduled testing date.  Schedule the visit at least three days after an infusion with Alphanate®. 
4. 
If subject develops an inhibitor, repeat assay at two week intervals until either the inhibitor falls to < 0.6 BU/mL or it is clear that the inhibitor titer will not fall. 
5. 
Perform only if subject exhibits clinical or subjective symptoms indicating possible viral infection. 
6. 
Perform only if a) there is a clinical indication that the normal dosage of Alphanate® is not providing adequate hemostasis or b) surgery. 
7. 
If an inhibitor titer is detected at a titer of greater than or equal to 10 BU/mL, collect a 10 mL sample of citrated plasma. For an inhibitor titer less than 10 
BU/mL, collect a 50 mL sample of citrated plasma. If a 50 mL is required, this sample can be obtained in multiple draws within a two-week period. 


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 8 of 13 
 
4. 
SAMPLE SIZE CONSIDERATIONS 
Assuming a maximum biannual rate of inhibitor incidence of three percent in multi-transfused 
hemophilia patients, the 95% one-sided confidence interval for this incidence rate in a sample of 50 
subjects is up to two percent.  Thus, with a sample of 50 evaluable subjects monitored for two years, if 
one subject develops an antibody inhibitor to Factor VIII with a titer higher than 0.6 modified BU/mL, 
that persists for at least one month, the incidence of inhibitors to Factor VIII will be judged to be higher 
than that for patients with hemophilia A who are treated with currently marketed Factor VIII 
concentrates.  If the sample size is increased to 80 subjects, no more than 1 subject may develop an 
antibody inhibitor to Factor VIII. 
5. 
ANALYSIS POPULATIONS 
5.1 
Safety Population 
The safety analysis population consists of all patients who were randomized and received at least one 
Alphanate® treatment; no treated patients will be excluded from the safety analysis population.  
6. 
CONSIDERATIONS FOR DATA ANALYSIS 
6.1 
Programming Environment 
All analyses will be conducted using SAS version 9.2. 
6.2 
Strata and Covariates 
There are no planned strata or covariate adjustments planned for in the analysis. 
6.3 
Subgroups 
There are no planned subgroup analyses. 
6.4 
Multiple Comparisons and Multiplicity 
There are no planned adjustments for multiplicity, especially since the analysis is primarily descriptive.  
6.5 
Significance Level 
Unless otherwise noted, all statistical analyses will be conducted with a significance level (α) of 0.05 and 
utilize two-sided testing. 
6.6 
Statistical Notation and Methodology 
Unless stated otherwise, the term “descriptive statistics” refers to the number of subjects (n), mean, 
median, standard deviation (SD), minimum, and maximum for continuous data and frequencies and 
percentages for categorical data.  Unless otherwise noted, all data collected during the study will be 


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 9 of 13 
 
included in data listings and will be sorted first by subject number and then by date within each subject 
number. 
7. 
DATA HANDLING METHODS 
7.1 
Missing Data 
Every effort will be made to obtain required data at each scheduled evaluation from all patients who 
have been enrolled.  There will be no imputation for missing data. 
7.2 
Visit Windows 
Study periods will be windowed as follows: 
• 
Post-Infusion # 1 – 7 to 14 days after infusion 
• 
Quarterly Visits - ± 10 days 
7.3 
Data Derivations 
Baseline values will be considered as the last non-missing assessment prior to first dose of study drug. 
On-treatment will be considered as all days between the first dose of study drug and until 1 day post 
Off-Study. 
8. 
STUDY POPULATION 
8.1 
Subject Disposition 
Subject disposition will be presented for all subjects.  Subjects who completed and discontinued from 
the study will be summarized with descriptive statistics.  Reasons for early discontinuation also will be 
presented with frequencies and percentages for all categories.   
8.2 
Protocol Deviations 
Protocol deviations will be presented in a data listing and include date of violation and description of the 
event.  
8.3 
Demographic Characteristics 
Demographic and baseline characteristic data will be summarized with descriptive statistics for age and 
race.   


Grifols Biologicals Inc. 
Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 10 of 13 
 
8.4 
Medical History 
Medical history will be presented in a data listing and include body system, diagnosis and the onset 
month and year as appropriate.  
8.5 
Concomitant Medications 
Concomitant medications will be summarized with descriptive statistics by drug using the WHO Drug 
dictionary.  Data listings will display all data by preferred term and verbatim name.  Prior medications 
will be excluded from the summary but be included in the listing. 
9. 
STATISTICAL ANALYSIS 
9.1 
Primary Analysis 
Analysis of the primary safety endpoint will be conducted on the Safety Population.  The primary safety 
endpoint, presence/absence of Factor VIII inhibitors, will be summarized with frequencies and 
percentages.   
9.2 
Secondary Analyses 
There are both efficacy and safety endpoints.  All secondary safety analyses will be conducted on the 
Safety Population. 
Secondary efficacy analyses will be summarized with categorical descriptive statistics for the following: 
• 
The amount of product used per year as part of at-home prophylaxis and therapy for bleeding 
episodes, and 
• 
A physician’s qualitative assessment of hemostasis (evaluated as “None”, “Moderate”, “Good”, 
or “Excellent” in response to therapy with Alphanate® received by the subject in a hospital or 
another location under the physician’s direct supervision). 
Secondary safety analyses will be summarized with categorical descriptive statistics for the following: 
• 
The frequency, nature, causality and severity of adverse events, 
• 
The occurrence of changes in biochemical parameters which would indicate renal or hepatic 
impairment, 
• 
The incidence of seroconversion to human immunodeficiency virus type 1 and 2 (HIV-1/HIV-2), 
hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) or parvovirus B19 in 
subjects seronegative for these viruses at the time of enrollment, 
9.2.1 Adverse Events 
All reported terms (Investigator descriptions) for adverse events (AEs) will be coded using Version 13.0 
of the Medical Dictionary for Regulatory Activities (MedDRA).  The incidence of treatment-emergent AEs 
(TEAEs) will be summarized as those events that occur on or after the date of the first dose of study 
drug. 


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Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 11 of 13 
 
AE data will be listed individually and summarized by body system organ class (SOC) and preferred terms 
within system organ class.  Each AE will be counted only once for a given subject.  If the same AE occurs 
on multiple occasions, the highest severity and least favorable relationship will be assumed.  If two or 
more AEs are reported as a unit, the individual terms will be reported as separate experiences.   
All AEs will be included in the data listings. 
9.2.2 Serious Adverse Events and Death 
A data listing of all Serious Adverse Events (SAEs), including treatment-emergent SAEs, will be 
presented.  SAEs with onset prior to receiving study medication or after discontinuation of study 
medication will be excluded from the descriptive summaries.   
A separate listing will be presented for all deaths as well as events leading to discontinuation.  Complete 
narratives will be included for deaths. 
9.3 
Additional Analyses 
9.3.1 Exposure to Study Drug 
Total exposure through the number of Factor VIII units infused will be summarized with descriptive 
statistics by dosing.  Frequencies and percentages will also be used to summarize the number of 
infusions at each dosing. 
Exposure to study drug will be listed for each subject to indicate the complete usage pattern including 
number of exposures at home and in clinic, reason for infusion, whether hemostasis was achieved (if 
evaluation available) and physician comments. 
9.3.2 Laboratory Evaluations 
Descriptive summaries of quantitative clinical laboratory results will be presented by nominal study visit 
for change from baseline.  Laboratory results will include Factor VIII findings, biochemistry, hematology, 
immunological status and viral serology. 
 A data listing will be included that displays all laboratory panels and test values. 
9.3.3 Vital Signs 
Vital signs will be summarized by nominal study visit and change from baseline.   
10. 
INTERIM AND END-OF-STUDY ANALYSES


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Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
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Page 12 of 13 
 
10.1 
Interim Analysis 
No interim efficacy analysis is planned for this study. 
10.2 
End-of-Study Analysis 
A final analysis is planned after the last patient completes or discontinues the study and the 
resulting clinical database has been cleaned, quality checked, and locked. 
11. 
ATTACHMENTS 
11.1 
Table of Contents for Data Displays 
Study Population 
Tables 
10.1. 
Summary of Subject Disposition 
10.2. 
Summary of Demographic Characteristics 
10.3. 
Summary of Concomitant Medications 
10.4. 
Summary of Inclusion/Exclusion Criteria Deviations 
 
Listings 
10.5. 
Listing of Subject Disposition 
10.6. 
Listing of Demographic Characteristics 
10.7. 
Listing of Prior and Concomitant Medications 
10.8. 
Listing of Prior and Current Vaccinations 
10.9. 
Listing of Inclusion/Exclusion Criteria Violations 
10.10. 
Listing of Protocol Violations 
10.11. 
Listing of Medical History 
 
Safety 
Tables 
12.1. 
Summary of Exposure to Study Drug 
12.2. 
Summary of Adverse Events by System Organ Class and Preferred Term 
12.3. 
Summary of Adverse Events by System Organ Class, Preferred Term, and 
Greatest Severity 
12.4. 
Summary of Adverse Events Related to Study Drug by System Organ Class and 
Preferred Term 


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Statistical Analysis Plan 
September 29, 2010 
Protocol GBI 04-01 
CONFIDENTIAL 
 
Page 13 of 13 
 
12.5. 
Summary of Serious Adverse Events by System Organ Class and Preferred Term 
12.6. 
Summary of Factor VIII: C Data 
12.7. 
Summary of Factor VIII Inhibitor Data 
12.8. 
Summary of Biochemistry Data 
12.9. 
Summary of Hematology Data 
12.10. 
Summary of Immunological Data 
12.11. 
Summary of Viral Serology Data 
12.12. 
Summary of Vital Signs 
12.13. 
Summary of Positive Factor VIII Inhibitor Tests 
12.14. 
Summary of Viral infection  
 
Listings 
12.15. 
Listing of Exposure to Study Drug 
12.16. 
Listing of Adverse Events 
12.17. 
Listing of Serious Adverse Events 
12.18. 
Listing of Deaths 
12.19. 
Listing of Factor VIII Results 
12.20. 
Listing of Abnormal Factor VIII Results 
12.21. 
Listing of Biochemistry Results 
12.22. 
Listing of Abnormal Biochemistry Results 
12.23. 
Listing of Hematology Results 
12.24. 
Listing of Abnormal Hematology Results 
12.25. 
Listing of Immunological Status Results 
12.26. 
Listing of Abnormal Immunological Status Results 
12.27. 
Listing of Viral Serology Results 
12.28. 
Listing of Abnormal Serology Results 
12.29. 
Listing of Inhibitor Monitoring and Viral Infection 
12.30. 
Listing of Vital Signs 
12.31. 
Listing of Physical Exams 
12.32. 
Listing of Abnormal Physical Exams