Conditions: Pyruvate Kinase Deficiency Anemia, Hemolytic
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 80
Healthy volunteers: no

Brief summary:
Study AG348-C-006 evaluated the efficacy and safety of orally administered AG-348 as compared with placebo in participants with pyruvate kinase (PK) deficiency, who were not regularly receiving blood transfusions. Participants were randomized 1:1 to receive either AG-348 or a matching placebo.

Detailed description:

Arms:
arm 1: Participants received a matching placebo to AG-348 tablets, for a period of 12 weeks as an optimized dose. This was followed by matching placebo further, for a period of 12 weeks as a fixed-dose. arm 2: Participants received AG-348 tablets, 5 milligrams (mg) twice daily (BID), administered orally, for 4 weeks as a starting dose, followed by two potential sequential dose level increases to 20 mg and 50 mg BID at Weeks 4 and 8 respectively as determined by the investigator based on safety and efficacy. The optimized dose for each participant was determined as 5 mg BID at Week 12, and participants then received that optimized dose for a period of 12 weeks as a fixed dose. arm 3: Participants received AG-348 tablets, 5 mg BID, administered orally, for 4 weeks as a starting dose, followed by two potential sequential dose level increases to 20 mg and 50 mg BID at Weeks 4 and 8 respectively as determined by the investigator based on safety and efficacy. The optimized dose for each participant was determined as 20 mg BID at Week 12, and participants then received that optimized dose for a period of 12 weeks as a fixed dose. arm 4: Participants received AG-348 tablets, 5 mg BID, administered orally, for 4 weeks as a starting dose, followed by two potential sequential dose level increases to 20 mg and 50 mg BID at Weeks 4 and 8 respectively as determined by the investigator based on safety and efficacy. The optimized dose for each participant was determined as 50 mg BID at Week 12, and participants then received that optimized dose for a period of 12 weeks as a fixed dose.

Interventions: intervention 1: Placebo intervention 2: AG-348
Intervention descriptions: intervention 1: Placebo matching AG-348 tablets, administered to maintain the blind. intervention 2: AG-348 tablets.

Protocol document:
SAP_002.pdf: 
 
Official Title: A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to 
Evaluate the Efficacy and Safety of AG-348 in not Regularly 
Transfused Adult Subjects With Pyruvate Kinase Deficiency 
NCT Number: NCT03548220 
Document Date(s): SAP Version 1.0 – 30 June 2020 
 
 


Study AG348-C-006 
 
Statistical Analysis Plan (v1.0) 
______________________________________________________________________________ 
Confidential  
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STATISTICAL ANALYSIS PLAN 
A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Efficacy 
and Safety of AG-348 in not Regularly Transfused Adult Subjects With Pyruvate Kinase 
Deficiency 
 
AG348-C-006 
 
 
 
 
 
 
 
 
 
Version: 1.0 
Date: 30-Jun-2020 
 
 
 
 
 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. 
Any distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal 
regulations or state law. Persons to whom the information is disclosed must know that it is confidential and that it 
may not be further disclosed by them. 
 
 
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Study AG348-C-006 
 
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TABLE OF CONTENTS 
STATISTICAL ANALYSIS PLAN ................................................................................................1 
TABLE OF CONTENTS .................................................................................................................3 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..................................................6 
1. 
VERSION HISTORY ...................................................................................................8 
2. 
INTRODUCTION ........................................................................................................8 
3. 
TRIAL OBJECTIVES AND ENDPOINTS .................................................................8 
3.1. 
Objectives .....................................................................................................................8 
3.1.1. 
Primary Objective .........................................................................................................8 
3.1.2. 
Secondary Objectives ...................................................................................................8 
3.1.3. 
Exploratory Objectives .................................................................................................8 
3.2. 
Endpoints ......................................................................................................................9 
3.2.1. 
Primary Endpoint ..........................................................................................................9 
3.2.2. 
Key Secondary Endpoint ..............................................................................................9 
3.2.3. 
Other Secondary Endpoints ..........................................................................................9 
3.2.4. 
Exploratory Endpoints ................................................................................................10 
4. 
STUDY DESIGN .......................................................................................................10 
5. 
ANALYSIS DATA SETS ..........................................................................................11 
6. 
GENERAL STATISTICAL CONSIDERATIONS ....................................................11 
6.1. 
Randomization, Blinding, Unblinding, and Crossover ...............................................11 
6.2. 
Sample Size Determination and Decision Rules ........................................................12 
6.2.1. 
Sample Size Determination ........................................................................................12 
6.2.2. 
Decision Rules ............................................................................................................15 
6.3. 
Definitions ..................................................................................................................15 
6.3.1. 
Study Drug and Study Treatment ...............................................................................15 
6.3.2. 
Start and End Dates of Study Treatment ....................................................................15 
6.3.3. 
Study Day ...................................................................................................................15 
6.3.4. 
Baseline .......................................................................................................................15 
6.3.5. 
On-Treatment Period and Optimized Dose ................................................................17 
6.4. 
General Methods .........................................................................................................17 
6.4.1. 
Data Handling After Cutoff Date ...............................................................................17 
6.4.2. 
Standard Derivations and Reporting Conventions .....................................................17 
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6.4.3. 
Pooling of Data Across Sites ......................................................................................18 
6.4.4. 
Continuous and Categorical Variables .......................................................................18 
6.4.5. 
Unscheduled Visits .....................................................................................................18 
6.5. 
Methods for Handling Missing Data ..........................................................................20 
6.5.1. 
Adverse Event and Concomitant Medication Start Dates ..........................................20 
6.5.2. 
Adverse Event and Concomitant Medication End Dates ...........................................20 
6.5.3. 
Exposure .....................................................................................................................21 
7. 
STATISTICAL ANALYSES .....................................................................................21 
7.1. 
Subject Disposition .....................................................................................................21 
7.2. 
Protocol Deviations ....................................................................................................22 
7.3. 
Demographic and Other Baseline Characteristics ......................................................22 
7.3.1. 
Demographics and Physical Measurements ...............................................................23 
7.3.2. 
Disease Characteristics ...............................................................................................23 
7.3.3. 
Medical History ..........................................................................................................24 
7.3.4. 
Prior Therapies ............................................................................................................24 
7.4. 
Exposure to Study Drug and Compliance ..................................................................24 
7.4.1. 
Treatment Duration and Exposure ..............................................................................24 
7.4.2. 
Dose Modifications .....................................................................................................25 
7.5. 
Concomitant Therapies ...............................................................................................25 
7.6. 
Efficacy Analyses .......................................................................................................25 
7.6.1. 
Primary Endpoint ........................................................................................................25 
7.6.2. 
Key Secondary Endpoint ............................................................................................27 
7.6.3. 
Additional Secondary Efficacy Endpoints ..................................................................28 
7.6.4. 
Subgroup Analyses .....................................................................................................30 
7.7. 
Safety Analysis ...........................................................................................................31 
7.7.1. 
Adverse Events ...........................................................................................................31 
7.7.2. 
Death ...........................................................................................................................33 
7.7.3. 
Clinical Laboratory Data ............................................................................................33 
7.7.4. 
Vital Signs and Physical Measurements .....................................................................36 
7.7.5. 
Electrocardiograms .....................................................................................................36 
7.7.6. 
DXA Scans .................................................................................................................38 
7.7.7. 
Menstrual Cycle Diary ................................................................................................38 
7.8. 
Exploratory Analysis ..................................................................................................38 
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7.8.1. 
Use of Iron Chelation Therapy ...................................................................................38 
7.8.2. 
Iron Markers ...............................................................................................................38 
7.8.3. 
Erythropoietic Activity Markers .................................................................................39 
7.8.4. 
Liver Iron Concentration (LIC) by Magnetic Resonance Imaging (MRI) .................39 
7.8.5. 
HRQOL Measurements ..............................................................................................39 
7.9. 
Interim Analysis ..........................................................................................................40 
8. 
REFERENCES ...........................................................................................................41 
9. 
APPENDICES ............................................................................................................42 
Appendix A. Sensitivity Analysis of Primary Endpoint on Hemoglobin Response 
Using Multiple Imputation Sample Code ...................................................................42 
 
LIST OF TABLES 
Table 1: 
Summary of Major Changes in Statistical Analysis Plan Amendments.......................8 
Table 2: 
Analysis Sets for Each Endpoint ................................................................................11 
Table 3: 
Subgroup Analyses Primary and the Key Secondary Endpoints ................................30 
 
 LIST OF FIGURES 
Figure 1: 
Overview of Study Design for Study AG348-C-006 .................................................10 
Figure 2: 
Testing Strategy ..........................................................................................................14 
 
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
AE 
Adverse event 
AESI 
Adverse event of special Interest 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
ANCOVA 
Analysis of Covariance 
AST 
Aspartate Aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BID 
Twice daily 
BMI 
Body mass index 
CGIC 
Clinician Global Impression of Change 
CI 
Confidence Interval 
CMH 
Cochran-Mantel-Haenszel  
CTCAE 
Common Terminology Criteria for Adverse Events 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
EOS 
End of study 
EOT 
End of treatment 
EPO 
Erythropoietin 
EQ-5D-5L 
European quality of life five-dimensional descriptive system 
FACT-An 
Functional Assessment of Cancer Therapy-Anemia 
FAS 
Full Analysis Set 
Hb 
Hemoglobin 
HLT 
High Level Term 
HRQOL 
Health-related quality of life 
I-DMC 
Independent data monitoring committee 
IXRS 
Interactive response system 
LDH 
Lactate dehydrogenase 
LFT 
Liver function test 
LIC 
Liver Iron Concentration 
LLN 
Lower limit of normal 
LS 
Least Square  
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Abbreviation 
Definition 
Max 
Maximum  
MedDRA 
Medical Dictionary for Regulatory Activities 
MI 
Multiple imputation 
Min 
Minimum  
MMRM 
Mixed-Effect Model Repeated Measure 
MRI 
Magnetic resonance imaging 
PGIC 
Patient Global Impression of Change 
PGIS 
Patient Global Impression of Severity 
PKD 
Pyruvate kinase deficiency 
PKDD 
Pyruvate kinase deficiency diary 
PKDIA 
Pyruvate kinase deficiency impact assessment 
PKR 
Pyruvate kinase isoform R 
PPS 
Per-Protocol Set 
PRO 
Patient Reported Outcome 
PT 
Preferred Term 
QD 
Once-daily 
QOD 
Every Other Day 
QTc 
Heart-rate corrected QT interval 
QTcB 
Heart rate-corrected QT interval using the Bazett’s formula 
QTcF 
Heart rate-corrected QT interval using the Fridericia’s formula 
RBC 
Red blood cell  
ROW 
Rest of the world 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SE 
Standard error 
SF-12 
12-item Short Form Healthy Survey 
SOC 
System organ class 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
WHO 
World Health Organization 
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1. 
VERSION HISTORY 
This statistical analysis plan (SAP) describes the analysis associated with protocol AG348-C-006 
Amendment 3, Version 4.0 (dated 14 August 2019). 
Table 1: 
Summary of Major Changes in Statistical Analysis Plan Amendments 
Version 
Version Date 
Summary of Changes 
1.0 
30-Jun-2020 
Original version. 
2. 
INTRODUCTION 
This SAP provides the detailed methodology for summary and statistical analyses of the data 
collected in Study AG348-C-006 except for pharmacokinetic data which will be described in a 
separate SAP. This document may modify the plans outlined in the protocol. 
The clinical study report (CSR) will include all data up to the End of Study (EOS) for all 
subjects, which is defined as the time at which all subjects have completed the study or have 
been lost to follow-up. 
3. 
TRIAL OBJECTIVES AND ENDPOINTS 
3.1. 
Objectives 
3.1.1. 
Primary Objective 
The primary objective of the study is to evaluate the efficacy of treatment with AG-348 
compared with placebo in increasing hemoglobin (Hb) concentrations. 
3.1.2. 
Secondary Objectives 
 To evaluate the safety of AG-348  
 To determine the effect of the study treatment regimens on markers of hemolysis, 
hematopoietic activity, and other indicators of clinical activity  
 To determine the effect of the study treatment regimens on health-related quality of 
life (HRQOL), as determined using patient-reported outcomes (PROs)  
 To evaluate the pharmacokinetics of AG-348 after oral administration 
 To evaluate the relationship between AG-348 pharmacokinetics and safety 
parameters 
3.1.3. 
Exploratory Objectives 
 To evaluate the relationship of AG-348 pharmacokinetics to indicators of clinical 
activity 
 To evaluate the pharmacodynamic (PD) markers of pyruvate kinase deficiency (PK 
deficiency) and how they are affected by study treatment 
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 To determine the effect of the study treatment regimens on: 
 Number of transfusion events and number of red blood cell (RBC) units 
transfused 
 Markers of iron metabolism and indicators of iron overload 
3.2. 
Endpoints 
3.2.1. 
Primary Endpoint 
The primary endpoint is the Hb response, defined as a ≥1.5 g/dL (0.93 mmol/L) increase in Hb 
concentration from baseline that is sustained at 2 or more scheduled assessments at Weeks 16, 
20, and 24 during the Fixed Dose Period. The individual subject’s baseline Hb concentration is 
defined as the average of all available Hb concentrations collected for that subject during the 
Screening Period up to the first dose of study treatment. 
3.2.2. 
Key Secondary Endpoint 
The key secondary endpoint is the average change from baseline in Hb concentration at Weeks 
16, 20, and 24.  
3.2.3. 
Other Secondary Endpoints 
 Maximal Hb concentration increase from baseline 
 Time to first achieve an increase in Hb concentration of 1.5 g/dL (0.93 mmol/L) or 
more from baseline  
 Average change from baseline at Weeks 16, 20, and 24 in markers of hemolysis: 
bilirubin, lactate dehydrogenase (LDH), and haptoglobin levels 
 Average change from baseline at Weeks 16, 20, and 24 in markers of hematopoietic 
activity: reticulocyte percentages 
 Change from baseline in HRQOL PRO scores: Pyruvate Kinase Deficiency Diary 
(PKDD) and Pyruvate Kinase Deficiency Impact Assessment (PKDIA) 
 Safety endpoints, including: the type, incidence, severity, and relationship to study 
treatment of adverse events (AEs) and serious adverse events (SAEs); number of 
discontinuations due to AEs; results of clinical laboratory tests over time (eg, serum 
chemistry, liver function tests [LFTs], hematology, lipids, sex steroids, urinalysis, 
coagulation); physical examination findings; dual-energy x-ray absorption (DXA) 
scans; vital signs; 12-lead electrocardiogram (ECG) data 
 Pharmacokinetic endpoints, including plasma concentrations over time and 
pharmacokinetic parameters of AG-348 (eg, area under the concentration-time curve 
[AUC], maximum concentration [Cmax], others as applicable) 
 Exposure-response relationship between safety parameters and AG-348 concentration 
and relevant AG-348 pharmacokinetic parameters 
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3.2.4. 
Exploratory Endpoints 
 Exposure-response (or pharmacokinetic-pharmacodynamic) relationship between 
relevant pharmacokinetic parameters and endpoints that are indicators of clinical 
activity 
 Change from baseline in additional markers of hematopoietic activity 
 Change from baseline in markers of iron metabolism and indicators of iron overload 
 Change from baseline in RBC-specific form of pyruvate kinase (PKR) protein level 
 Relationship between baseline PKR protein level and Hb response status 
 Change from baseline in HRQOL PRO scores: European quality of life five-
dimensional descriptive system (EQ-5D-5L) 
 Change from baseline in PKR flux assay results 
 Proportion of subjects requiring transfusions and the total number of RBC units 
transfused 
4. 
STUDY DESIGN  
AG348-C-006 is a Phase 3, randomized, multicenter, double-blind, placebo-controlled study 
consisting of a Dose Optimization Period (Part 1) followed by a Fixed Dose Period (Part 2). An 
overview of the study design is provided in Figure 1. This study will evaluate the efficacy and 
safety of orally administered AG-348 (hereinafter referred to as mitapivat) as compared with 
placebo in subjects with PK deficiency who are not regularly receiving blood transfusions.  
Figure 1: 
Overview of Study Design for Study AG348-C-006 
 
Abbreviations: BID = twice daily 
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1Stratified by average of screening Hb concentrations (Hb <8.5 g/dL vs Hb ≥8.5 g/dL [5.28 mmol/L]) and the PKLR 
gene mutation category (missense/missense vs missense/non-missense). In rare instances where PKLR gene 
mutation category cannot be made definitively (eg, if a subject harbors 3 mutant PKLR alleles), the subject will be 
assigned to the missense/non missense category. 
2A subject’s Screening Period duration may be extended beyond 42 days upon the Medical Monitor’s, or designee’s, 
approval.  
5. 
ANALYSIS DATA SETS 
Only subjects who sign informed consent will be included in the analysis sets below. The 
following analysis sets will be evaluated and used for presentation of the data: 
 The Full Analysis Set (FAS) will include all subjects who are randomized. Subjects 
will be classified according to the randomized treatment arm. 
 The Safety Analysis Set will include all subjects who receive at least 1 dose of study 
treatment. Subjects will be classified according to the treatment actually received. If a 
subject randomized to placebo receives at least one dose of mitapivat then the subject 
will be classified to the mitapivat arm. 
 The Per-Protocol Set (PPS) is a subset of the FAS. Subjects who meet any of the 
following criteria will be excluded from the PPS: 
o Do not receive at least 1 dose of the randomized treatment  
o Do not have Hb assessments at Weeks 16, 20, and 24 during the Fixed Dose 
Period  
Table 2 summarizes the use of the analysis sets.  
Table 2: 
Analysis Sets for Each Endpoint 
Endpoints 
Full Analysis Set 
Per Protocol Analysis Set 
Safety Analysis Set 
Demographic and other baseline 
characteristics 
 
 
 
Disposition 
 
 
 
Major protocol deviations 
 
 
 
Exposure and concomitant therapies 
 
 
 
Efficacy   
 
 (primary endpoint only) 
 
Safety 
 
 
 
6. 
GENERAL STATISTICAL CONSIDERATIONS 
6.1. 
Randomization, Blinding, Unblinding, and Crossover 
Eligible subjects will be randomized in a 1:1 ratio to mitapivat or matching placebo. 
Randomization assignment will be implemented by an interactive response system (IXRS) and 
stratified by:  
 The average of screening Hb concentrations (<8.5, ≥8.5 g/dL [5.28 mmol/L])   
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 The PKLR gene mutation category (missense/missense, missense/nonmissense). In 
rare instances where PKLR gene mutation category cannot be made definitively (eg, 
if the subject harbors 3 mutant PKLR alleles), the subject will be assigned to the 
missense/nonmissense category. 
This is a double-blind study; the subject, Investigators, and site personnel will all be blinded to 
the subject’s study treatment assignment until database lock, if that subject is not entering the 
open label extension study AG348-C-011. If the subject expresses his/her intention to enter the 
extension study, the subject, Investigators, and site personnel will be unblinded to the subject’s 
study treatment assignment, but only after the subject completes the Week 24 assessments. The 
Sponsor study team will be blinded to study treatment assignment until the database has been 
locked. The subject, Investigators, site personnel, and Sponsor study team will not have access to 
the pharmacokinetic data until database lock.  
In the event that a subject’s treatment assignment is unblinded to the subject, Investigator, and/or 
the Sponsor study team, either accidentally or in the case of emergency unblinding, the subject 
will be allowed to continue study treatment. Although the protocol specified that data from the 
unblinded subject after unblinding would be excluded from efficacy analyses all data will be 
included in all analyses following the intention-to-treat principle, irrespective of unblinding. 
Crossover is not permitted in this study. 
6.2. 
Sample Size Determination and Decision Rules 
6.2.1. 
Sample Size Determination 
The following statistical hypothesis will be tested to address the primary objective 
H01: 𝑂𝑑𝑑𝑠 𝑅𝑎𝑡𝑖𝑜= 1 vs H11: 𝑂𝑑𝑑𝑠 𝑅𝑎𝑡𝑖𝑜≠1 
where the odds ratio of Hb response rate of the mitapivat arm over that of the placebo arm is 
tested. 
Approximately 76 subjects will be randomized in the study in a 1:1 ratio to mitapivat or matched 
placebo. Assuming a response rate of 35% in the mitapivat arm and 5% in the placebo arm, 76 
subjects (38 per arm) are needed to have 90% power to reject H01 based on a 2-sided Fisher’s 
Exact test with 0.05 significance level. The sample size calculation was performed in EAST© 
Cytel Inc. Version 6.4.   
In addition, the following statistical hypotheses will be tested to address the secondary 
objectives: 
H02: 𝛽𝑡2 −𝛽𝑐2 = 0 vs H12: 𝛽𝑡2 −𝛽𝑐2 ≠0 
H03: 𝛽𝑡3 −𝛽𝑐3 = 0 vs H13: 𝛽𝑡3 −𝛽𝑐3 ≠0 
H04: 𝛽𝑡4 −𝛽𝑐4 = 0 vs H14: 𝛽𝑡4 −𝛽𝑐4 ≠0 
H05: 𝛽𝑡5 −𝛽𝑐5 = 0 vs H15: 𝛽𝑡5 −𝛽𝑐5 ≠0 
H06: 𝛽𝑡6 −𝛽𝑐6 = 0 vs H16: 𝛽𝑡6 −𝛽𝑐6 ≠0 
H07: 𝛽𝑡7 −𝛽𝑐7 = 0 vs H17: 𝛽𝑡7 −𝛽𝑐7 ≠0 
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H08: 𝛽𝑡8 −𝛽𝑐8 = 0 vs H18: 𝛽𝑡8 −𝛽𝑐8 ≠0 
where  
𝛽𝑡2 and 𝛽𝑐2 are the average of mean change from baseline in Hb at Weeks 16, 20, and 
24 during the Fixed Dose Period in the mitapivat arm and the placebo arm, 
respectively.  
𝛽𝑡3 and 𝛽𝑐3 are the average of mean change from baseline in indirect bilirubin at 
Weeks 16, 20, and 24 during the Fixed Dose Period in the mitapivat arm and the 
placebo arm, respectively. 
𝛽𝑡4 and 𝛽𝑐4 are the average of mean change from baseline in reticulocyte percentage 
at Weeks 16, 20, and 24 during the Fixed Dose Period in the mitapivat arm and the 
placebo arm, respectively. 
𝛽𝑡5 and 𝛽𝑐5 are the average of mean change from baseline in LDH at Weeks 16, 20, 
and 24 during the Fixed Dose Period in the mitapivat arm and the placebo arm, 
respectively.  
𝛽𝑡6 and 𝛽𝑐6 are the average of mean change from baseline in haptoglobin at Weeks 
16, 20, and 24 during the Fixed Dose Period in the mitapivat arm and the placebo 
arm, respectively. 
𝛽𝑡7 and 𝛽𝑐7 are the mean change from baseline in PKDD weekly mean score at Week 
24 during the Fixed Dose Period in the mitapivat arm and the placebo arm, 
respectively. 
𝛽𝑡8 and 𝛽𝑐8 are the mean change from baseline in PKDIA score at Week 24 during 
the Fixed Dose Period in the mitapivat arm and the placebo arm, respectively. 
The sample size of 76 subjects (38 per arm) also provides >80% power to detect a difference of 
1.4 g/dL between mitapivat and placebo in the average of mean change from baseline in Hb at 
Weeks 16, 20, and 24, based on a 2-sample t-test, assuming a standard deviation (SD) of 1.5 
g/dL (98% power) or 2.1 g/dL (81% power). 
To control the overall type I error in the study at the 2-sided 5% level, the fixed sequence testing 
procedure (Mauer, 1995) will be used to adjust for multiple statistical testing of the primary and 
secondary efficacy endpoints. These endpoints will be tested following the testing strategy 
outlined in Figure 2. 
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Figure 2: 
Testing Strategy  
 
Abbreviations: CMH = Cochran-Mantel-Haenszel test; MMRM = Mixed-Effect Model Repeated Measure; PKDD = 
Pyruvate Kinase Deficiency Diary; PKDIA = Pyruvate Kinase Deficiency Impact Assessment.  
For PKDD, PKDIA, LDH, and haptoglobin endpoints, the Hochberg testing procedure 
(Hochberg, 1988) will be used with 2-sided α=0.05 if all 4 endpoints higher in the hierarchy 
achieved statistical significance. Four 2-sided p-values for PKDD, PKDIA, LDH, and 
haptoglobin endpoints will be ordered from largest to smallest and compared to a set of critical 
values. The comparison will be conducted sequentially by comparing the largest p-value to α, 
then the second-largest p-value to α/2, then the third largest p-value to α/3, and the fourth largest 
p-value to α/4, until a p-value for an endpoint is smaller than the corresponding critical value, 
whereupon the Hochberg procedure provides a conclusion of statistically significant effect of 
mitapivat compared to placebo for that endpoint and all endpoints with smaller p-values. 
No interim analyses for efficacy are planned. 
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6.2.2. 
Decision Rules 
The study will have demonstrated that mitapivat is statistically significantly superior to placebo 
if the 2-sided p-value for Hb response is <0.05 in favor of the mitapivat arm.  
To protect the integrity of the study and to preserve the type I error at or below 2-sided 
α=0.05, the testing strategy outlined in Section 6.2.1 will be followed and statistical significance 
will be achieved for mitapivat compared to placebo for each secondary endpoint that can be 
tested per this strategy at the associated significance level.  
6.3. 
Definitions 
6.3.1. 
Study Drug and Study Treatment 
Both study drug and study treatment are defined as mitapivat or matched placebo.  
6.3.2. 
Start and End Dates of Study Treatment 
The start of study treatment (mitapivat or matched placebo) is the earliest date/time of 
administration of a non-zero dose of the study treatment.  
The end of study treatment (mitapivat or matched placebo) is the latest date/time of 
administration of a non-zero dose of the study treatment on or before the EOS date. 
6.3.3. 
Study Day 
The study day for assessments or events occurring on or after the start of study treatment (eg, AE 
onset, laboratory assessment) will be calculated as: 
Study day = Date of the assessment or event − start of study treatment + 1.  
The study day for assessments occurring before the start of study treatment (eg, lab assessment 
during the Screening Period, medical history) will be negative and calculated as: 
Study day = Date of the assessment or event − start of study treatment. 
There is no study day 0. The study day will be displayed in data listings.  
6.3.4. 
Baseline 
Efficacy Evaluations  
For efficacy and exploratory laboratory parameters [Hb, hemolysis markers (indirect bilirubin, 
LDH, haptoglobin), reticulocyte percentage, iron markers, and erythropoietic markers], baseline 
is defined as the average of all screening assessments within 45 (42+3) days before the date of 
randomization (including assessments on the date of randomization) for subjects randomized and 
not dosed or before the start of study treatment for subjects randomized and dosed. Assessments 
collected within 61 days after a transfusion will be excluded from the baseline derivation.  
Baseline for efficacy laboratory parameters will be derived based on central laboratory data; if no 
central laboratory data are available before the start of study treatment, then local laboratory data 
will be used to derive the baseline.  
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For HRQOL assessments (except for PKDD), the last measurement before the start of study 
treatment will be used as the baseline. For PKDD, baseline of weekly mean score is defined as 
the average of daily scores collected within 7 days before the start of study treatment. 
Baseline Characteristics 
For summaries of baseline characteristics based on the FAS, baseline will be defined as follows: 
 For subjects randomized and not dosed: the last assessment on or before the date of 
randomization  
 For subjects randomized and dosed: the last assessment on or before the start of study 
treatment 
Safety Evaluations 
For alanine aminotransferase (ALT) and aspartate aminotransferase (AST), baseline is defined as 
the average of all screening assessments collected within 45 (42+3) days before the start of study 
treatment. Baseline will be derived based on central laboratory data; if no central laboratory data 
are available before the start of study treatment, then local laboratory data will be used to derive 
the baseline.  
For other laboratory assessments: 
 Prior to deriving the baseline, 
o  If there are multiple records with the same assessment day and time from the 
same laboratory, the average value will be used  
o If there are multiple records with the same assessment day and time from different 
laboratories, the value from the central laboratory will be used   
 The baseline will then be the last value on or before the start of study treatment. 
Triplicate ECGs are collected in the study; the baseline for each ECG measurement is the 
average of the last predose replicate measurements on or before the start of study treatment. 
Unscheduled assessments will not be included in the calculation of the average. QTcP will be 
derived based on RR and QT. The average of the replicate measurements will be determined 
after the derivation of the individual parameter at each time point.  
For all other safety parameters, the last assessment on or before the start of study treatment will 
be used as the baseline.   
If, per protocol, an assessment (efficacy, baseline characteristic, or safety) is to be performed on 
study day 1, before the first dose of study treatment, and the assessment time, time of first dose 
of study treatment, or both, is missing (or not collected), it will be assumed that the assessment is 
performed before study treatment administration. Unscheduled assessments will be used in the 
determination of baseline; however, an unscheduled assessment on study day 1 will be 
considered to have been obtained after study treatment administration. 
If no assessment meets the definition of baseline for an evaluation (efficacy, baseline 
characteristic, or safety), the baseline will be set to missing. 
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6.3.5. 
On-Treatment Period and Optimized Dose 
The on-treatment period starts on the date of start of study treatment and ends 28 days after the 
end of study treatment. 
Within the on-treatment period the following dosing periods are defined: 
 Dose Optimization Period starts on the date of start of study treatment and ends on 
the date of the dose administered at the Week 12 Visit or the earliest date of EOS, the 
end of the on-treatment period, and the first day of the dose taper prescription if a 
subject discontinued the study before reaching the Week 12 Visit 
 Fixed Dose Period starts 1 day after the end of the Dose Optimization Period and 
ends on the first day of the dose taper prescription if the subject enters the dose taper 
period or the date 4 days after the end of study treatment otherwise 
 Dose Taper Period starts 1 day after the first day of the dose taper prescription and 
ends on the date of the earlier date of EOS and the end of on-treatment period. This 
period is only applicable to subjects who went through the dose taper 
Data listings will include all assessments and events, with those that occur outside of the 
on-treatment period flagged. 
The optimized dose is defined as the dose prescribed at the Week 12 visit (5 mg BID, 20 mg 
BID, or 50 mg BID) or the last prescribed dose if the subject discontinues study drug before the 
Week 12 visit.  
6.4. 
General Methods 
6.4.1. 
Data Handling After Cutoff Date 
Not applicable. 
6.4.2. 
Standard Derivations and Reporting Conventions 
The following conversion factors will be used to convert days into weeks, months, or years:   
1 week=7 days, 1 month=30.4375 days, and 1 year=365.25 days. 
The following derivations will be implemented: 
 Age (years)=(year of given informed consent − year of birth), since only year of birth 
is collected in the eCRF.  
The integer part of the calculated age will be used for reporting purposes.  
 Body mass index (BMI; kg/m2)=weight (kg)/height (m)2 
 Duration (in days) from a reference date (eg, randomization date, start date of study 
treatment) = 
o date of event − reference date + 1, if the date of the event is on or after the 
reference date  
o date of event − reference date, if the date of the event is before the reference date 
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Reporting conventions will be as follows:  
 Mean and median will be displayed to one more decimal place than the raw data. 
 SD will be displayed to two more decimal places than the raw data. 
 Percentages will be displayed to 1 decimal place (however, percentages 
corresponding to 0 counts will be reported as 0 rather than 0.0 and 100 percent will be 
reported as 100 rather than 100.0). 
 p-values will be reported with 4 decimal places; all p-values should be specified to be 
1-sided or 2-sided. 
 Unless otherwise specified, rounding will be performed to the closest integer / first 
decimal using the common mid-point between the two consecutive values, eg, 5.11 to 
5.14 will be rounded to 5.1, and 5.15 to 5.19 will be rounded to 5.2. 
o Non-zero percentages that are < 0.1 before rounding will be displayed as “< 0.1”, 
eg, 0.09 will be reported as < 0.1 rather than as 0.1. 
o P-values <0.0001 before rounding will be displayed as “<0.0001”, eg, a p-value 
of 0.00009 will be displayed as <0.0001 rather than as 0.0001. 
6.4.3. 
Pooling of Data Across Sites 
In order to provide overall estimates of treatment effects, data will be pooled across sites. The 
“site” factor will not be considered in statistical models or subgroup analyses given the high 
number of participating sites in contrast to the anticipated small number of subjects randomized 
at each site. 
6.4.4. 
Continuous and Categorical Variables 
Continuous variables will be summarized using descriptive statistics, ie, number of non-missing 
values, mean, SD, median, quartiles, minimum, and maximum. Time-to-event endpoints in the 
presence of censoring will be estimated using Kaplan-Meier (KM) methodology. 
Categorical variables will be summarized by frequency distributions (number and percentage of 
subjects within a given category in the analysis data set). Unless otherwise specified, the 
calculation of percentages will include the “missing” category. Therefore, counts of missing 
observations will be included in the denominator and presented as a separate category. For 
summaries by visit, percentages will be based on the number of subjects with data available for 
that visit, unless otherwise specified. 
6.4.5. 
Unscheduled Visits 
Generally, data collected at unscheduled visits will be included and summarized for both safety 
and efficacy analyses in the same manner as the data collected at scheduled visits. Data collected 
at unscheduled visits will be included in by-subject listings together with the data collected at 
scheduled visits.  
Summaries of outliers (eg, worst value, worst change from baseline, worst Common 
Terminology Criteria for Adverse Events [CTCAE] grade) during the on-treatment period for 
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safety endpoints such as laboratory measurements and ECG parameters will include data from 
both scheduled and unscheduled visits. 
Individual longitudinal plots for laboratory measurements during the on-treatment period will 
include data from both scheduled and unscheduled visits.   
Descriptive statistics (mean, SD, median, quartiles, minimum, maximum) by nominal visit will 
only be provided for DXA scans results and liver iron concentrations (LIC) by MRI. Data 
collected at unscheduled and scheduled postbaseline visits will be mapped to scheduled visits 
using analysis visit windows, and then values at scheduled postbaseline visits will be derived 
based on the rules described below. 
For efficacy and exploratory endpoints [Hb, hemolysis markers (indirect bilirubin, LDH, and 
haptoglobin), reticulocyte percentage, iron markers, erythropoietic markers, and HRQOL], data 
collected at unscheduled and scheduled postbaseline visits will be mapped to scheduled visits 
using analysis visit windows, and then values at scheduled postbaseline visits will be derived 
based on the rules described below. Descriptive statistics by nominal visit and longitudinal plots 
during the on-treatment period for efficacy endpoints such as Hb concentration will be provided 
using the derived values at scheduled visits.  
Analysis Visit Windows  
For the evaluation of Hb, hemolysis markers (indirect bilirubin, LDH, and haptoglobin), 
reticulocyte percentage, iron markers, erythropoietic markers, and HRQOL endpoints, the 
analysis visit windows will be derived based on the target study day for the scheduled visits as 
follows. Note that based on the scheduled of assessments, a Week 4 Visit, for example, will have 
a target study day of 1+(4×7)=29. 
 Visit windows will be implemented for scheduled visits after Day 1. 
 For analysis visit(n):  
o Start day of visit window =1+end day of window for visit(n−1). If n=1, start day 
of the visit window is study day 2  
o End day of visit window = [(target day for analysis visit(n) + target day for 
analysis visit(n+1))/2]–1 for Week < 24. The end day of visit window at Week 24 
is the end of the Fixed Dose Period for Hb, hemolysis markers (indirect bilirubin, 
LDH, and haptoglobin), reticulocyte percentage, iron markers, erythropoietic 
markers, and HRQOL endpoints 
 For DXA scan results and LIC by MRI, the analysis visit window for the Week 24 
Visit will start on study day 86 and end on the EOS. Results outside this window will 
not be summarized. 
Derivation of Values at Scheduled Postbaseline Visits Based on Analysis Visit Windows 
For efficacy laboratory parameters (Hb, hemolysis markers, reticulocyte percentage, iron 
markers, and erythropoietic markers), any assessments obtained within 61 days after a 
transfusion will be excluded. In addition: 
 Central laboratory assessment(s) (scheduled or unscheduled) within the visit windows 
will be used 
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 If no central laboratory value is within the visit window, local laboratory 
assessment(s) within the visit window will be used 
For HRQOL data, assessments (scheduled or unscheduled) within the visit windows and on or 
before the end of the Fixed Dose Period will be used.  
If multiple assessments are identified within a visit window for a parameter, the following rules 
will be applied: 
 The assessment measured closest to the target study day of the scheduled visit will be 
used  
 If there are multiple assessments equidistant to the target study day  
o the average value will be used for efficacy laboratory parameters 
o the later assessment will be used for HRQOL endpoints 
6.5. 
Methods for Handling Missing Data 
6.5.1. 
Adverse Event and Concomitant Medication Start Dates 
If the end date is non-missing and the imputed start date is after the end date, the end date will be 
used as the start date. 
(1) Missing day only 
 If the month and year are the same as the month and year of the date of the start of 
study treatment, the date of the start of study treatment will be used. 
 If the month and year are before the month and year of the date of the start of study 
treatment, the last day of the month will be used. 
 If the month and year are after the month and year of the date of the start of study 
treatment, the first day of the month will be used. 
(2) Missing day and month 
 If the year is the same as the year of the date of the start of study treatment, the date 
of the start of study treatment will be used. 
 If the year is before the year of the date of the start of study treatment, 31 December 
will be used. 
 If the year is after the year of the date of the start of study treatment, 01 January will 
be used. 
(3) Missing day, month, and year 
 The date of the start of study treatment will be used. 
6.5.2. 
Adverse Event and Concomitant Medication End Dates 
If the start date is non-missing and the imputed end date is before the start date, the start date will 
be used as the end date. If an imputation for an AE end date results in an AE end date that is after 
the EOS date, the AE will be considered as ongoing at the EOS date. 
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(1) Missing day only 
 The last day of the month will be used. 
(2) Missing day and month 
 31 December will be used. 
(3) Missing day, month, and year 
 The event will be regarded as ongoing. 
6.5.3. 
Exposure 
No imputation will be done for the date of the first dose of study drug. 
If the date of the last dose of study drug is missing or partially missing, it will be imputed as 
follows (separately for each study drug): 
 If the last date of study drug is completely missing and there is no End of Treatment 
Disposition eCRF page for the study drug AND there is no death date, the subject 
should be considered to be ongoing and the EOS date for the analysis will be used as 
the last dosing date. 
 If the last date of study drug is completely or partially missing and there is EITHER 
an End of Treatment Disposition eCRF page for the study drug OR a death date (on 
or before the EOS date, then the imputed last dose date is: 
= Last day of the year, if only the year is available and Year < Year of min (EOT 
date, death date)  
= Last day of the month, if both the year and month are available and Year = Year of 
min (EOT date, death date) and Month < Month of min (EOT date, death date) 
= min (EOT date, death date), for all other cases 
7. 
STATISTICAL ANALYSES 
7.1. 
Subject Disposition 
For all subjects screened in the study, the following will be summarized: 
 Number of subjects screened in the study  
 Frequency (number and percentage) of subjects who discontinued the study before 
randomization, overall and by reason for discontinuation. Percentages will be 
calculated based on the number of subjects screened in the study.    
In addition, the frequency of subjects in each of the analysis sets described in Section 5 will be 
summarized by treatment arm. Percentages will be calculated only for analysis sets that are a 
subset of the FAS or a subset of the safety analysis set. 
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The following summaries will be presented by treatment arm based on the FAS:  
 Frequency of subjects in each randomization strata and combination of randomization 
strata (per IXRS)  
 Frequency of subjects randomized in each geographic region, country, and site 
 Frequency of subjects randomized but not treated, overall and by reason for 
discontinuation 
 Frequency of subjects with study drug ongoing 
 Frequency of subjects who discontinued study drug, overall and by the reason for 
discontinuation of study drug 
 Frequency of subjects who completed the study 
 Frequency of subjects ongoing in the study 
 Frequency of subjects who discontinued the study, overall and by the reason for study 
discontinuation 
The frequency of subjects with disposition reason, in each epoch, due to reasons associated with 
COVID-19 will further be summarized under the main reason for discontinuation. 
In addition, a cross-tabulation of subjects randomized (mitapivat, matched placebo, none) vs 
subjects who have received at least one dose of study drug (mitapivat, matched placebo, none) 
will be presented.  
Disposition for all screened subjects and randomization data will be provided in by-subject 
listings. 
7.2. 
Protocol Deviations 
All major protocol deviations that impact the safety of the subjects, the conduct of the study, or 
the evaluation of the study results will be reported by treatment arm based on the FAS. These 
will include: 
 Subjects randomized despite not satisfying the eligibility criteria 
 Subjects who develop withdrawal criteria whilst on the study but are not withdrawn 
 Subjects who receive a study drug different from that assigned at randomization  
 Subjects who are randomized under the wrong stratification factor(s) 
 Subjects who receive an excluded concomitant medication 
In addition, for each category of major protocol deviations, those related to COVID-19 will be 
summarized. 
Major protocol deviations will be provided in a by-subject listing. 
7.3. 
Demographic and Other Baseline Characteristics 
The following summaries will be presented by treatment arm, and overall based on the FAS, 
unless otherwise specified. 
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7.3.1. 
Demographics and Physical Measurements 
Demographic characteristics and physical measurements at baseline will be summarized as 
follows: 
 Demographic characteristics  
o Sex: Male, Female (child bearing potential status will be summarized for female 
subjects) 
o Race: American Indian or Alaska Native, Asian, Black or African American, 
Native Hawaiian or Other Pacific Islander, White, Other, Unknown 
o Ethnic origin: Hispanic or Latino, Not Hispanic or Latino, Not reported 
o Age (years): summary statistics  
o Age categories:  
 <65, ≥65 years 
 <35, ≥35 years 
 Physical measurements 
o Height (cm) 
o Weight (kg) 
o BMI (kg/m2) 
Demographic data for all screened subjects will be provided in a by-subject listing.  
7.3.2. 
Disease Characteristics 
The following baseline characteristics of the underlying disease will be summarized based on the 
data entered in the eCRF:  
 Baseline Hb concentration [both continuously and by categories (<8.5 g/dL, ≥8.5 
g/dL)] 
 Prior splenectomy status (Yes, No; if Yes, age of splenectomy) 
 Prior chelation status (Yes, No); the status is “Yes” if a subject has received chelation 
therapy within 52 weeks (364 days) prior to the first dose of study treatment. 
 Prior cholecystectomy status (Yes, No; if Yes, age of cholecystectomy) 
 DXA scan results by location (femoral total and adjusted spine): Bone mineral 
density (BMD) and corresponding T-scores and Z-scores. Frequency of subjects with 
T-scores in 3 categories (≤−2.5, >−2.5-<−1.0, ≥−1.0) 
Disease characteristics will be provided in by-subject listings. 
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7.3.3. 
Medical History 
Medical and surgical history will be summarized in frequency tabulations according to the 
Medical Dictionary for Regulatory Activities (MedDRA) latest version by System Organ Class 
(SOC) and Preferred Term (PT).  
Medical history and surgical history of splenectomy will be provided in by-subject listings.    
7.3.4. 
Prior Therapies 
The following summaries will be presented by treatment arm and overall based on the safety 
analysis set. 
Prior medications are defined as medications (from the Prior and Concomitant Medications 
eCRF) that are started before the start of study treatment.  
All non-study medications will be coded according to the Anatomical Therapeutic Chemical 
(ATC) code and PT using the latest version of the World Health Organization (WHO) Drug 
Dictionary. All prior medications will be summarized in frequency tabulations according to the 
WHO ATC third level and PT.  
Prior medications will be provided in a by-subject listing. 
Prior transfusions are collected from the Transfusion History eCRF.  
Transfusion history will be provided in a by-subject listing.  
7.4. 
Exposure to Study Drug and Compliance 
7.4.1. 
Treatment Duration and Exposure 
The frequency of subjects with an optimized dose of 5 mg BID, 20 mg BID or 50 mg BID will 
be summarized by treatment arm. 
Duration of exposure will be summarized as a continuous variable as well as in categories 
(>0-≤4, >4-≤8, >8-≤12, >12-≤20, >20-≤24, and >24 weeks), where  
Duration of exposure = end date of study drug – start date of study drug +1. 
Study drug compliance will be summarized based on percentage of tablets taken, where  
 Percentage of tablets taken = 100×(total number of tablets administered)/(total 
number of tablets intended) 
 Total number of tablets administered = total number of tablets dispensed − tablets 
returned 
 Duration of prescription = end date of prescription – start date of prescription +1 
 Number of tablets intended during each prescription: for each new prescription, 
prescribed dosing frequency × duration of the prescription ×3. Prescribed dosing 
frequency takes value of 0.5, 1, and 2 for every other day (QOD), once daily (QD) 
and twice daily (BID), respectively. 
 Total number of tablets intended = sum of number of tablets intended over all 
prescriptions. 
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Percentage of tablets taken will be summarized by treatment arm. The frequency of subjects 
whose compliance is <80%, 80-100%, >100-120%, and >120% will be summarized. 
7.4.2. 
Dose Modifications 
The summary of dose modifications will include:  
 The frequency of subjects with at least 1 dose reduction  
 Summary of reasons for dose reduction  
Dose reduction is defined as the prescribed dose being decreased from the previous dose 
collected in the Prescribed Dose eCRF. Dose prescriptions and modifications will be provided in 
a by-subject listing.  
7.5. 
Concomitant Therapies 
The following summaries will be presented by treatment arm based on the safety analysis set. 
Concomitant medications are defined as non-study medications (from the Prior and 
Concomitant Medications eCRF) that are started during the on-treatment period or are started 
before the start of the study treatment and end or remain ongoing during the on-treatment period.  
All non-study medications will be coded according to ATC code and PT using the latest version 
of the WHO Drug Dictionary. All concomitant medications will be summarized in frequency 
tabulations according to WHO ATC third level and PT. 
Concomitant procedures are defined as procedures (from the Prior and Concomitant 
Procedures eCRF) that are started during the on-treatment period or are started before the start of 
the study treatment and end or remain ongoing during the on-treatment period. 
The concomitant procedures will be coded by the latest version of MedDRA by SOC and PT and 
will be summarized in frequency tabulations by SOC and PT.  
Concomitant transfusions are collected in the “On Study Transfusions” eCRF page.  
Concomitant transfusions will be provided in a by-subject listing.  
7.6. 
Efficacy Analyses 
The following analyses will be based on the FAS using the IXRS randomization stratification 
factors, unless otherwise specified.  
7.6.1. 
Primary Endpoint 
The primary endpoint is Hb Response, defined as a ≥1.5 g/dL increase in Hb concentration from 
baseline that is sustained at 2 or more scheduled assessments at Weeks 16, 20, and 24 during the 
Fixed Dose Period. Subjects with missing Hb assessment(s) over Weeks 16, 20, and 24 and who 
did not obtain 2 assessments with ≥1.5 g/dL increase from baseline will be considered as non-
responders. The details for derivation of Hb at baseline and postbaseline visits are provided in 
Sections 6.3.4 and 6.4.5. 
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7.6.1.1. 
Primary Analysis 
Subjects' Hb response status (Yes vs No) will be analyzed using a logistic regression model. The 
model includes treatment as an independent variable and adjusts for the randomization 
stratification factors. The estimated odds ratio between the mitapivat arm and the placebo arm 
with the corresponding 95% confidence interval (CI) and the 2-sided p-value will be provided.  
If the logistic regression model fails to converge, or its maximum likelihood estimate does not 
exist due to quasi-complete separation, the non-parametric CMH test will be used for the primary 
analysis. 
The frequency of subjects with Hb response (Hb responders) will be presented by treatment arm, 
and within each treatment arm by optimized dose. If the number of subjects with an optimized 
dose is small (<5% of the randomized subjects), the results by optimized dose may not be 
interpretable.  
7.6.1.2. 
Sensitivity Analyses 
Cochran-Mantel-Haenszel Test 
Hb response rate will be compared between the mitapivat arm and the placebo arm based on the 
CMH test without continuity correction and based on the randomization stratification factors. 
The common odds ratio will be provided with the corresponding 95% CI and the 2-sided p-value. 
Logistic Regression based on PPS 
The primary analysis will be repeated based on the PPS.  
Multiple Imputation (MI) 
The Hb response status is indeterminate if a subject has missing Hb assessment(s) over weeks 
16, 20, and 24 in the following scenarios:  
 Missing 1 visit and only 1 out of the 2 remaining assessments has ≥1.5 g/dL change 
from baseline; or 
 Missing ≥2 visits 
In the primary analysis, these subjects will be considered non-responders. If >10% of 
randomized subjects are non-responders due to missing Hb assessments, a sensitivity analysis 
based on multiple imputation (MI) will be performed. 
Missing Hb concentrations at Weeks 16, 20, and 24 will be multiply imputed for each treatment 
arm using the Markov Chain Monte Carlo (MCMC) method following the missing-at-random 
(MAR) assumption. In the imputation model, treatment arm, the randomization stratification 
factors, baseline Hb, and all scheduled postbaseline visits will be included. One hundred imputed 
datasets will be generated using the SAS PROC MI procedure. Hb response rate will be 
compared between the mitapivat arm and the placebo arm in each imputed dataset using the 
CMH test. CMH statistics will be normalized using the Wilson-Hilferty transformation (Wilson, 
1931) (Goria, 1992), and then combined following Rubin’s rules (Rubin, 1987) to generate a p-
value for the combined CMH test. Sample code is provided in Appendix A (Ratitch B., 2013).  
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7.6.2. 
Key Secondary Endpoint 
7.6.2.1. 
Definition 
The key secondary endpoint is the average change from baseline in Hb concentration at 
Weeks 16, 20, and 24 during the Fixed Dose Period.  
7.6.2.2. 
Primary Analysis 
The average change from baseline in Hb concentrations at Week 16, 20, and 24 will be compared 
between the mitapivat arm and the placebo arm by the Mixed-effect Model Repeat Measurement 
(MMRM) method. The model will include change from baseline in Hb as the dependent 
variable; baseline Hb as a covariate, treatment, visit, and treatment-by-visit interaction as fixed 
factors; and subject as the random effect with adjustment for the randomization stratification 
factors.  
The MMRM model will be based on the restricted maximum likelihood method assuming an 
unstructured covariance structure to model the within-subject errors. If the model does not 
convergence, the compound symmetry structure will be used instead. A Kenward-Roger 
approximation will be used for the denominator degrees of freedom. 
The estimated treatment difference between the mitapivat arm and the placebo arm in the 
average change from baseline at Weeks 16, 20, and 24 based on the LS Means will be provided 
with 95% CI and the 2-sided p-value. The estimated change from baseline for each treatment arm 
and the estimated treatment difference at each visit will be provided with 95% CI. Two-sided p-
values will be provided for the estimated treatment difference at each visit. A longitudinal plot of 
estimated change from baseline (+/-SE) at each visit by treatment arm (mitapivat vs Placebo) 
will be provided. 
In addition, the following summaries and figures will be provided based on Hb data collected up 
to the end of the Fixed Dose Period:  
 Summary of Hb concentrations by visit for each treatment arm 
 Summary of Hb data by visit and prior splenectomy status for each treatment arm 
 A waterfall plot of the subject-level average Hb over Weeks 16, 20, and 24 with 
treatment arm and optimized dose, PKLR gene mutation category, baseline Hb 
concentration (<8.5 g/dL, ≥8.5 g/dL), and Hb response status indicated for each 
subject. 
7.6.2.3. 
Sensitivity Analysis 
Although the primary analysis using the MMRM model does not use formal imputation for 
missing Hb concentrations at Weeks 16, 20, and 24, it assumes missing at random (MAR) when 
handling missing data. To evaluate the robustness of the results, an ANCOVA model will be 
fitted with the average change from baseline in Hb at Weeks 16, 20 and 24 as the response 
variable; fixed effects include baseline Hb concentration, treatment arm and the randomization 
stratification factors. The estimated treatment difference between the mitapivat arm and the 
placebo arm will be provided with the 95% CIs and the 2-sided p-values. 
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7.6.3. 
Additional Secondary Efficacy Endpoints 
By-subject longitudinal plots will be presented with Hb concentrations, hemolysis markers, and 
reticulocyte percentage at baseline and scheduled visits, and prescribed dose over time; the plots 
will further include treatment arm and optimized dose, age, sex, race, PKLR gene mutation 
category, baseline Hb concentration, prior splenectomy status, baseline chelation status, and 
postbaseline chelation status. 
7.6.3.1. 
Maximum Hb Change from Baseline   
Maximum Hb change from baseline up to the end of the Fixed Dose Period will be summarized 
by treatment arm. 
A waterfall plot of the subject-level maximum Hb change from baseline will be provided with 
treatment arm and optimized dose, PKLR gene mutation category, baseline Hb concentration 
(<8.5 g/dL, ≥8.5 g/dL) and Hb response status indicated for each subject; reference lines will be 
included at 1.5 g/dL and 1.0 g/dL.   
7.6.3.2. 
Hemolysis Markers 
Each hemolysis marker (indirect bilirubin, LDH, and haptoglobin) will be analyzed using the 
same method as described for Hb in Section 7.6.2.2 The estimated treatment difference between 
the mitapivat arm and the placebo arm in the average change from baseline at Weeks 16, 20, and 
24 based on the LS Means will be provided with 95% CI and the 2-sided p-value. The estimated 
change from baseline for each treatment arm and the estimated treatment difference at each visit 
will be provided with 95% CI. Two-sided p-values will be provided for the estimated treatment 
difference at each visit. A longitudinal plot of estimated change from baseline (+/-SE) at each 
visit will be provided for each treatment arm. 
In addition, for each hemolysis marker, the following summaries and figures will be provided 
based on data collected up to the end of the Fixed Dose Period:  
 Summary of data by visit for each treatment arm 
 Summary of data by visit and prior splenectomy status for each treatment arm  
 A longitudinal plot of mean value (+/-SD) at each visit by treatment arm and prior 
splenectomy status 
 Summary of data by visit and baseline Hb concentration (<8.5 g/dL, ≥ 8.5 g/dL) for 
each treatment arm 
 A longitudinal plot of mean value (+/-SD) at each visit by treatment arm and baseline 
Hb concentration (<8.5 g/dL, ≥ 8.5 g/dL) 
7.6.3.3. 
Hematopoietic Activity Marker 
The hematopoietic activity marker reticulocyte percentage will be analyzed using the same 
methodology as described for Hb in Section 7.6.2.2.  
The summaries and figures listed for hemolysis markers in Section 7.6.3.2. will also be provided 
for reticulocyte percentage.  
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7.6.3.4. 
Pyruvate Kinase Deficiency Diary (PKDD) 
PKDD is a self-administered, daily, 7-item PRO measure of the core signs and symptoms of 
PKD in adults. The PKDD daily scores will be calculated and provided by the instrument 
developer (Endpoint Outcomes) based on subjects’ response to the PKDD questionnaire. For 
summary and analysis, the daily PKDD scores summarized into weekly mean scores.  
The change from baseline in PKDD weekly mean score at Week 24 will be evaluated and 
compared between the mitapivat arm and the placebo arm using the MMRM method. The model 
will include change from baseline in weekly mean scores as the dependent variable, baseline 
PKDD weekly mean score as a covariate, treatment, week, treatment-by-week interactions, and 
the stratification factors as fixed factors; and subject as the random effect. 
The MMRM model will be based on the restricted maximum likelihood method assuming an 
unstructured covariance structure to model the within-subject errors. If the model has 
convergence issue, the compound symmetry structure will be used instead. A Kenward-Roger 
approximation will be used for the denominator degrees of freedom. 
The estimated treatment effect of change from baseline in PKDD weekly mean score for each 
arm and the treatment difference between the mitapivat arm and the placebo arm at each week 
will be provided with corresponding 95% CIs. Two-sided p-values will be provided for the 
estimated treatment difference at each week.  
In addition, PKDD weekly mean scores up to the end of the Fixed Dose Period will be 
summarized by treatment arm at each visit. 
7.6.3.5. 
Pyruvate Kinase Deficiency Impact Assessment (PKDIA) 
PKDIA is a 12-item PRO measure of the impacts of PKD experienced by adults. The PKDIA 
score at each visit will be calculated and provided by the instrument developer (Endpoint 
Outcomes) based on subjects’ response to the PKDIA questionnaire.  
The change from baseline in PKDIA score at Week 24 will be evaluated and compared between 
the mitapivat arm and the placebo arm using the MMRM method. The model will include change 
from baseline in PKDIA score as the dependent variable, baseline PKDIA score as a covariate, 
treatment arm, visit, treatment-by-visit interaction, and the randomization stratification factors as 
fixed factors, and subject as the random effect. 
The MMRM model will be based on the restricted maximum likelihood method assuming an 
unstructured covariance structure to model the within-subject errors. If the model has 
convergence issue, the compound symmetry structure will be used instead. A Kenward-Roger 
approximation will be used for the denominator degrees of freedom. 
The estimated treatment effect of change from baseline in PKDIA score for each treatment arm 
and the treatment difference between the mitapivat arm and the placebo arm at each week will be 
provided with corresponding 95% CIs. Two-sided p-values will be provided for the estimated 
treatment difference at each visit.  
In addition, the following summaries will be provided for PKDIA scores up to the end of the 
Fixed Dose Period by treatment arm at each visit, including: 
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 Summaries of scores for each item. For questions 9a, 11a and 12, the frequency of 
subjects with answers in each category will be summarized. For questions 9b and 
11b, scores from patients who answered “Yes” will be summarized. For all the other 
questions, response will be treated as continuous variables and summarized   
 PKDIA score and change from baseline 
7.6.4. 
Subgroup Analyses 
Subgroup analyses to be performed for the primary endpoint (Hb response) and the key 
secondary endpoint (average change from baseline in Hb at Weeks 16, 20, and 24) based on the 
FAS are presented in Table 3. 
 Table 3: 
Subgroup Analyses Primary and the Key Secondary Endpoints  
Subgroup  
Categories 
Randomization stratification factor (per 
IXRS): Average of screening Hb 
concentration 
<8.5 g/dL, ≥8.5 g/dL 
Randomization stratification factor (per 
IXRS): PKLR gene mutation  
Missense/Missense, Missense/Non-missense 
Baseline Hb concentration 
<8.5 g/dL, ≥8.5 g/dL 
Age at screening  
<35, ≥35 years 
Sex  
Female, Male 
Race  
White, Asian, Black or African American, Other (Other includes 
American Indian or Alaska Native, Native Hawaiian or other 
Pacific Islander, and not reported) 
Geographic region 
North America, Western Europe, ROW 
Prior splenectomy status 
Yes, No 
Prior cholecystectomy status 
Yes, No 
Prior chelation status  
Yes, No 
Efficacy analyses in subgroups will be purely exploratory and are intended to evaluate the 
consistency of treatment effect. If there is a low number of subjects within a category (≤10% of 
the subjects in the FAS), the categories will be pooled (if 3 or more categories are pre-specified 
for the subgroup) or the subgroup will not be analyzed (if only 2 pre-specified categories in the 
subgroup). 
For each category within each subgroup the following unstratified analyses will be performed 
and presented in a forest plot, separately for the primary endpoint and the key secondary 
endpoint: 
 Hb response will be summarized for each treatment arm (number of responders and 
response rate), and the odds ratio and 95% exact confidence interval will be provided.  
 The average of change from baseline in Hb at Weeks 16, 20 and 24 during the Fixed 
Dose Period will be summarized for each treatment arm using LS means and SE, and 
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the difference between the mitapivat arm and the placebo arm will be presented with 
the associated 95% CI based on the MMRM model.  
7.7. 
Safety Analysis 
Summaries of safety data will be presented by treatment arm based on the safety analysis set.  
7.7.1. 
Adverse Events  
Treatment-emergent adverse events (TEAEs) are AEs with a first onset date during the on-
treatment period or worsening from baseline. All summaries described below will be based on 
TEAEs, if not otherwise specified.  
All AEs will be listed by subject and AEs with onset outside of the on-treatment period will be 
flagged in the listings. Unless otherwise specified, TEAEs will be summarized according to the 
latest version of MedDRA by SOC and/or PT, severity (based on CTCAE v4.03 grading), 
seriousness, and relation to study treatment in decreasing frequency based on the frequencies 
observed for the mitapivat arm.  
Each subject will be counted only once within each SOC or PT. If a subject experiences multiple 
TEAEs under the same PT within a SOC for the same summary period, only the TEAE assessed 
as related or with the worst severity, as applicable, will be included in the summaries of 
relationship and severity. If a subject has TEAEs with missing and non-missing grades, the 
maximum of the non-missing grades will be displayed. No imputation of missing grades will be 
performed. 
The following will be summarized: 
 TEAEs by SOC and PT 
 TEAEs by SOC, PT, and worst grade 
 Most common TEAEs and Grade ≥3 TEAEs by PT; these will include TEAEs (any 
grade) reported in ≥10% of subjects in either treatment arm or Grade ≥3 TEAEs 
reported in ≥5% of subjects in either treatment arm. These thresholds may be changed 
based on the observed data without an amendment to this SAP. 
 Treatment-related TEAEs by SOC and PT  
 Treatment-related TEAEs by SOC, PT, and worst grade 
 Grade ≥3 TEAEs, by SOC and PT 
 Treatment-related Grade ≥3 TEAEs, by SOC and PT  
 Serious TEAEs by SOC and PT 
 Treatment-related Serious TEAEs, by SOC and PT  
 TEAEs leading to discontinuation of study drug, by SOC and PT  
 TEAEs leading to interruption of study drug, by SOC and PT  
 TEAEs leading to dose reduction, by SOC and PT 
 TEAEs leading to death, by SOC and PT 
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 Treatment-related TEAEs leading to death, by SOC and PT  
In addition, the following will be summarized by treatment arm and prescribed dose at TEAE 
onset. 
 First occurrence of TEAEs by PT 
 First occurrence of serious TEAEs by PT 
 TEAEs by PT 
 Serious TEAEs by PT 
7.7.1.1. 
Adverse Events of Special Interest 
Transaminase increase is an AESI for mitapivat and will be reported by the investigator in the 
AESI eCRF page if there is a transaminase increase of >2.5×baseline or an increase in AST or 
ALT to Grade ≥2 in severity, whichever is lower.   
Additional TEAEs of interest for mitapivat are as follows:  
 AEs of endocrinological interest (identified based on the criteria outlined in the 
mitapivat program specified Safety Search Criteria) 
 Insomnia (PTs under HLT of “Disturbances in Initiating and Maintaining Sleep” or 
identified based on the criteria outlined in the mitapivat program specified Safety 
Search Criteria) 
The following will be summarized for AESIs and the additional TEAEs of interest: 
 AESIs/TEAEs of interest by PT 
 AESIs/TEAEs of interest by PT and worst grade 
 Grade ≥3 AESIs/TEAEs of interest by PT 
 AESIs/TEAEs of interest leading to discontinuation of study drug by PT  
 Serious AESIs/TEAEs of interest by PT  
 AESIs/TEAEs of interest leading to death by PT 
In addition, the following will be summarized by treatment arm and prescribed dose at TEAE 
onset for the additional AE of interest “Insomnia”: 
 First occurrence of TEAEs by PT 
 First occurrence of serious TEAEs by PT 
 TEAEs by PT 
 Serious TEAEs by PT 
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7.7.1.2. 
Adverse Events Associated with COVID-19 
The selection of AEs associated with COVID-19 will be based on the MedDRA MSSO list of 
PTs. The following will be summarized: 
 TEAEs associated with COVID-19, by SOC and PT 
 Grade ≥3 TEAEs associated with COVID-19, by SOC and PT 
 Serious TEAEs associated with COVID-19, by SOC and PT 
 TEAEs associated with COVID-19 leading to discontinuation of study drug, by SOC 
and PT  
 TEAEs associated with COVID-19 leading to interruption of study drug, by SOC and 
PT  
 TEAEs associated with COVID-19 leading to dose reduction, by SOC and PT 
 TEAEs associated with COVID-19 leading to death, by SOC and PT 
7.7.2. 
Death 
The frequency of subjects in the safety analysis set who died will be tabulated based on 
information from the EOS eCRF. Deaths will be summarized for the following categories:  
 On-treatment death: Deaths within 28 days after the last dose of study treatment (ie, 
deaths during the on-treatment period) 
 Post-treatment death: Deaths more than 28 days after the last dose of study treatment 
(ie, deaths after the end of the on-treatment period) 
 Overall: All deaths 
In addition, deaths related to COVID-19 will be summarized. 
Deaths for all screened subjects will be provided in a by-subject listing. 
7.7.3. 
Clinical Laboratory Data 
Clinical laboratory test results will be expressed in SI units. Preferred unit (g/dL) will also be used 
for Hb in efficacy analysis. 
For each laboratory test (chemistry, hematology, coagulation) performed in the study, a by-
subject listing of laboratory test results will be presented with the corresponding CTCAE grades 
(if applicable), laboratory normal ranges, and flags for values below lower limit of normal (LLN) 
or above upper limit of normal (ULN).   
Parameters with CTCAE grades available:  
Clinical laboratory test results will be graded according to CTCAE v4.03 as applicable. Grading 
will be derived based on the numerical thresholds defined by the CTCAE criteria. Non-numerical 
qualifiers will not be taken into consideration in the derivation of CTCAE grading.  
Laboratory test results classified according to CTCAE will be described using the worst grade. 
For parameters graded with 2 separate toxicity criteria, such as potassium 
(hypokalemia/hyperkalemia), the toxicities will be summarized separately. Low direction 
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toxicity (eg, hypokalemia) grades at baseline and postbaseline will be set to 0 when the variables 
are derived for summarizing high direction toxicity (eg, hyperkalemia), and vice versa. 
The frequency of subjects with laboratory toxicities during the on-treatment period will be 
tabulated as follows. The denominator used to calculate percentages for each laboratory test is 
the number of subjects evaluable for CTCAE grading for that parameter (ie, those subjects for 
whom a Grade of 0, 1, 2, 3 or 4 can be derived). 
 The summary of laboratory parameters by CTCAE grade will include the number and 
percentage of subjects with Grade 1, 2, 3, 4; Grade 3-4; and Any Grade (Grades 1-4) 
during the on-treatment period. The highest CTCAE grade during the on-treatment 
period is considered the worst grade 
 The shift table will summarize baseline CTCAE grade versus worst CTCAE grade 
during the on-treatment period. The highest CTCAE grade during the on-treatment 
period is considered the worst grade  
 Newly occurring or worsening laboratory abnormalities (Any Grade, Grade 3-4) 
during the on-treatment period will also be summarized 
Parameters with CTCAE grades not available: 
Results of laboratory tests that are not part of CTCAE will be presented according to the 
following categories: below the LLN, within normal limits, and above the ULN according to the 
laboratory normal ranges.   
Shift tables will display the frequency of subjects with shifts from baseline missing, <LLN, 
normal, or >ULN to each of <LLN, normal or >ULN during the on-treatment period. 
7.7.3.1. 
Hematology 
For WBC differential counts [total neutrophil, lymphocyte, monocyte, eosinophil, and basophil 
counts], the absolute value will be used when reported. When only percentages are available 
(relevant primarily for neutrophils and lymphocytes, because the CTCAE grading is based on the 
absolute counts), the absolute value is derived as follows:  
Derived differential absolute count=(WBC count)×(Differential %value/100) 
If the range for the differential absolute count is not available (ie, the range is only available for 
the percentage) then Grade 1 will be attributed as follows:  
 Lymphocyte count decreased:   
o derived absolute count does not meet Grade 2-4 criteria, and 
o % value <% LLN value, and 
o derived absolute count ≥800/mm3 
 Neutrophil count decreased:  
o derived absolute count does not meet Grade 2-4 criteria, and 
o % value<% LLN value, and 
o derived absolute count ≥1,500/mm3 
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7.7.3.2. 
Chemistry 
Liver function tests: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), 
alkaline phosphatase (ALP), and total bilirubin are used to assess possible drug-induced liver 
toxicity. The ratios of test result to ULN will be calculated and categorized for these parameters 
during the on-treatment period.    
The summary of liver function tests will include the following categories. The frequency of 
subjects with each of the following during the on-treatment period will be summarized by 
treatment arm: 
 ALT >3×ULN, ALT >5×ULN, ALT >10×ULN, ALT >20×ULN 
 AST >3×ULN, AST >5×ULN, AST >10×ULN, AST >20×ULN 
 (ALT or AST) >3×ULN, (ALT or AST) >5×ULN, (ALT or AST) >10×ULN, 
(ALT or AST) >20×ULN 
 total bilirubin >2×ULN 
 Concurrent ALT >3×ULN and total bilirubin >2×ULN 
 Concurrent AST >3×ULN and total bilirubin >2×ULN 
 Concurrent (ALT or AST) >3×ULN and total bilirubin >2×ULN  
 Concurrent (ALT or AST) >3×ULN and total bilirubin >2×ULN and ALP ≥2×ULN 
 Concurrent (ALT or AST) >3×ULN and total bilirubin >2×ULN and (ALP <2×ULN 
or missing) 
Concurrent measurements are those occurring on the same date. 
Categories will be cumulative, ie, a subject with an AST >10×ULN will also appear in the 
categories >5×ULN and >3×ULN. Liver function test elevation and possible Hy’s Law cases 
will be summarized using frequency counts and percentages.  
An evaluation of Drug-Induced Serious Hepatotoxicity (eDISH) plot will be created, with 
different symbols for different treatment arms, by graphically displaying: 
 Peak serum ALT (/ULN) vs peak total bilirubin (/ULN) including reference lines at 
ALT=3×ULN and total bilirubin=2×ULN  
 Peak serum AST (/ULN) vs peak total bilirubin (/ULN) including reference lines at 
AST=3×ULN and total bilirubin=2×ULN  
In addition, the following individual longitudinal plots and by-subject listings will be provided: 
 Individual longitudinal plot of ALT including subjects with at least one ALT during 
the on-treatment period >2.5×baseline or worsening to CTCAE Grade ≥2 during the 
on-treatment period  
 Individual longitudinal plot of AST including subjects with at least one AST during 
the on-treatment period >2.5×baseline or worsening to CTCAE Grade ≥2 during the 
on-treatment period 
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 Listing of all total bilirubin, ALT, AST, and ALP values for subjects with a 
postbaseline total bilirubin >2×ULN, ALT >3×ULN, or AST >3×ULN 
 Listing of all total bilirubin, indirect bilirubin, ALT, AST and ALP values for subjects 
with a postbaseline ALT >ULN or AST >ULN 
In addition, a shift table from baseline to the worst CTCAE grade of ALT and AST during the 
on-treatment period will be provided. For each subject:  
 If the worst CTCAE grade of ALT is worse than that of AST during the on-treatment 
period, the baseline CTCAE grade of ALT will be used  
 If the worst CTCAE grade of AST is worse than that of ALT during the on-treatment 
period, the baseline CTCAE grade of AST will be used  
 
If AST and ALT have the same worst CTCAE grade during the on-treatment period, 
the lower baseline CTCAE grade of ALT and AST will be used 
For calcium, CTCAE grading is based on corrected calcium and ionized calcium. Corrected 
Calcium is calculated from albumin and calcium as follows: 
 
Corrected calcium (mmol/L)=measured total calcium (mmol/L)+0.02×[40−serum 
albumin (g/L)]     
7.7.3.3. 
Sex Steroid Tests 
For sex steroid test results, shift tables will display the frequency of subjects with shifts from 
baseline missing, < LLN, normal, > ULN to each of < LLN, normal or > ULN during the on-
treatment period.   
In addition, individual longitudinal plots will be provided for each sex hormone by sex.  
7.7.3.4. 
Pregnancy Test 
Pregnancy test results will be presented in a by-subject listing. 
7.7.4. 
Vital Signs and Physical Measurements 
All physical measurements and vital sign assessments (height, weight, BMI, systolic blood 
pressure, diastolic blood pressure, pulse rate, temperature) will be presented in a by-subject listing.  
7.7.5. 
Electrocardiograms 
All ECG summaries and listings will be based on the central reading results. 
ECG summaries will include all ECG assessments from the on-treatment period. QTcB and 
QTcF interval will be derived based on RR and QT interval (see below), if not collected in the 
eCRF. The average of the replicate measurements should be determined after the derivation of 
the individual parameter at each time point. 
Selecting Primary QT Interval Correction for Heart Rate 
The analysis of QT interval data is complicated by the fact that the QT interval is highly 
correlated with heart rate. Because of this correlation, formulas are routinely used to obtain a 
corrected QT interval, denoted QTc, which is independent of heart rate. This QTc is intended to 
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represent the QT interval at a standardized heart rate. Several correction formulas have been 
proposed in the literature. For this analysis several of those methods of correction will be used, 
as described below. The QT interval corrected for heart rate by the Bazett’s formula, QTcB, is 
defined as  
RR
QT
QTcB 
, 
and the QT interval corrected for heart rate by the Fridericia’s formula, QTcF, is defined as  
3 RR
QT
QTcF 
 , 
where RR represents the RR interval of the ECG, in seconds.   
Although Bazett’s correction is the historical standard, it does not perform well when heart rate 
fluctuates. Fridericia’s formula may perform better under these conditions. If QTcB and QTcF 
methods do not adequately correct for heart rate and there are a sufficient number of subjects 
(>30) with baseline ECGs, an alternative correction (QTcP) to achieve the goal of getting 
uncorrelated QTc and RR is based on a linear regression method which yields, theoretically, 
uncorrelated QTc and RR. 
Linear regression method: 
 Fit a model QT (ms)=a+b×RR (sec) to baseline data  
 Use the estimated slope,  𝑏̂, to correct QT 
 Corrected QT for heart rate will be derived as follows: 
QTcP (ms)=QT (ms)+ 𝑏̂×[1−RR (sec)] 
Data will be summarized using QTcF and QTcB. However, if QTcF is not appropriate for the 
data set because of an observed large correlation between corrected QT and heart rate using the 
baseline assessments, the results will also be summarized using QTcP.   
ECG Summaries 
The following analyses will be performed for each applicable ECG parameter (RR, PR, QRS, 
QT, and QTc) during the on-treatment period. The denominator to calculate percentages for each 
category is the number of subjects evaluable for the category. 
 Pearson correlation between QT and RR interval, QTc (QTcF, QTcB, and, if 
applicable, QTcP) and RR interval using individual (non-averaged) baseline 
assessments  
 Frequency of subjects with notable ECG values, defined as those in the following 
categories:            
o QT/QTc interval increase from baseline >30 ms, >60 ms 
o QT/QTc interval > 450 ms, > 480 ms, > 500 ms 
o PR interval >200 ms 
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o QRS duration >120 ms  
All ECG assessments and qualitative ECG abnormalities will be presented in by-subject listings.  
7.7.6. 
DXA Scans 
DXA scan results including bone mineral density (BMD), T-scores, Z-scores during the on-
treatment period will be summarized by treatment arm, location (total femur and adjusted spine), 
and visit. For T-scores, shift from baseline to Week 24 by category (≤-2.5, >-2.5 to <-1.0, ≥-1.0) 
will be provided.   
All DXA scan results will be presented in a by-subject listing.  
7.7.7. 
Menstrual Cycle Diary  
Menstrual cycle diary data collected from women of childbearing potential during the on-
treatment period will be summarized by treatment arm and by regular contraceptive status (oral 
contraceptives or depot injection). The following summaries will be included: 
 Total number of menstrual cycles reported 
 Total number of abnormal menstrual cycles in the following categories: heavier, 
lighter, longer, shorter, sooner and later than usual.  
Menstrual cycle diary data will be presented in a by-subject listing with regular contraceptive 
status flagged. 
7.8. 
Exploratory Analysis 
Unless otherwise specified, summaries for exploratory endpoints will be presented by treatment 
arm based on the full analysis set.  
7.8.1. 
Use of Iron Chelation Therapy 
For each subject, chelation status (Yes vs No) prior to and during the on-treatment period will be 
determined based on data collected in the Prior and Concomitant Medications eCRF. Shift in 
chelation status will be summarized.  
7.8.2. 
Iron Markers 
For iron markers including serum iron, serum ferritin, total iron-binding capacity, transferrin 
saturation and hepcidin, the following summaries will be provided for each parameter based on 
data collected up to the end of the Fixed Dose Period:  
 Summaries at each visit 
 Longitudinal plots of mean value (+/-SD) at each visit by treatment arm 
In addition, by-subject longitudinal plots will be presented with iron markers at baseline and 
scheduled visits, and prescribed dose over time; the plots will further include treatment arm and 
optimized dose, age, sex, race, PKLR gene mutation category, baseline Hb concentration, prior 
splenectomy status, baseline chelation status, and postbaseline chelation status. 
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Iron markers including non-transferrin bound iron (NTBI), C-reactive protein (CRP) will be 
presented in a by-subject listing. 
7.8.3. 
Erythropoietic Activity Markers 
Erythropoietic markers include erythropoietin (EPO), soluble transferrin receptor, 
erythroferrone, and absolute reticulocyte count.  
By-subject longitudinal plots will be presented with erythropoietic markers at baseline and 
scheduled visits, and prescribed dose over time; the plots will further include treatment arm and 
optimized dose, age, sex, race, PKLR gene mutation category, baseline Hb concentration, prior 
splenectomy status, baseline chelation status, and postbaseline chelation status 
Erythropoietic markers will be presented in a by-subject listing. 
7.8.4. 
Liver Iron Concentration (LIC) by Magnetic Resonance Imaging (MRI) 
Liver MRI results will be summarized at each visit. 
7.8.5. 
HRQOL Measurements 
7.8.5.1. 
EQ-5D-5L 
For EQ-5D-5L results, the following descriptive summaries will be provided at each visit, 
including: 
 Index score and change from baseline. 
 VAS score and change from baseline. 
 Frequency of subjects in each level for each of the 5 dimensions.  
7.8.5.2. 
SF-12 
For SF-12 results, the following descriptive summaries will be provided at each visit, including: 
 Physical Component Summary (PCS) score and change from baseline. 
 Mental Component Summary (MCS) score and change from baseline. 
 Frequency of subjects with answers in each category for each question.  
7.8.5.3. 
FACT-An Subscale 
For FACT-An subscale results, the following descriptive summaries will be provided at each 
visit. 
 Subscale score and change from baseline. 
 Maximum change from baseline in subscale score. 
 Frequency of subjects with at least 4 points increase from baseline in subscale score.  
 Frequency of subjects with answers in each category for each question.  
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7.8.5.4. 
PGIC, PGIS and CGIC 
For PGIC, PGIS and CGIC results, the frequency of subjects with answers in each category for 
each question will be summarized at each visit.   
7.9. 
Interim Analysis 
There are no interim analyses for efficacy planned for this study.  
Safety data will be reviewed regularly by an I-DMC. The last I-DMC data review will be no later 
than 4 months after the last subject is randomized in the study. Details are provided in the 
I-DMC Charter. 
 
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8. 
REFERENCES 
Goria, M. N. (1992). On the Fourth Root Transformation of Chi-Square. Australian Journal of 
Statistics, 34(1), 55-64. 
Hochberg, Y. (1988). A Sharper Bonferroni Procedure for Multiple Tests of Significance. 
Biometrika, 75(4), 800-802. 
Mauer, W. H. (1995). Multiple Comparisons in Drug Clinical Trials and Preclinical Assays: a-
priori Ordered Hypotheses. Biometrie in der Chemisch-harmazeutischen Industrie, 6, 3-
18. 
Ratitch B., L. I. (2013). Combining Analysis Results from Multply Imputed Categorical Data. 
SAS Conference Proceedings: Pharmaceutical SAS User Group. 
Rubin, D. B. (1987). Multiple Imputation for Nonresponse in Surveys. New York: John Wiley & 
Sons. 
Wilson, E. B. (1931). The Distribution of Chi-Square. Proceedings of the National Academy of 
Sciences of the United States of America, 17(12), 684-688. 
 
 
 
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9. 
APPENDICES 
Appendix A. Sensitivity Analysis of Primary Endpoint on Hemoglobin 
Response Using Multiple Imputation Sample Code 
The sensitivity analysis specified in Section 7.6.1.2 based on MI will be performed in the following 
3 steps with the sample code provided. 
 
Step 1. 100 datasets will be imputed.  
proc MI data=adhb_in seed=12345 NIMPUTE= 100 out=adhb_mi; 
     by trt; 
     em maxiter=5000 converge=1e-4 itprint outem=outem; 
     var mutcat hgbcat wk0 wk4 wk8 wk12 wk16 wk20 wk24; 
     mcmc chain=multiple initial=EM;  
run; 
 
Step 2. For each imputed dataset, Hb response status will be derived and analyzed using the 
CMH test. 
proc freq data=adhb_mi(where=(paramcd='HGBRESP')); 
     table mutcat*hgbcat*trt*aval /cmh; 
     by _imputation_; 
     ods output cmh=cmh; 
run; 
 
Step 3. Apply Wilson-Hilferty transformation (Wilson, 1931) (Goria, 1992) to the CMH 
statistics. Combine results and compute two-sided p-value following Rubin’s rules (Rubin, 1987) 
(Ratitch B., 2013) 
Data cmh_wh; 
Set cmh(WHERE=(AltHypothesis="General Association"));  
    cmh_value_wh=((VALUE/DF)**(1/3) - (1-2/(9*DF)))/SQRT(2/(9*DF));  
    cmh_sterr_wh = 1.0;  
RUN;  
 
PROC MIANALYZE DATA=cmh_wh;  
     ODS OUTPUT PARAMETERESTIMATES=mian_cmh_wh;  
     MODELEFFECTS cmh_value_wh;  
     STDERR cmh_sterr_wh;  
RUN;  
 
DATA mian_cmh_wh_p;  
SET mian_cmh_wh;  
    pval = Probt;  
RUN; 
 
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Prot_000.pdf: 
Study AG348-C-006 
 
Protocol Amendment 3, Version 4.0 
 
Confidential 
 
Agios Pharmaceuticals, Inc. 
 
 
 
 
CLINICAL STUDY PROTOCOL AG348-C-006 
 
A PHASE 3, RANDOMIZED, DOUBLE-BLIND, 
PLACEBO-CONTROLLED STUDY TO EVALUATE THE 
EFFICACY AND SAFETY OF AG-348 IN NOT 
REGULARLY TRANSFUSED ADULT SUBJECTS WITH 
PYRUVATE KINASE DEFICIENCY 
Study Sponsor 
Agios Pharmaceuticals, Inc. 
88 Sidney Street, Cambridge, MA 02139-4169, USA 
Phone: +1 617-649-8600 
Study Medical Director 
, MD, MS 
 
Agios Pharmaceuticals, Inc. 
Independent Medical Monitor 
, MD, FACP 
Agios Pharmaceuticals, Inc. 
Phone: 
 (landline) or 
 (mobile) 
Email: 
 
Medical Monitor 
, RN, 
 
Agios Pharmaceuticals, Inc. 
Phone: 
 
Email
 
 
, MD, PhD, 
 
 
 
Covance Clinical and Periapproval Services Limited 
Phone: 
 
Email
 
EudraCT Number 
2017-003823-31 
NCT Number 
NCT03548220 
Document Version (Date) 
Revised 
Original Protocol, Version 1.0 (30 November 2017) 
Amendment 1, Version 2.0 (26 February 2018) 
Amendment 2, Version 3.0 (15 August 2018) 
Amendment 3, Version 4.0 (14 August 2019) 
This study will be conducted according to the protocol and in compliance with Good Clinical Practices 
(GCP) as described in the International Council for Harmonisation (ICH) of Technical Requirements for 
Pharmaceuticals for Human Use guidelines, the ethical principles stated in the Declaration of Helsinki, 
and other applicable regulatory requirements. 


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CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc.  
Any distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal 
regulations or state law. Persons to whom the information is disclosed must know that it is confidential and that it 
may not be further disclosed by them. 




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INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios Pharmaceuticals, Inc. (Agios/the 
Sponsor) or its designated representative(s) concerning this study, which has not been previously 
published, will be kept in strict confidence. This documentation includes the study protocol, 
Investigator’s Brochure (IB), case report forms (CRFs), and other scientific data. 
This study will not commence without the prior written approval of a properly constituted 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC). No changes will be 
made to the study protocol without the prior written approval of Agios and the IRB/IEC, except 
where necessary to eliminate an immediate hazard to the subject. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
 
 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
(DD MMM YYYY) 
 
 
 
 
 
 
Investigational site or name of institution and location (printed) 
 
 
 


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2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Title of Study:  
A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Efficacy and Safety 
of AG-348 in Not Regularly Transfused Adult Subjects With Pyruvate Kinase Deficiency 
Study Center(s):  
This is a multicenter study that will be conducted in multiple countries. 
Phase of Development: 
3 
Objectives: 
Primary: 

To evaluate the efficacy of treatment with AG-348 compared with placebo in increasing
hemoglobin (Hb) concentrations
Secondary: 

To evaluate the safety of AG-348

To determine the effect of the study treatment regimens on markers of hemolysis,
hematopoietic activity, and other indicators of clinical activity

To determine the effect of the study treatment regimens on health-related quality of life
(HRQoL), as determined using patient-reported outcomes (PROs)

To evaluate the pharmacokinetics of AG-348 after oral administration

To evaluate the relationship between AG-348 pharmacokinetics and safety parameters
Exploratory: 

To evaluate the relationship of AG-348 pharmacokinetics to indicators of clinical activity

To evaluate the pharmacodynamic markers of pyruvate kinase deficiency (PK deficiency)
and how they are affected by study treatment

To determine the effect of the study treatment regimens on:
 
Number of transfusion events and number of red blood cell (RBC) units transfused
 
Markers of iron metabolism and indicators of iron overload
Methodology: 
Overview: 
This is a Phase 3, randomized, multicenter, double-blind, placebo-controlled study consisting of a Dose 
Optimization Period (Part 1) followed by a Fixed Dose Period (Part 2) (see study schema below).  
After a Screening Period, eligible subjects will be randomized 1:1 to receive either AG-348 or 
matching placebo. The term “study treatment” is used throughout the protocol to define both the active 
(AG-348) drug and placebo. Randomization will be stratified by the average of screening 
Hb concentrations (<8.5 vs ≥8.5 g/dL [5.28 mmol/L]) and the PKLR gene mutation category 
(missense/missense vs missense/non-missense). In rare instances in which PKLR gene mutation 


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category cannot be made definitively (eg, if a subject harbors 3 mutant PKLR alleles), the subject will 
be assigned to the missense/non-missense category. 
Following randomization to either AG-348 or matching placebo, subjects will enter the Part 1, a 
12-week period starting on Day 1 of the study. The goal of the Dose Optimization Period is to 
maximize a subject’s increase in Hb while maintaining an acceptable safety profile. All subjects will 
receive an initial dose of 5 mg twice daily (BID) of study treatment with 2 potential sequential steps for 
dose level increase (ie, from 5 to 20 mg BID and from 20 to 50 mg BID; no increases beyond 50 mg 
BID will be allowed). The first dose of study treatment on Day 1 should be taken at the study site 
following all Day 1 assessments (with the exception of electronic diary [eDiary] assessments). 
Subjects will be assessed for safety and efficacy (as defined by Hb increase) every 4 weeks during 
Part 1, to determine if their dose should be increased, maintained at the current level, or decreased. At 
the Week 4 and Week 8 Visits, study treatment dose should be increased to the next dose level if the 
subject has met both of the following criteria: 
 
The subject is tolerating the study treatment, and  
 
The subject’s Hb concentration on the day of the visit based on local laboratory results is 
lower than 2.5 g/dL (1.55 mmol/L) below the upper limit of normal (ULN), as applies to 
men and women. 
Dose re-escalation or re-introduction should be avoided after the Week 8 Visit, but may be permitted, 
after discussion with the Independent Medical Monitor, or designee. 
At the Week 12 Visit, if the subject has tolerated the study treatment, the subject will remain at his/her 
current dose level. If the Investigator deems it necessary to reduce the study treatment for safety 
reasons, the subject’s dose may be reduced to 1 of the 2 available lower dose levels (ie, 5 mg BID, 
20 mg BID). If the subject is already receiving 5 mg BID and/or cannot tolerate BID dosing, another 
regimen may be allowed after discussion with, and approval by, the Independent Medical Monitor, or 
designee. 
If questions arise about whether the dose level of a given subject should be increased, maintained, or 
decreased, the Independent Medical Monitor, or designee, should be contacted. 
At any time during the study, the Investigator can discontinue, interrupt, or reduce the subject’s 
dose of study treatment for reasons related to safety. 
Following Part 1, each subject will remain on his/her individually optimized dose and enter the 12-
week Fixed Dose Period (Part 2). For the purposes of dosing during the Fixed Dose Period, the dose 
the subject is being administered at the Week 12 Visit will be considered the subject’s optimized dose 
and will be the dose the subject receives during Part 2. 
All subjects who remain on study during Part 2 through the Week 24 Visit may be eligible for an 
open-label extension study, in which all subjects will receive AG-348. 
All subjects who discontinue or interrupt study treatment during the study should undergo the 
recommended dose taper, unless an emergency situation justifies discontinuing or interrupting the 
study treatment abruptly. Whether or not the recommended dose taper is performed, subjects who 
discontinue or interrupt study treatment should be monitored as clinically indicated for signs of 
withdrawal hemolysis and worsening of anemia. 
Visit Schedule and Analysis Time Points: 
A study schema is presented in the figure below.  
The Screening Period will last up to 42 days (a subject’s Screening Period duration may be extended 
beyond 42 days upon the Medical Monitor’s, or designee’s, approval). During Part 1, all subjects will 
attend visits on Day 1, Week 2, Week 4, Week 6, Week 8, Week 10, and Week 12. During Part 2, all 
subjects will attend visits every 4 weeks through Week 24 (ie, Week 16, Week 20, and Week 24).  


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Subjects who discontinue study prior to the Week 24 Visit should attend the End of Study Visit, but do 
not need to complete the Follow-Up Visit. Subjects who continue the study through the Week 24 Visit 
on study treatment but who do not continue on into an extension study, will attend the Follow-up Visit. 
Subjects who continue on into an extension study at the Week 24 Visit will not be required to attend 
the Follow-up Visit. 
All adverse events (AEs) will be monitored until resolution of the AE to baseline, the AE is considered 
stable within the context of the trial, the subject is lost to follow-up, or until 28 days after the last dose 
of study treatment unless the subject is enrolled in an extension study, in which case ongoing AEs will 
be reported in the extension study database following consenting of the subject. All serious adverse 
events (SAEs) will be followed until final outcome of the SAE is known, the subject is lost to 
follow-up, or the subject enrolls in an extension study, in which case the SAE follow-up information 
will be reported in the extension study database.  
Study Schema 
 
Abbreviations: BID = twice daily. 
1Stratified by average of screening Hb concentrations (Hb <8.5 g/dL vs Hb ≥8.5 g/dL [5.28 mmol/L]) and the 
PKLR gene mutation category (missense/missense vs missense/non-missense) 
2 A subject’s Screening Period duration may be extended beyond 42 days upon the Medical Monitor’s, or 
designee’s, approval. 
Number of Subjects (Planned):  
Approximately 76 
Diagnosis and Main Criteria for Inclusion: 
Inclusion Criteria: 
For enrollment into this study, subjects must meet all of the following criteria during the Screening 
Period: 
1. Have provided signed written informed consent prior to performing any study procedure, 
including screening procedures. 
2. Be aged 18 years or older. 


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3. Have documented clinical laboratory confirmation of PK deficiency, defined as documented 
presence of at least 2 mutant alleles in the PKLR gene, of which at least 1 is a missense 
mutation, as determined per the genotyping performed by the central genotyping laboratory.  
4. Have an Hb concentration less than or equal to 10.0 g/dL (6.21 mmol/L) regardless of gender 
(average of at least 2 Hb measurements [separated by a minimum of 7 days] during the 
Screening Period). 
5. Be considered not regularly transfused, defined as having had no more than 4 transfusion 
episodes in the 12-month period up to the first day of study treatment and no transfusions in 
the 3 months prior to the first day of study treatment.  
6. Have received at least 0.8 mg oral folic acid daily for at least 21 days prior to the first dose of 
study treatment, to be continued daily during study participation. 
7. Have adequate organ function, as defined by: 
a. Serum aspartate aminotransferase (AST) ≤2.5 × ULN (unless the increased AST is 
assessed by the Investigator as due to hemolysis and/or hepatic iron deposition) and 
alanine aminotransferase (ALT) ≤2.5 × ULN (unless the increased ALT is assessed by the 
Investigator as due to hepatic iron deposition). 
b. Normal or elevated levels of serum bilirubin. In subjects with serum bilirubin >ULN, the 
elevation must not be associated with choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. Elevated bilirubin attributed to hemolysis with or without 
Gilbert’s syndrome is not exclusionary. 
c. Estimated glomerular filtration rate (GFR) ≥60 mL/min/1.73 m2, measured GFR 
≥60 mL/min, or calculated creatinine clearance (CrCL; Cockcroft-Gault) ≥60 mL/min. 
d. Absolute neutrophil count ≥1.0 × 109/L (based on an average of at least 2 measurements 
[separated by a minimum of 7 days] during the Screening Period). 
e. Platelet count ≥100 × 109/L in the absence of a spleen, or platelet count ≥50 × 109/L in the 
presence of a spleen and in the absence of any other cause of thrombocytopenia (based on 
an average of at least 2 measurements [separated by a minimum of 7 days] during the 
Screening Period). 
f. Activated partial thromboplastin time and international normalized ratio ≤1.25 × ULN, 
unless the subject is receiving therapeutic anticoagulants. 
8. For women of reproductive potential, have a negative serum pregnancy test during the 
Screening Period. Women of reproductive potential are defined as sexually mature women 
who have not undergone a hysterectomy, bilateral oophorectomy, or tubal occlusion; or who 
have not been naturally postmenopausal (ie, who have not menstruated at all for at least the 
preceding 12 months prior to signing informed consent and have an elevated 
follicle-stimulating hormone level indicative of menopause during the Screening Period). 
9. For women of reproductive potential as well as men with partners who are women of 
reproductive potential, be abstinent as part of their usual lifestyle, or agree to use 2 forms of 
contraception, 1 of which must be considered highly effective, from the time of giving 
informed consent, during the study, and for 28 days following the last dose of study treatment 
for women and 90 days following the last dose of study treatment for men. A highly effective 
form of contraception is defined as combined (estrogen and progestin containing) hormonal 
contraceptives (oral, intravaginal, or transdermal) known to be associated with inhibition of 
ovulation; progestin-only hormonal contraceptives (oral, injectable, or implantable) known to 
be associated with inhibition of ovulation; intrauterine device; intrauterine hormone releasing 
system; bilateral tube occlusion; or vasectomized partner. The second form of contraception 
can include an acceptable barrier method, which includes male or female condoms with or 
without spermicide, and cervical cap, diaphragm, or sponge with spermicide. Women of 


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reproductive potential using hormonal contraception as a highly effective form of 
contraception must also utilize an acceptable barrier method while enrolled in the study and for 
at least 28 days after their last dose of study treatment. 
10. Be willing to comply with all study procedures for the duration of the study. 
Exclusion Criteria: 
Subjects who meet any of the following criteria during screening will not be enrolled in the study: 
1. Are homozygous for the R479H mutation or have 2 non-missense mutations, without the 
presence of another missense mutation, in the PKLR gene as determined per the genotyping 
performed by the central genotyping laboratory.  
2. Have a significant medical condition that confers an unacceptable risk to participating in the 
study, and/or that could confound the interpretation of the study data. Such significant medical 
conditions include, but are not limited to the following: 
a. Poorly controlled hypertension (defined as systolic blood pressure [BP] >150 mmHg or 
diastolic BP >90 mmHg) refractory to medical management.  
b. History of recent (within 6 months prior to signing informed consent) congestive heart 
failure; myocardial infarction or unstable angina pectoris; hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism. 
c. Cardiac dysrhythmias judged as clinically significant by the Investigator. 
d. Heart-rate corrected QT interval-Fridericia's method (QTcF) >450 msec (average of 
triplicate electrocardiograms [ECGs]) with the exception of subjects with right or left 
bundle branch block. 
e. Clinically symptomatic cholelithiasis or cholecystitis. Prior cholecystectomy is not 
exclusionary. Subjects with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved. 
f. History of drug-induced cholestatic hepatitis. 
g. Iron overload sufficiently severe to result in a clinical diagnosis by the Investigator of 
cardiac (eg, clinically significant impaired left ventricular ejection fraction), hepatic 
(eg, fibrosis, cirrhosis), or pancreatic (eg, diabetes) dysfunction. 
h. Have a diagnosis of any other congenital or acquired blood disorder, or any other 
hemolytic process, except mild allo-immunization as a consequence of transfusion therapy. 
Genetic findings that in isolation are predicted to be insufficient to explain the observed 
clinical phenotype may be allowed (eg, heterozygous status for certain recessive red blood 
cell disorders). 
i. 
Positive test for hepatitis B surface antigen or hepatitis C virus (HCV) antibody (Ab) with 
signs of active hepatitis B or C virus infection. If the subject is positive for HCVAb, a 
reverse transcriptase-polymerase chain reaction test will be conducted. Subjects with 
hepatitis C may be rescreened after receiving appropriate hepatitis C treatment. 
j. 
Positive test for human immunodeficiency virus (HIV)-1 or -2 Ab. 
k. Active infection requiring the use of parenteral antimicrobial agents or Grade ≥3 in 
severity (per National Cancer Institute Common Terminology Criteria for Adverse Events) 
within 2 months prior to the first dose of study treatment. 
l. 
Diabetes mellitus judged to be under poor control by the Investigator or requiring 
>3 antidiabetic agents, including insulin (all insulins are considered 1 agent); use of insulin 
per se is not exclusionary.  
m. History of any primary malignancy, with the exception of: curatively treated 
nonmelanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 


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other primary tumor treated with curative intent, no known active disease present, and no 
treatment administered during the last 3 years. 
n. Unstable extramedullary hematopoiesis that could pose a risk of imminent neurologic 
compromise. 
o. Current or recent history of psychiatric disorder that, in the opinion of the Investigator or 
Medical Monitor, or designee, could compromise the ability of the subject to cooperate 
with study visits and procedures. 
3. Have a splenectomy scheduled during the study treatment period or have undergone 
splenectomy within 12 months prior to signing informed consent. 
4. Are currently enrolled in another therapeutic clinical trial involving ongoing therapy with any 
investigational or marketed product or placebo. Prior and subsequent participation in the 
PK Deficiency Natural History Study (NHS) (NCT02053480) or PK Deficiency Registry is 
permitted, however, concurrent participation is not. Therefore, subjects enrolling in this current 
study will be expected to temporarily suspend participation in the NHS or Registry.  
5. Have exposure to any investigational drug, device, or procedure within 3 months prior to the 
first dose of study treatment. 
6. Have had any prior treatment with a pyruvate kinase activator. 
7. Have a prior bone marrow or stem cell transplant. 
8. Are currently pregnant or breastfeeding. 
9. Have a history of major surgery within 6 months of signing informed consent. Note that 
procedures such as laparoscopic gallbladder surgery are not considered major in this context. 
10. Are currently receiving medications that are strong inhibitors of cytochrome P450 (CYP)3A4, 
strong inducers of CYP3A4, strong inhibitors of P-glycoprotein (P-gp), or digoxin (a P-gp 
sensitive substrate medication) that have not been stopped for a duration of at least 5 days or a 
timeframe equivalent to 5 half-lives (whichever is longer) prior to the first dose of study 
treatment. 
11. Are currently receiving hematopoietic stimulating agents (eg, erythropoietins, granulocyte 
colony stimulating factors, thrombopoietins) that have not been stopped for a duration of at 
least 28 days prior to the first dose of study treatment. 
12. Have a history of allergy to sulfonamides if characterized by acute hemolytic anemia, 
drug-induced liver injury, anaphylaxis, rash of erythema multiforme type or Stevens-Johnson 
syndrome, cholestatic hepatitis, or other serious clinical manifestations. 
13. Have a history of allergy to AG-348 or its excipients (microcrystalline cellulose, 
croscarmellose sodium, sodium stearyl fumarate, and mannitol). 
14. Are currently receiving anabolic steroids, including testosterone preparations, within 28 days 
prior to the first dose of study treatment. 


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Investigational Product, Dosage, and Mode of Administration:  
AG-348 will be administered orally BID as tablets of different sizes for the 5, 20, and 50 mg dose 
levels. Subjects will be receiving 1 of 3 potential doses, each of which is supplied as a different sized 
tablet. Thus, to maintain blinding, each dose of study treatment will be supplied as 3 different sized 
tablets: 1 tablet will be the active drug and the other 2 tablets will be placebo for subjects who are 
randomized to active, and all 3 tablets will be placebo for subjects who are randomized to placebo. 
Doses of AG-348 may be taken with or without food.  
Subjects should be advised not to discontinue or interrupt dosing without first speaking with the 
treating Investigator except in case of medical emergency; abrupt discontinuation or interruption of 
AG-348 may result in withdrawal hemolysis. If a subject needs to discontinue or interrupt study 
treatment at any time during the study, guidance is provided. 
Reference Therapy, Dosage, and Mode of Administration: 
Placebo will be administered as 3 visually matched tablets of different sizes, and dosed following the 
same guidelines and procedures as active treatment. 
Duration of Study Treatment (ie, AG-348/Matching Placebo):  
The duration of study treatment will be 24 weeks (not including the recommended dose taper). 
End of Study: 
End of Study is defined as the time at which all subjects have completed the study or are lost to 
follow-up. 
Study Endpoints: 
Primary Endpoint: 
The primary endpoint is the hemoglobin response (HR), defined as a ≥1.5 g/dL (0.93 mmol/L) increase 
in Hb concentration from baseline that is sustained at 2 or more scheduled assessments at Weeks 16, 
20, and 24 during the Fixed Dose Period. The individual subject’s baseline Hb concentration is defined 
as the average of all available Hb concentrations collected for that subject during the Screening Period 
up to the first dose of study treatment. 
Key Secondary Endpoint: 
 
Average change from baseline in Hb concentration at Weeks 16, 20, and 24 
Other Secondary Endpoints: 
 
Maximal Hb concentration increase from baseline 
 
Time to first achieve an increase in Hb concentration of 1.5 g/dL (0.93 mmol/L) or more from 
baseline  
 
Average change from baseline at Weeks 16, 20, and 24 in markers of hemolysis: bilirubin, 
lactate dehydrogenase, and haptoglobin levels 
 
Average change from baseline at Weeks 16, 20, and 24 in markers of hematopoietic activity: 
reticulocyte percentages 
 
Change from baseline in HRQoL PRO scores: Pyruvate Kinase Deficiency Diary and Pyruvate 
Kinase Deficiency Impact Assessment 
 
Safety endpoints, including: the type, incidence, severity, and relationship to study treatment of 
AEs and serious adverse events (SAEs); number of discontinuations due to AEs; results of 
clinical laboratory tests over time (eg, serum chemistry, liver function test, hematology, lipids, 
sex steroids, urinalysis, coagulation); physical examination findings; dual-energy x-ray 
absorption (DXA) scans, vital signs; 12-lead ECG data 


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 
Pharmacokinetic endpoints, including plasma concentrations over time and pharmacokinetic 
parameters of AG-348 (eg, area under the concentration × time curve [AUC], maximum [peak] 
concentration [Cmax], others as applicable) 
 
Exposure-response relationship between safety parameters and AG-348 concentration and 
relevant AG-348 pharmacokinetic parameters 
Exploratory Endpoints: 
 
Exposure-response (or pharmacokinetic-pharmacodynamic) relationship between relevant 
pharmacokinetic parameters and endpoints that are indicators of clinical activity 
 
Change from baseline in additional markers of hematopoietic activity 
 
Change from baseline in markers of iron metabolism and indicators of iron overload 
 
Change from baseline in the red blood cell-specific form of pyruvate kinase (PKR) protein 
level 
 
Relationship between baseline PKR protein level and Hb response status 
 
Change from baseline in HRQoL PRO scores: European quality of life five-dimensional 
descriptive system (EQ-5D-5L)  
 
Change from baseline in PKR flux assay results 
 
Proportion of subjects requiring transfusions and the total number of RBC units transfused 
Randomization, Blinding, Handling of Restricted Data, and Unblinding: 
Randomization: Eligible subjects will be randomized 1:1 to receive either AG-348 or matching 
placebo. The randomization will be stratified by the average of screening Hb concentrations 
(<8.5 vs ≥8.5 g/dL [5.28 mmol/L]) and the PKLR gene mutation category (missense/missense vs 
missense/non-missense). The randomization assignment will be double-blinded to minimize any 
potential assessment bias. In rare instances where PKLR gene mutation category cannot be made 
definitively (eg, if a subject harbors 3 mutant PKLR alleles), the subject will be assigned to the 
missense/non-missense category. 
Blinding: The subject, Investigators, and site personnel will be blinded to the subject’s study treatment 
allocation until database lock, if that subject is not entering an extension study. If the subject expresses 
his/her intention to enter an extension study, the subject, Investigators, and site personnel will be 
unblinded to the subject’s study treatment allocation, but only after the subject completes the Week 24 
assessments. The Sponsor study team will be blinded to study treatment allocation until the database 
has been locked. 
Handling of Restricted Data: The following data will be considered restricted data: RBC parameters, 
hemolysis parameters, and hormone data. The Investigators will have access to these restricted data, 
with the exception of the hormone data, for their own subjects. Subjects should not have access to their 
own restricted data, to reduce the potential for bias in the PRO measurements. The Sponsor study team 
will not have access to the restricted data. The Independent Medical Monitor, or designee, will have 
access to the restricted data to be able to provide guidance to the Investigators and perform periodic 
review of these data. 
Unblinding: In the event of a medical emergency or pregnancy in a female subject, or in the female 
sexual partner of a male subject, in which knowledge of the treatment allocation is critical to the 
subject’s management, the blind for that subject may be broken by the Investigator. Prior to unblinding, 
Investigators are encouraged to discuss a plan to break the blinding code with the Independent Medical 
Monitor, or designee. 
 
 


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Statistical Methods: 
Analysis Sets: 
 
Full Analysis Set (FAS): All subjects who are randomized. Subjects will be analyzed according 
to the treatment to which they were randomized, regardless of any errors in dosing. The FAS 
will be the primary analysis set for the efficacy endpoints.  
 
Per Protocol Set (PPS): All subjects who are randomized and dosed and have Hb assessments 
at Weeks 16, 20, and 24 during the Fixed Dose Period. Subjects will be analyzed according to 
the treatment to which they were randomized, regardless of any errors in dosing. The PPS will 
be used for a sensitivity analysis for the primary endpoint and the key secondary endpoint. 
 
Safety Analysis Set (SAS): All subjects who receive at least 1 dose of study treatment. 
Subjects will be analyzed according to the actual treatment they received (eg, if a subject 
received any active treatment, the subject will be grouped to the active arm). The SAS will be 
the primary analysis set for safety analyses. 
Statistical Methods: 
The study data will be analyzed and reported when all subjects have completed their Week 24 Visit or 
Follow-up Visit (if applicable) or have discontinued the study. The statistical analysis details will be 
provided in the Statistical Analysis Plan (SAP), which will be finalized before the database lock and 
study unblinding. 
Study data will be summarized for disposition, demographics, and baseline characteristics 
(including genotype) based on the FAS. 
In the primary efficacy analysis, subjects’ HR status will be analyzed using a logistic regression model. 
The model will include the HR response (Yes vs No) as the dependent variable and treatment as the 
independent variable, adjusting for stratification factors, including the average of screening 
Hb concentrations (<8.5 vs ≥8.5 g/dL) and the PKLR gene mutation category (missense/missense vs 
missense/non-missense). The primary analysis will be based on the FAS. In the primary analysis, 
subjects who discontinue the study before having at least 2 Hb concentration assessments in the Fixed 
Dose Period will be considered as not achieving an HR. The primary result obtained from the logistic 
model will be the estimated odds ratio between the active arm vs the placebo arm, along with the 95% 
confidence interval, and the 2-sided P value. If the 2-sided P value is less than the required alpha 
(0.05), then the null hypothesis will be rejected and statistical significance will be claimed for the 
active arm (ie, AG-348). When the logistic model fails to converge or its maximum likelihood estimate 
does not exist due to quasi-complete separation, the non-parametric method 
(ie, Cochran-Mantel-Haenszel test [CMH]) will be used. A sensitivity analysis based on the PPS will 
be conducted to evaluate the impact of missing assessments due to early study discontinuation. 
Additional sensitivity and supportive analyses will be specified in the SAP.  
The average change from baseline in Hb concentrations at Weeks 16, 20, and 24 will be analyzed and 
compared between the active arm and the placebo arm by the linear Mixed-Effect Model Repeat 
Measurement (MMRM) method. The model will include the change in Hb at each visit as the 
dependent variable; treatment, visit, and treatment-by-visit interaction as fixed effects; and subject as a 
random effect with adjustment for stratification factors, including the average of screening Hb 
concentrations (<8.5 vs ≥8.5 g/dL) and the PKLR gene mutation category (missense/missense vs 
missense/non-missense). Based on the least square estimate (LS Means), the average change from 
baseline of Hb concentrations at Weeks 16, 20, and 24 will be compared between the active arm and 
the placebo arm. In addition the Hb concentrations and their change from baseline at each visit over 
time will also be summarized by visit. 
Safety will be summarized using descriptive statistics. Summaries will be produced for all 
treatment-emergent adverse events (TEAEs), related TEAEs (those considered by the Investigator as 
related to study treatment) TEAEs Grade ≥3 in severity, TEAEs leading to treatment 


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discontinuation/interruption, SAEs, adverse events of special interest (AESIs), and death. In addition, 
listings containing individual subject data for all TEAEs leading to treatment discontinuation, TEAEs 
leading to treatment interruption, serious AEs, AESIs, and death will be provided separately.  
For clinical laboratory values, vital signs, and ECG assessments, both actual values and changes from 
baseline will be summarized by visit using summary statistics. The number and percentage of subjects 
with transaminase increases of >2.5 × baseline or increases to Grade ≥2, will be summarized.  
Interim Analysis and Data Monitoring Committees: 
No interim analysis is planned in this study. An Independent Data Monitoring Committee will be 
established to review study data periodically and provide safety oversight for subjects in the study. 
Sample Size: 
Assuming a response rate of 35% in the active arm and 5% in the placebo arm, 76 subjects 
(38 per arm) are needed to have 90% power to detect a treatment effect in HR rate based on a 2-sided 
Fisher’s Exact test at the 0.05 significance level.  


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3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES  
 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................5 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............15 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................21 
5. 
INTRODUCTION ......................................................................................................24 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................24 
5.1.1. 
Epidemiology and Prevalence ....................................................................................24 
5.1.2. 
Biochemistry and Genetics .........................................................................................24 
5.1.3. 
Clinical Characteristics ...............................................................................................25 
5.2. 
Investigational Product (AG-348) ..............................................................................26 
5.2.1. 
Proposed Mechanism of Action of AG-348 ...............................................................26 
5.2.2. 
Summary of AG-348 Nonclinical Data With Potential Clinical Interest ...................26 
5.2.3. 
Summary of AG-348 Clinical Data ............................................................................27 
5.3. 
Study Rationale ...........................................................................................................29 
5.3.1. 
Justification of Study Design ......................................................................................30 
5.3.2. 
Rationale for Dose ......................................................................................................31 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................33 
6.1. 
Objectives ...................................................................................................................33 
6.1.1. 
Primary Objective .......................................................................................................33 
6.1.2. 
Secondary Objectives .................................................................................................33 
6.1.3. 
Exploratory Objectives ...............................................................................................33 
6.2. 
Endpoints ....................................................................................................................33 
6.2.1. 
Primary Endpoint ........................................................................................................33 
6.2.2. 
Key Secondary Endpoint ............................................................................................33 
6.2.3. 
Other Secondary Endpoints ........................................................................................34 
6.2.4. 
Exploratory Endpoints ................................................................................................34 
7. 
INVESTIGATIONAL PLAN .....................................................................................35 
7.1. 
Study Design ...............................................................................................................35 
7.1.1. 
Overview of Study Design ..........................................................................................35 
7.1.2. 
Screening Period .........................................................................................................36 


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7.1.3. 
Part 1: Dose Optimization Period ...............................................................................37 
7.1.4. 
Part 2: Fixed Dose Period ...........................................................................................38 
7.1.5. 
Study Discontinuation and Follow-up ........................................................................39 
7.2. 
Number of Subjects ....................................................................................................39 
7.3. 
Criteria for Study Termination ...................................................................................39 
8. 
SELECTION AND WITHDRAWAL OF SUBJECTS ..............................................41 
8.1. 
Inclusion Criteria ........................................................................................................41 
8.2. 
Exclusion Criteria .......................................................................................................42 
8.3. 
Subject Withdrawal Criteria .......................................................................................44 
8.4. 
Subject Replacement ..................................................................................................45 
8.5. 
End of Study ...............................................................................................................45 
9. 
TREATMENT OF SUBJECTS ..................................................................................46 
9.1. 
Description of Study Treatment ..................................................................................46 
9.1.1. 
Study Treatment ..........................................................................................................46 
9.1.2. 
Study Treatment Packaging and Labeling ..................................................................46 
9.1.3. 
Study Treatment Storage ............................................................................................46 
9.1.4. 
Study Treatment Administration ................................................................................46 
9.1.5. 
Study Treatment Accountability .................................................................................47 
9.1.6. 
Study Treatment Handling and Disposal ....................................................................47 
9.2. 
Randomization ............................................................................................................47 
9.3. 
Blinding ......................................................................................................................48 
9.3.1. 
Handling of Restricted Data .......................................................................................48 
9.4. 
Unblinding ..................................................................................................................48 
9.5. 
Criteria for Dose Modification and Study Treatment Discontinuation or 
Interruption for Adverse Events or Excessive Hb Response ......................................49 
9.5.1. 
Excessive Hb Response ..............................................................................................49 
9.5.2. 
Occurrence of Study Treatment Related Adverse Events (Except for 
Excessive Hb Response) .............................................................................................49 
9.5.3. 
Study Treatment Discontinuation or Interruption With Recommended Dose 
Taper Regimen ............................................................................................................51 
9.6. 
Treatment Compliance ................................................................................................52 
9.7. 
Prior and Concomitant Medications ...........................................................................52 
9.7.1. 
Prohibited Medications ...............................................................................................52 


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9.7.2. 
Allowed Concomitant Medications ............................................................................53 
9.8. 
Prior and Concomitant Non-Drug Therapies ..............................................................54 
9.8.1. 
Prior and Concomitant Transfusions ..........................................................................54 
10. 
STUDY ASSESSMENTS ..........................................................................................55 
10.1. 
Informed Consent .......................................................................................................55 
10.2. 
Study Eligibility ..........................................................................................................55 
10.3. 
Demographics .............................................................................................................55 
10.4. 
Medical/Surgical History ............................................................................................55 
10.4.1. 
Prior Transfusions .......................................................................................................56 
10.4.2. 
Iron Overload-Related History ...................................................................................56 
10.5. 
Pyruvate Kinase Deficiency Specific Assessments ....................................................56 
10.5.1. 
PKLR Genotyping .......................................................................................................56 
10.5.2. 
PK Enzyme Assay ......................................................................................................56 
10.6. 
Assessment of Efficacy ...............................................................................................56 
10.6.1. 
Hemoglobin Laboratory Assessment ..........................................................................56 
10.6.2. 
Additional Laboratory Assessments for Efficacy .......................................................56 
10.7. 
Assessments of Safety ................................................................................................56 
10.7.1. 
Vital Signs ..................................................................................................................56 
10.7.2. 
Physical Examination .................................................................................................56 
10.7.3. 
Prior and Concomitant Medications ...........................................................................57 
10.7.4. 
Safety Laboratory Assessments ..................................................................................57 
10.7.5. 
Electrocardiogram .......................................................................................................57 
10.7.6. 
Dual-Energy X-ray Absorptiometry Scans .................................................................57 
10.7.7. 
Menstrual Cycle Diary ................................................................................................57 
10.8. 
Laboratory Assessments .............................................................................................57 
10.8.1. 
Safety Laboratory Assessments ..................................................................................58 
10.8.2. 
Other Laboratory Assessments ...................................................................................59 
10.9. 
Other Assessments ......................................................................................................59 
10.10. 
Pharmacokinetic Assessments ....................................................................................59 
10.10.1. 
Blood Sample Collection ............................................................................................59 
10.10.2. 
Sample Analysis .........................................................................................................60 
10.11. 
Pharmacodynamic Assessments .................................................................................60 
10.12. 
Health-Related Quality of Life Assessments and CGIC ............................................61 


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10.12.1. 
Pyruvate Kinase Deficiency-Specific HRQoL Assessments ......................................61 
10.12.2. 
European Quality of Life Five-Dimensional Descriptive System ..............................61 
10.12.3. 
Patient Global Impression of Severity ........................................................................61 
10.12.4. 
12-Item Short Form Healthy Survey, Version 2 .........................................................62 
10.12.5. 
Functional Assessment of Cancer Therapy Anemia – “Additional Concerns” ..........62 
10.12.6. 
Patient Global Impression of Change .........................................................................62 
10.12.7. 
Clinician Global Impression of Change .....................................................................62 
10.13. 
Schedule of Assessments ............................................................................................62 
11. 
ADVERSE EVENTS..................................................................................................69 
11.1. 
Reporting Period for Adverse Events and Serious Adverse Events ...........................69 
11.2. 
Definition of Adverse Events .....................................................................................69 
11.2.1. 
Adverse Event .............................................................................................................69 
11.2.2. 
Serious Adverse Event ................................................................................................69 
11.2.3. 
Adverse Events Occurring Secondary to Other Events ..............................................70 
11.2.4. 
Pre-existing Medical Conditions ................................................................................70 
11.2.5. 
Abnormal Laboratory Values .....................................................................................70 
11.2.6. 
Adverse Events of Special Interest .............................................................................71 
11.3. 
Procedures for Reporting Adverse Events, Adverse Events of Special 
Interest, and Serious Adverse Events .........................................................................72 
11.3.1. 
Intensity ......................................................................................................................72 
11.3.2. 
Relationship to Study Treatment ................................................................................73 
11.4. 
Pregnancy Reporting ..................................................................................................73 
12. 
STATISTICAL METHODS .......................................................................................75 
12.1. 
Analysis Sets ...............................................................................................................75 
12.1.1. 
Full Analysis Set .........................................................................................................75 
12.1.2. 
Per Protocol Set ..........................................................................................................75 
12.1.3. 
Safety Analysis Set .....................................................................................................75 
12.2. 
Statistical Analysis ......................................................................................................75 
12.2.1. 
General Methods .........................................................................................................75 
12.2.2. 
Background Characteristics ........................................................................................76 
12.2.3. 
Study Treatment Exposure and Compliance ..............................................................77 
12.2.4. 
Efficacy Analysis ........................................................................................................78 
12.2.5. 
Safety Analysis ...........................................................................................................80 


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12.3. 
Interim and Independent Data Monitoring Committee Analyses ...............................82 
12.3.1. 
Interim Analysis ..........................................................................................................82 
12.3.2. 
Independent Data Monitoring Committee Analysis ...................................................82 
12.4. 
Sample Size and Power ..............................................................................................82 
12.4.1. 
Sample Size for the Primary Endpoint .......................................................................82 
12.4.2. 
Power for the Key Secondary Endpoint .....................................................................82 
13. 
ADMINISTRATIVE REQUIREMENTS ..................................................................83 
13.1. 
Good Clinical Practices ..............................................................................................83 
13.2. 
Ethical Considerations ................................................................................................83 
13.3. 
Subject Information and Informed Consent ...............................................................83 
13.4. 
Subject Confidentiality ...............................................................................................83 
13.5. 
Protocol Compliance ..................................................................................................84 
13.6. 
Data Management .......................................................................................................84 
13.7. 
Source Documentation and Electronic Case Report Form Completion .....................84 
13.8. 
Direct Access to Source Data .....................................................................................85 
13.9. 
Record Retention ........................................................................................................85 
13.10. 
Liability and Insurance ...............................................................................................85 
13.11. 
Publication of Study Findings and Use of Information ..............................................85 
14. 
REFERENCES ...........................................................................................................86 
 


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LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Considered Related to Study 
Treatment (Except for Excessive Hb Response), Study AG348-C-006 .....................50 
Table 2: 
Recommended Dose Taper Regimen, Study AG348-C-006 ......................................52 
Table 3: 
Schedule of Assessments, Study AG348-C-006 ........................................................63 
 
LIST OF FIGURES 
Figure 1: 
Glycolysis in Red Blood Cells of Patients With Pyruvate Kinase Deficiency ...........25 
Figure 2: 
Overview of Design for Study AG348-C-006 ............................................................36 
 


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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
Ab 
Antibody 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the plasma concentration × time curve 
AUC0-12 
Area under the plasma concentration × time curve from 0 to 12 hours 
AUC0-last 
Area under the plasma concentration × time curve from time 0 to the time of the 
last measurable concentration 
BID 
Twice daily 
BP 
Blood pressure 
CGIC 
Clinician Global Impression of Change 
CI 
Confidence interval 
Cmax 
Maximum (peak) concentration 
CrCL 
Creatinine clearance 
CRP 
C-reactive protein 
CTCAE 
Common Terminology Criteria for Adverse Events 
CYP 
cytochrome P450 enzymes 
2,3-DPG 
2,3-Diphosphoglycerate 
DXA 
Dual-energy X-ray absorptiometry 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
eDiary 
Electronic diary 
EPO 
Erythropoietin 
EQ-5D-5L 
European quality of life five-dimensional descriptive system  
FACT-An 
Functional Assessment of Cancer Therapy Anemia 
FAS 
Full Analysis Set 
FSH 
Follicle-stimulating hormone 


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Abbreviation 
Definition 
GCP(s) 
Good Clinical Practice(s) 
GFR 
Glomerular filtration rate 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
hCG 
Human chorionic gonadotropin 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
HR 
Hemoglobin response 
HRQoL 
Health-related quality of life 
IB 
Investigator’s Brochure 
ICF 
Informed consent form 
ICH 
International Council for Harmonisation 
IDMC 
Independent Data Monitoring Committee 
IEC 
Independent Ethics Committee 
IMP 
Investigational medicinal product 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
IXRS 
Interactive response system 
LDH 
Lactate dehydrogenase 
LFT 
Liver function test 
LIC 
Liver iron concentration 
LS Means  
Least square estimate 
MAD 
Multiple ascending dose  
MCV 
Mean corpuscular volume 
MMRM 
Mixed-Effect Model Repeat Measurement 
MRI 
Magnetic resonance imaging 
NCI 
National Cancer Institute 
NHS 
Natural History Study 
NTBI 
Non-transferrin bound iron 
PD 
Pharmacodynamic(s) 
PE 
Physical examination 
PEP 
Phosphoenolpyruvate 


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Abbreviation 
Definition 
PGIC 
Patient Global Impression of Change 
PGIS 
Patient Global Impression of Severity 
P-gp 
P-glycoprotein 
PK deficient/cy 
Pyruvate kinase deficient/cy 
PKDD 
Pyruvate Kinase Deficiency Diary 
PKDIA 
Pyruvate Kinase Deficiency Impact Assessment 
PKL 
Liver-specific form of pyruvate kinase 
PKM 
Pyruvate kinase muscle isozyme 
PKR 
Red blood cell-specific form of pyruvate kinase 
PPS 
Per Protocol Set 
PRO 
Patient-reported outcome 
QD 
Once daily 
QOD 
Every other day 
QoL 
Quality of Life 
QTcB 
Heart rate-corrected QT interval by Bazett’s method 
QTcF 
Heart rate-corrected QT interval by Fridericia’s method 
RBC 
Red blood cell 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SAS 
Safety Analysis Set 
SD 
Standard deviation 
SF-12v2 
12-item Short Form Health Survey, Version 2 
Study treatment 
AG-348 or matching placebo 
TEAE 
Treatment-emergent adverse event 
TIBC 
Total iron-binding capacity 
Tmax 
Time to maximum (peak) concentration 
ULN 
Upper limit of normal 
WT 
Wild-type 


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5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in life-long, 
nonspherocytic hemolytic anemia. It is an autosomal recessive disease with a variable clinical 
presentation, ranging from mild to life-threatening, which can be associated with severe, 
debilitating comorbidities.  
5.1.1. 
Epidemiology and Prevalence 
Epidemiological data for PK deficiency are scarce; however, the current estimated diagnosed 
prevalence of patients with PK deficiency in the United States (US) and European Union 5 
(EU5) (ie, France, Germany, Italy, Spain, the United Kingdom) is approximately 2,400 cases 
(Carey et al, 2000; de Medicis et al, 1992). As with many rare genetic diseases, true prevalence 
of PK deficiency is not well understood (Beutler and Gelbart, 2000). Most recent estimates have 
been cited at approximately 1:20,000-1:485,000 (Beutler and Gelbart, 2000; Carey et al, 2000; 
Hirono et al, 2014; Zanella et al, 2007). 
5.1.2. 
Biochemistry and Genetics 
In normal cells, pyruvate kinase enzymatically catalyzes the metabolic conversion of 
phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) into pyruvate and adenosine 
triphosphate (ATP) as the final step in glycolysis. It is believed that PK deficiency leads to 
insufficient ATP production, resulting in red blood cell (RBC) hemolysis due to an impaired 
ability to maintain cellular membrane homeostasis (van Wijk and van Solinge, 2005). Indeed, PK 
deficiency has been reported to be associated with reduced RBC survival, as well as with 
impaired RBC maturation (Aizawa et al, 2003). 
Pyruvate kinase deficiency is the second most common of the glycolytic enzymopathies after 
glucose-6-phosphate dehydrogenase deficiency. Red blood cells from patients with PK 
deficiency are characterized by changes in metabolism associated with defective glycolysis, 
including a deficiency in ATP levels. Levels of 2,3-diphosphoglycerate (2,3-DPG), PEP, and 
other glycolytic intermediates upstream of the reaction catalyzed by the RBC-specific form of 
pyruvate kinase (PKR) have been reported to be elevated in patients with PK deficiency, 
reflecting the inhibition of glycolysis at the PKR step (Oski and Bowman, 1969) (see Figure 1). 
Red blood cells from patients with PK deficiency show less efficient utilization of glucose than 
the RBCs of normal healthy individuals (Tanaka et al, 1962). 


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Figure 1: 
Glycolysis in Red Blood Cells of Patients With Pyruvate Kinase Deficiency 
 
Abbreviations: 1,3-DPG = 1,3-diphosphoglycerate; 2,3-DPG = 2,3-diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-biphosphate; 
PEP = phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase. 
Note: Not all steps in glycolysis are shown. 
Pyruvate kinase deficiency is an autosomal recessive disease (patients must have 2 mutated 
alleles, usually as compound heterozygotes), and most patients with PK deficiency present with 
unique combinations of poorly characterized or private mutations. In addition to genetic 
heterogeneity, PK deficiency exhibits considerable phenotypic variability. For example, the US 
Pennsylvanian Amish community represents a subgroup of PK deficient patients with a relatively 
homogeneous genetic background (eg, homozygous R479H mutation, restricted marriage pool) 
and uniform lifestyle. Yet, despite these similarities, there is still considerable phenotypic 
variation within this community in terms of disease severity (Grace et al, 2015). 
A non-drug study protocol to obtain critical information regarding the natural history of PK 
deficiency and the range and incidence of related symptoms, treatments, and complications—
known as the PK Deficiency Natural History Study (NHS) (Boston Children’s Hospital and 
Agios, 2017) (NCT02053480)—has been developed by Boston Children’s Hospital (Boston, 
Massachusetts, US) and is funded and supported by Agios. This multicenter, global NHS is 
designed as a longitudinal cohort study with retrospective, baseline, and annual collection of data 
over a 2-year period. The study has thus far identified 123 mutations in 255 subjects (Bianchi et 
al, 2017). Fifty mutations, or approximately 40% of the identified mutations, had not been 
previously described and were only identified as a consequence of the systematic genotyping 
effort for participants in this NHS. This highlights the evolving understanding of the genetic 
basis of PK deficiency. 
An attempt has been made to impose a system of classification onto this large collection of 
potential genotypes by dividing the genotypes into 2 classifications and 3 groups (Bianchi et al, 
2015). This classification showed that 79 of the 123 mutations (64.2%) were missense mutations, 
which are single nucleotide changes that result in amino acid substitutions in the PKR enzyme. 
The effects of these missense mutations can include a loss of catalytic efficiency and/or a loss of 
protein stability of the enzyme. Non-missense mutations include those that cause premature 
truncations of the enzyme, deletions or frameshifts, or mutations that affect splicing of the 
enzyme. Many of these non-missense mutations are predicted to be null alleles of PKR, resulting 
in a lack of functional protein expression (Bianchi et al, 2017). 
5.1.3. 
Clinical Characteristics 
The natural history of untreated PK deficiency is characterized by life-long hemolytic anemia 
and subsequent associated comorbidities, which can include a need for transfusions, 
susceptibility to infections after splenectomy, worsening anemia during pregnancy, and 
symptoms associated with chronic hemolytic anemia (Rider et al, 2011). Some patients with PK 
deficiency may present with severe hemolytic anemia in early infancy that requires immediate 


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care. Unconjugated bilirubin is also often chronically elevated in patients with PK deficiency; 
thus, pigmented gallstones are common in both children and adults with the disease. 
Additionally, iron overload is progressive and can ultimately lead to life-threatening symptoms. 
There are no generally agreed-upon definitions of “mild”, “moderate”, and “severe” disease, 
because multiple factors – such as the degree of anemia, the level of bilirubin and severity of 
jaundice, and complications (including iron overload, transfusion need, and subjective feelings of 
fatigue and low energy level) – must be taken into account before an evaluation can be made.  
There are no guidelines for transfusion management in patients with PK deficiency. Most adults 
with the disease have been splenectomized and require only sporadic or ad hoc transfusions, 
which are usually administered in the context of an acute hemolytic episode triggered by 
infection, trauma, or stress (Grace et al, 2018; Grace et al, 2016; Zanella et al, 2007; Zanella et 
al, 2005). These patients are considered “not regularly transfused.” Some adult patients who have 
not been splenectomized require regular transfusions. A minority of adult patients with PK 
deficiency still require regular transfusions after splenectomy. There is no clear definition of 
what constitutes a regularly transfused patient with PK deficiency; data from the PK Deficiency 
NHS point to a wide range of transfusion frequencies in adults with the disease. 
5.2. 
Investigational Product (AG-348) 
5.2.1. 
Proposed Mechanism of Action of AG-348 
AG-348 is a potent, broad-spectrum activator of PKR, 1 of 4 pyruvate kinase isoenzymes 
expressed in human tissues from 2 separate genes. Both PKR and the liver-specific form of 
pyruvate kinase (PKL) are splice isoforms of the PKLR gene, while pyruvate kinase muscle 
isozyme (PKM)1 and PKM2 are both expressed from the PKM gene. AG-348 is an allosteric 
activator of the PKR, PKL, and PKM2 isoenzymes, with similar activity for each. AG-348 acts 
by directly binding to the PKR tetramer and allosterically enhancing its affinity for PEP. 
As described in Section 5.1.2, the activity of the glycolytic pathway is disrupted in patients with 
PK deficiency. This disruption results in significantly reduced RBC lifespan and manifests 
clinically as nonspherocytic hemolytic anemia. In patients with PK deficiency, RBCs and their 
progenitors are characterized by changes in metabolism associated with defective glycolysis, 
including a buildup of PEP and the intermediate 2,3-DPG, and lowered levels of ATP. It is 
hypothesized that AG-348 restores the ability of RBCs to convert PEP + ADP to pyruvate + ATP 
and thereby normalizes RBC metabolism in patients with PK deficiency. 
5.2.2. 
Summary of AG-348 Nonclinical Data With Potential Clinical Interest 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate wild-type (WT) PKR and anemia-associated PKR mutants in vitro, ex vivo, 
and in vivo. 
Biochemical studies showed that AG-348 is a potent, broad-spectrum activator of recombinant 
PKR with low nanomolar potency against both WT and mutant enzymes. The effect of AG-348 
on PKR activity and a number of downstream pathway markers was evaluated in both human 
and murine RBCs and whole blood. AG-348 dose-response curves in human and murine RBCs 
showed increased PKR activity. AG-348 dose-response curves also showed increased ATP 
levels. 


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The effects of AG-348 on PKR activity and RBC metabolism were also assessed in blood 
samples from subjects with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK-deficient patients with different mutations in the 
PKR enzyme. Finally, a series of 3 in vivo pharmacology studies conducted in C57BL/6 mice 
confirmed the in vitro potency of AG-348 in increasing WT PKR enzyme activity and in 
modulating the levels of the downstream markers, ATP, and 2,3-DPG. Based on the data from 
these studies, a strong pharmacokinetic/pharmacodynamic (PD) relationship was established 
between AG-348 area under the plasma concentration × time curve from 0 to 12 hours (AUC0-12) 
and ATP/2,3-DPG AUC0-12 ratio.  
AG-348 was evaluated for its potential to inhibit binding and enzymatic activity in a panel of 
89 receptors, ion channels, and enzymes. AG-348 is a histamine H3 antagonist/inverse agonist 
and an aromatase inhibitor. Findings consistent with aromatase inhibition have been observed in 
the reproductive organs of male and female rats in toxicology studies of up to 6 months duration 
at exposures as low as 10,900 ng•hr/mL. No findings consistent with aromatase inhibition have 
been observed in monkeys in toxicology studies of up to 9 months duration. Effects consistent 
with aromatase inhibition and antagonism/inverse agonism at the H3 receptor have been 
observed in clinical studies (Section 5.2.3.3). 
Emesis was observed in monkey toxicology studies, and dose-dependent emesis was observed in 
a dedicated safety pharmacology study in ferrets.  
Based on animal studies, AG-348 may affect fertility in males and females. In animals, these 
effects were reversible after discontinuation of AG-348. AG-348 may also affect the ability to 
maintain pregnancy. 
Further details on these and other nonclinical studies, including nonclinical pharmacokinetics, 
are in the AG-348 Investigator’s Brochure (IB). 
5.2.3. 
Summary of AG-348 Clinical Data 
AG-348 has been evaluated in 4 clinical pharmacology studies in healthy subjects (3 completed 
and 1 ongoing) and 1 ongoing Phase 2, open-label, efficacy, and safety study (AG348-C-003, 
referred to as DRIVE-PK) in adult subjects with PK deficiency. 
The ongoing DRIVE-PK study is an open-label study intended for adult subjects with 
PK deficiency who are considered transfusion independent (per protocol definition) with 
screening hemoglobin (Hb) concentration ≤12 g/dL or ≤11 g/dL for males and females, 
respectively. The study is designed to evaluate the safety, tolerability, and potential indicators of 
clinical activity of 2 dose levels of AG-348 (50 and 300 mg twice daily [BID]) administered for 
up to 24 weeks in the Core Period, and beyond 24 weeks in the Extension Period. The 
DRIVE-PK study is also intended to evaluate the pharmacokinetics of AG-348, the PD response 
(ATP and 2,3-DPG levels) after administration of AG-348, and additional PD biomarkers. 
A brief overview of AG-348 pharmacokinetic and PD data is provided in Section 5.2.3.1 and 
Section 5.2.3.2, respectively. An overview of the available safety from these studies as well as 
preliminary efficacy results from the DRIVE-PK study is included in Section 5.2.3.3 and 
Section 5.2.3.4, respectively. Please refer to the AG-348 IB for additional details on all clinical 
studies and results. 


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5.2.3.1. 
Summary of AG-348 Pharmacokinetics 
The pharmacokinetic profile of AG-348 has been well characterized in the Phase 1 
single-ascending dose (SAD) study (AG348-C-001) and Phase 1 multiple-ascending dose 
(MAD) study (AG348-C-002), conducted in healthy adult subjects. The pharmacokinetics of 
AG-348 increased in a dose-proportional manner across tested doses in the SAD study and at 
lower doses in the MAD study. At higher dose levels in the MAD study, a less than 
dose-proportional increase was observed, attributed to the cytochrome P450 (CYP)3A4 
induction effect of AG-348. The effective half-life of AG-348 has been estimated to be 
approximately 3 to 6 hours.  
A capsule formulation was used in the SAD and MAD studies and the DRIVE-PK study. A 
tablet formulation has subsequently been introduced into DRIVE-PK and will also be used in this 
study. Prior to introducing the tablet formulation in clinical studies, a relative bioavailability 
study (AG348-C-005) was conducted in healthy subjects to compare the pharmacokinetics of the 
2 formulations (ie, capsules and tablets). Systemic exposure to AG-348 appeared similar between 
formulations with an area under the plasma concentration × time curve (AUC) ratio of 1.05 and a 
maximum (peak) concentration (Cmax) ratio of 1.19 for the tablet formulation compared with the 
capsule formulation. These results suggest that no dose adjustments are required with the tablet 
formulation. Therefore, when the tablet formulation is used, the same dose as that of the capsule 
can be used in ongoing and future clinical studies. 
Additionally, in the ongoing Phase 2 DRIVE-PK study, conducted in adult subjects with 
PK deficiency, the pharmacokinetics of AG-348 in plasma have been evaluated. To date, 
pharmacokinetic data of AG-348 in adult subjects with PK deficiency were found to be similar to 
that observed in healthy adult subjects. 
Please refer to the AG-348 IB for detailed information regarding the pharmacokinetics of 
AG-348. 
5.2.3.2. 
Summary of AG-348 Pharmacodynamics 
In the SAD and MAD studies, the concentration of 2,3-DPG decreased in a dose-dependent 
manner and returned to levels close to baseline by 72 hours following the final dose of AG-348. 
In the SAD study, after a single dose of AG-348, a minimal increase in the concentration of ATP 
was observed at 24 to 120 hours postdose. In contrast to the SAD study, significant increases in 
ATP were observed in the MAD study, and the concentration of ATP remained elevated through 
120 hours after the final dose of AG-348. 
In the DRIVE-PK study, conducted in adult subjects with PK deficiency, the PD responses of 
ATP and 2,3-DPG in whole blood have also been evaluated. In this study, no consistent pattern 
of decrease in concentration of 2,3-DPG or increase in ATP has been observed. The reason for 
this is not completely clear, but may be due in part to changes in reticulocyte concentrations in 
response to treatment with AG-348. 
Refer to the AG-348 IB for detailed information regarding the PD properties of AG-348. 
5.2.3.3. 
Summary of AG-348 Clinical Safety Data 
Overall, AG-348 has been generally well tolerated among healthy adult subjects and adult 
subjects with PK deficiency. Important identified risks associated with administration of AG-348 


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in clinical studies include bone mineral density (BMD) decrease (including osteoporosis and 
osteopenia due to aromatase inhibition), withdrawal hemolysis, and insomnia (not clinically 
serious ie, not Grade 3 or Grade 4).  
Potential risks of AG-348 administration include anaphylactoid reaction, aromatase inhibition, 
gastrointestinal disturbances, transaminase increases, and triglyceride increase. Transaminase 
increases are adverse events of special interest (AESIs) for AG-348. Please refer to 
Section 11.2.6 for additional information on AESIs and the AG-348 IB for a more detailed 
overview of available safety data. 
5.2.3.4. 
Summary of AG-348 Efficacy Data 
In the ongoing Phase 2 DRIVE-PK study in adult subjects with PK deficiency, the efficacy of 
AG-348 is primarily analyzed via evaluation of changes in Hb concentrations.  
As of a data cutoff of 14 July 2017, a preliminary analysis indicates that of the 52 subjects who 
received AG-348 during the Core Period, 26 subjects (50.0%) achieved maximum increases in 
Hb >1 g/dL (Grace et al, 2017). Of the 42 subjects with ≥1 missense mutation, 25 subjects 
(59.5%) had an Hb increase >1.0 g/dL. The majority of Hb increases were rapid and sustained. 
The median time to the first observation of an Hb increase >1 g/dL above baseline was 10 days 
(range 7 to 187 days). 
Overall, treatment with AG-348 has resulted in Hb responses that are rapid in onset, robust, and 
sustained with prolonged treatment with AG-348. In summary, this clinical evidence from Study 
AG348-C-003 suggests that treatment with AG-348 has the ability to provide a significant 
clinical benefit to patients.  
5.3. 
Study Rationale 
The totality of the preliminary data from the Phase 2 DRIVE-PK study, combined with the data 
from the 2 completed Phase 1 SAD and MAD studies, support continued clinical development of 
AG-348 for the treatment of patients with PK deficiency. In order to evaluate the efficacy and 
safety of AG-348 across the disease spectrum of PK deficiency, 2 studies investigating the 
treatment of AG-348 in subjects with PK deficiency will be conducted. This protocol, 
AG348-C-006, will be conducted in adult subjects with PK deficiency who are not regularly 
receiving transfusions, while a separate study, AG348-C-007, will be conducted in adult subjects 
with PK deficiency who are regularly receiving transfusions.  
A clear and serious unmet medical need exists for patients with PK deficiency. At present, there 
are no approved, disease-specific therapeutic agents for the treatment of patients with PK 
deficiency; rather, available treatment options are supportive only. Many adult patients 
underwent splenectomy in childhood as a means to reduce hemolysis and eliminate the need for 
regular transfusions. However, some patients, despite undergoing splenectomy, still have 
Hb concentration below the lower limit of normal and have symptoms associated with anemia, 
such as shortness of breath, fatigue, lower energy, lower exercise tolerance, and other symptoms 
that affect quality of life (QoL).  
Treatment with AG-348 has the potential to correct the underlying pathology of PK deficiency 
by activating PKR and increasing glycolytic pathway activity in RBCs to reduce hemolysis and 
provide clinical benefit to subjects with PK deficiency.  


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5.3.1. 
Justification of Study Design 
This is a 2-part, Phase 3, randomized, multicenter, double-blind, placebo-controlled study in 
which subjects will be randomized 1:1 to receive either AG-348 or matching placebo. The 
randomized controlled trial design was chosen as the most robust design for the evaluation of 
safety and efficacy of AG-348, with the best potential for minimizing bias.  
The randomization will be stratified by the average of screening Hb concentrations 
(<8.5 vs ≥8.5 g/dL [5.28 mmol/L]) and the PKLR gene mutation category (missense/missense vs 
missense/non-missense). These stratification factors were chosen because both the average of 
screening Hb concentrations and the PKLR gene mutation category are related to the Hb 
response based on preliminary data from the Phase 2 DRIVE-PK study.  
In Part 1 (Dose Optimization Period), subjects will follow a dose titration schema (as described 
in Section 5.3.2.1) to identify their individually optimized dose of study treatment 
(ie, AG-348/matching placebo). In Part 2 (Fixed Dose Period), subjects will continue treatment 
with their individually optimized dose. Subjects will be treated in this study for 24 weeks 
(not including the recommended dose taper), including 12 weeks for the Dose Optimization 
Period and 12 weeks on the Fixed Dose Period. Additional details on the study design are 
provided in Section 7.1. See Section 5.3.1.1, Section 5.3.1.2, and Section 5.3.2 for further 
justification regarding specific aspects of the study design. 
5.3.1.1. 
Justification of Subject Population 
This study will enroll adult subjects with PK deficiency and moderate to severe anemia 
(Hb ≤10 g/dL [6.21 mmol/L]), who are not regularly receiving transfusions. For the purpose of 
this study, the following definition will be used for subjects who are not regularly receiving 
transfusions: no more than 4 transfusion episodes in the 12-month period up to the first day of 
study treatment and no transfusions in the 3 months prior to the first day of study treatment.  
As mentioned in Section 5.1.3, there are no published guidelines on the use of transfusion, and 
the appropriate transfusion frequency in PK deficient patients who may need transfusions. 
Hemolysis in patients with PK deficiency can be acutely exacerbated by infection, surgery, 
pregnancy, and other forms of stress. Since several such episodes can occur per year, it is 
considered that up to 4 transfusion episodes in the preceding 12 months reflects such stressors 
rather than a true dependency on regular transfusions. Because the Hb concentration, which 
informs the primary endpoint of the study, is influenced by transfusions in the preceding several 
months, the most recent transfusion must have occurred at least 3 months prior to the first day of 
study treatment. 
Additional inclusion criteria for this study have been added to increase the likelihood that the 
subject population will achieve a clinical benefit from the study treatment. This study will 
require an average screening Hb concentration of ≤10 g/dL (6.21 mmol/L), to ensure that all 
subjects enrolled in the study have a moderate-to-severe degree of anemia.  
Subjects who are homozygous for the R479H mutation or have 2 non-missense mutations, 
without the presence of another missense mutation, in the PKLR gene will not be included in this 
study. It is expected that non-missense mutations are more likely to result in loss of functional 
protein expression, as opposed to the missense mutations, which result in a single amino acid 
substitution. Genotype-response analysis in the DRIVE-PK study demonstrated in general that 


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all 10 PK-deficient subjects with 2 non-missense alleles did not respond to treatment with 
AG-348. Additionally, all 5 subjects with the R479H/R479H genotype (drawn from the 
 
) in the DRIVE-PK study were non-responders.  
5.3.1.2. 
Justification of Study Objectives  
The primary endpoint of this study will be the assessment of the Hb response (HR). Additionally, 
Hb concentrations over time and maximal Hb changes will be assessed as secondary efficacy 
measures.  
Hemoglobin response will be defined as a ≥1.5 g/dL (0.93 mmol/L) increase in Hb concentration 
from baseline that is sustained at 2 or more scheduled assessments at Weeks 16, 20, and 24 
during the Fixed Dose Period. A sustained Hb increase of ≥1.5 g/dL represents a clinically 
meaningful benefit as suggested by the standard use of splenectomy and transfusions for patients 
with PK deficiency. Splenectomy increases Hb by 1-3 g/dL and is considered an effective and 
standard treatment in PK deficiency (Grace et al, 2015; Zanella et al, 2007; Zanella et al, 2005), 
while transfusions, typically of 2 units of packed RBCs, increase Hb by about 2 g/dL in adults 
(Weinstein, 2012). There is abundant literature suggesting that such an increase in Hb delivers an 
immediate subjective benefit in patients (Ryblom et al, 2015). In particular, the FACT-An 
(Functional Assessment of Cancer Therapy Anemia) instrument has shown that transfusions 
result in an Hb increase that is above the value defined as a minimal clinically important 
difference (MCID) (Cella et al, 2002). Importantly, a substantial positive correlation between 
post-transfusion Hb concentrations and FACT-An scores has been established. 
Pyruvate kinase deficiency is a chronic hemolytic anemia. The hemolysis itself leads to 
morbidity (eg, jaundice, gall stones). Another important adverse consequence of the disease is 
the risk of iron overload, worsened by frequent transfusions and splenectomy. Altogether, these 
factors negatively impact patients’ QoL. Therefore, this study is designed to investigate multiple 
additional objective and subjective measures in PK deficiency, among them hematopoietic 
activity, hemolysis, iron overload, and QoL.  
5.3.2. 
Rationale for Dose 
To assist with dose selection, an exposure-response analysis was conducted using 
pharmacokinetic, efficacy, and safety data from the DRIVE-PK study.  
Briefly, a sequential population pharmacokinetic-efficacy model was developed using increase in 
Hb as the efficacy endpoint, while a binary logistic regression approach incorporating the safety 
endpoints of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total and free 
testosterone, estrone, estradiol, insomnia, and hot flush was used for the analysis of 
exposure-safety relationship. Insomnia was the only safety event that was found to be significant 
in the logistic regression analysis.  
Following model development, simulations were conducted to select 3 dose levels for evaluation 
in Phase 3 studies:  
1. The low dose level was identified as a dose at which patients were likely to have an Hb 
increase of ≥1.5 g/dL (0.93 mmol/L) from baseline without exceeding the upper limit of 
normal (ULN) Hb (efficacy criterion), with a minimal probability of occurrence of 
Grade ≥1 insomnia (safety criterion).  


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2. Since the mid dose (20 mg BID) involves an intra-patient dose level increase, this dose 
level was selected such that it would result in a reasonable increase (approximately 2- to 
2.5-fold) in exposure compared to the predicted exposure at the low dose level. 
3. Since the high dose (50 mg BID) also involves an intra-patient dose level increase, this 
dose level was selected such that it would result in a reasonable increase (approximately 
2- to 2.5-fold) in exposure compared to the predicted exposure at the mid dose level. 
Using these criteria and simulations from the population pharmacokinetic-efficacy-safety 
analyses, doses of 5 mg BID, 20 mg BID, and 50 mg BID were selected for the Dose 
Optimization Period.  
5.3.2.1. 
Justification of Individual Dose Optimization 
In the DRIVE-PK study of AG-348 in PK deficiency, subjects were randomized to receive doses 
of either 50 mg or 300 mg BID of AG-348. The preliminary results of this study indicated that 
efficacy could be achieved at lower doses than 300 mg in the majority of subjects. More 
specifically, several subjects had to have their randomized dose level reduced because of excess 
increases in Hb or the occurrence of adverse events (AEs), such as insomnia, headache, or 
nausea. Dose reductions led to the resolution of most of these AEs and the maintenance of a 
satisfactory but not excessive level of Hb. In the pivotal studies, Hb overshoots will be avoided 
by gradually titrating the dose upward in each subject.  
For this reason, all subjects will start on an initial dose of study treatment of 5 mg BID with 
2 potential sequential steps for dose level increases (ie, from 5 to 20 mg BID and from 20 to 
50 mg BID), depending on safety and Hb change. 
Preliminary data from the DRIVE-PK study indicated that subjects with PK deficiency who 
respond to activation of the PKR protein typically do so within 2-3 weeks. Therefore, every 
4 weeks during Part 1, subjects will be assessed for safety and efficacy (as defined by Hb 
increase) to determine if their dose should be increased, maintained at the current level, or 
decreased. 
Individual dose optimization is incorporated in this study to allow each subject to gradually 
increase his/her dose of AG-348 in order to identify a dose that confers maximum benefit with 
minimum risk to that subject. This approach is made possible by the rapid responses to activation 
of PKR that were observed in the DRIVE-PK study and that are consistent with the mechanism 
of action of this class of drugs. 
Details on maintaining the study blind during the dose-optimization part of the study can be 
found in Section 9.3. 


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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Objectives 
6.1.1. 
Primary Objective 
The primary objective of the study is to evaluate the efficacy of treatment with AG-348 
compared with placebo in increasing Hb concentrations. 
6.1.2. 
Secondary Objectives 
 To evaluate the safety of AG-348 
 To determine the effect of the study treatment regimens on markers of hemolysis, 
hematopoietic activity, and other indicators of clinical activity 
 To determine the effect of the study treatment regimens on health-related quality of 
life (HRQoL), as determined using patient-reported outcomes (PROs) 
 To evaluate the pharmacokinetics of AG-348 after oral administration 
 To evaluate the relationship between AG-348 pharmacokinetics and safety 
parameters 
6.1.3. 
Exploratory Objectives 
 To evaluate the relationship of AG-348 pharmacokinetics to indicators of clinical 
activity 
 To evaluate the PD markers of PK deficiency and how they are affected by study 
treatment 
 To determine the effect of the study treatment regimens on: 
 Number of transfusion events and number of RBC units transfused 
 Markers of iron metabolism and indicators of iron overload 
6.2. 
Endpoints 
6.2.1. 
Primary Endpoint 
The primary endpoint is the HR, defined as a ≥1.5 g/dL (0.93 mmol/L) increase in Hb 
concentration from baseline that is sustained at 2 or more scheduled assessments at Weeks 16, 
20, and 24 during the Fixed Dose Period. The individual subject’s baseline Hb concentration is 
defined as the average of all available Hb concentrations collected for that subject during the 
Screening Period up to the first dose of study treatment. 
6.2.2. 
Key Secondary Endpoint 
The key secondary endpoint is the average change from baseline in Hb concentration at 
Weeks 16, 20, and 24.  


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6.2.3. 
Other Secondary Endpoints 
 Maximal Hb concentration increase from baseline 
 Time to first achieve an increase in Hb concentration of 1.5 g/dL (0.93 mmol/L) or 
more from baseline  
 Average change from baseline at Weeks 16, 20, and 24 in markers of hemolysis: 
bilirubin, lactate dehydrogenase (LDH), and haptoglobin levels 
 Average change from baseline at Weeks 16, 20, and 24 in markers of hematopoietic 
activity: reticulocyte percentages 
 Change from baseline in HRQoL PRO scores: Pyruvate Kinase Deficiency Diary 
(PKDD) and Pyruvate Kinase Deficiency Impact Assessment (PKDIA) 
 Safety endpoints, including: the type, incidence, severity, and relationship to study 
treatment of AEs and serious adverse events (SAEs); number of discontinuations due 
to AEs; results of clinical laboratory tests over time (eg, serum chemistry, liver 
function tests (LFTs), hematology, lipids, sex steroids, urinalysis, coagulation); 
physical examination (PE) findings; dual-energy x-ray absorption (DXA) scans; vital 
signs; 12-lead electrocardiogram (ECG) data 
 Pharmacokinetic endpoints, including plasma concentrations over time and 
pharmacokinetic parameters of AG-348 (eg, AUC, Cmax, others as applicable) 
 Exposure-response relationship between safety parameters and AG-348 concentration 
and relevant AG-348 pharmacokinetic parameters 
6.2.4. 
Exploratory Endpoints 
 Exposure-response (or pharmacokinetic-pharmacodynamic) relationship between 
relevant pharmacokinetic parameters and endpoints that are indicators of clinical 
activity 
 Change from baseline in additional markers of hematopoietic activity 
 Change from baseline in markers of iron metabolism and indicators of iron overload 
 Change from baseline in PKR protein level 
 Relationship between baseline PKR protein level and Hb response status 
 Change from baseline in HRQoL PRO scores: European quality of life 
five-dimensional descriptive system (EQ-5D-5L) 
 Change from baseline in PKR flux assay results 
 Proportion of subjects requiring transfusions and the total number of RBC units 
transfused 


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7. 
INVESTIGATIONAL PLAN 
7.1. 
Study Design 
7.1.1. 
Overview of Study Design 
This is a Phase 3, randomized, multicenter, double-blind, placebo-controlled study consisting of 
a Dose Optimization Period (Part 1) followed by a Fixed Dose Period (Part 2) as shown in 
Figure 2. This study will evaluate the efficacy and safety of orally administered AG-348 as 
compared with placebo in subjects with PK deficiency who are not regularly receiving blood 
transfusions. Blinding and unblinding procedures are detailed in Section 9.3 and Section 9.4, 
respectively. 
Approximately 76 subjects will be randomized 1:1 to receive either AG-348 or matching 
placebo. The term “study treatment” is used throughout the protocol to define both the active 
(AG-348) drug and placebo. Randomization will be stratified by the average of screening 
Hb concentrations (<8.5 vs ≥8.5 g/dL [5.28 mmol/L]) and the PKLR gene mutation category 
(missense/missense vs missense/non-missense). In rare instances where PKLR gene mutation 
category cannot be made definitively (eg, if a subject harbors 3 mutant PKLR alleles), the subject 
will be assigned to the missense/non-missense category. 
Each subject’s course of participation will be composed of the following periods: 
Screening Period: Prior to randomization, there will be a Screening Period of up to 42 days in 
which a subject’s eligibility for the study will be determined and confirmed. A subject’s 
Screening Period duration may be extended beyond 42 days upon the Medical Monitor’s, or 
designee’s, approval. 
Dose Optimization Period (Part 1): Following randomization, subjects will enter Part 1 of the 
study. During Part 1, subjects will receive scheduled dose increases depending on their response 
to study treatment (safety and efficacy), as detailed in Section 7.1.3, to determine their 
individually optimized dose level.  
Fixed Dose Period (Part 2): Following Part 1, subjects will proceed to Part 2 and continue study 
treatment on their individually optimized dose. For the purposes of dosing during the Fixed Dose 
Period, the dose the subject is being administered at the Week 12 Visit will be considered the 
subject’s optimized dose and will be the dose the subject receives during Part 2. All subjects who 
remain on study during Part 2 through the Week 24 Visit may be eligible for an open-label 
extension study, in which all subjects will receive AG-348. 
Discontinuation and follow-up: Subjects who discontinue the study prior to the Week 24 Visit 
should attend the End of Study Visit 28±4 days after the last study visit that the subject attended, 
or 28±4 days after the last dose of study treatment (including the recommended dose taper), 
whichever is later. The End of Study Visit will be identical to the Week 24 Visit with these 
exceptions: no study treatment will be dispensed, and if the subject has not already done so, the 
subject should return their electronic diary (eDiary) and remaining study treatment. These 
subjects do not need to complete the Follow-Up visit. 
Subjects who continue the study through the Week 24 Visit on study treatment but do not 
continue on into an extension study, will attend the Follow-up Visit 28±4 days after the last dose 


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of study treatment. Subjects who continue on into an extension study at the Week 24 Visit will 
not be required to attend the Follow-up Visit. 
All subjects who discontinue or interrupt study treatment during the study should undergo the 
recommended dose taper (see Section 9.5.3), unless an emergency situation justifies 
discontinuing or interrupting the study treatment abruptly. Whether or not the recommended 
dose taper is performed, subjects who discontinue or interrupt study treatment should be 
monitored as clinically indicated for signs of withdrawal hemolysis and worsening of anemia. 
The maximum total duration that a subject can receive study treatment in this study is 24 weeks 
(not including the recommended dose taper).  
The Screening Period, Part 1, Part 2, and follow-up are described in more detail in 
Sections 7.1.2, 7.1.3, 7.1.4, and 7.1.5, respectively.  
Figure 2: Overview of Design for Study AG348-C-006 
 
Abbreviations: BID = twice daily. 
1Stratified by average of screening Hb concentrations (Hb <8.5 g/dL vs Hb ≥8.5 g/dL [5.28 mmol/L]) and the PKLR 
gene mutation category (missense/missense vs missense/non-missense) 
2 A subject’s Screening Period duration may be extended beyond 42 days upon the Medical Monitor’s, or 
designee’s, approval. 
7.1.2. 
Screening Period 
Following signing of the informed consent form (ICF), subjects will enter the Screening Period 
to determine eligibility. Screening assessments will be performed after the signing of the ICF and 
within 42 days prior to Day 1 of the study. A subject’s Screening Period duration may be 
extended beyond 42 days upon the Medical Monitor’s, or designee’s, approval. 
The Investigator will determine whether each subject meets all the inclusion criteria and none of 
the exclusion criteria (see Section 8). Eligibility of each subject will be confirmed by the Medical 


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Monitor, or designee. A subject cannot be randomized (and cannot receive his/her first dose of 
study treatment) until eligibility is confirmed.  
Subjects will receive an eDiary at screening to document dosing, HRQoL assessments, and 
menstrual cycle (for menstruating women) throughout the study.  
For each subject, the following information will be collected during screening (see 
Section 10.13): 
 Pyruvate kinase deficiency specific assessments: PKR enzyme assay, PKR 
genotyping, and dates of all transfusions received during the 12-month period prior to 
the first day of study treatment 
 General assessments: Medical/surgical history (as detailed in Section 10.4), 
demographics, prior medications, PE, 12-lead ECG, vital signs, hepatitis B surface 
antigen (HBsAg), hepatitis C virus (HCV) antibody (Ab), human immunodeficiency 
virus (HIV)-1 and -2 Ab, and follicle-stimulating hormone (FSH) (to confirm 
post-menopausal status, as applicable) or serum human chorionic gonadotropin (hCG) 
pregnancy test (in women of reproductive potential) 
 Patient-reported outcome assessments: PKDD daily and PKDIA, FACT-An, SF12v2, 
EQ-5D-5L, and PGIS all on the first day of screening (+1 day) and then at weekly 
intervals (±1 day) until Part 1 Day 1 
 Assessment of iron overload: Liver magnetic resonance imaging (MRI) to assess liver 
iron concentration (LIC); iron panel and related markers 
 Other: DXA scans 
 Safety and efficacy laboratory markers: Hematology; LDH and haptoglobin 
 At least 2 hematology labs will be collected (at least 7 days apart) 
 Safety laboratory markers: Serum chemistry, LFTs, coagulation studies, urinalysis, 
and lipids  
 Other laboratory markers: Markers of erythropoietic activity 
7.1.3. 
Part 1: Dose Optimization Period  
On Day 1, the following additional conditions must be met before a subject, who is considered 
eligible after completing screening, can receive the first dose of study treatment: 
 The subject must not have received a transfusion since completing screening 
(see Section 8.1, Inclusion Criterion 5). 
 Women of reproductive potential must have a negative urine or serum pregnancy test 
(see the Schedule of Assessments Section 10.13). 
Following randomization to either AG-348 or matching placebo, subjects will enter the Dose 
Optimization Period (Part 1), a 12-week period starting on Day 1 of the study. The goal of the 
Dose Optimization Period is to maximize a subject’s increase in Hb while maintaining an 
acceptable safety profile. All subjects will receive an initial dose of 5 mg BID of study treatment 
with 2 potential sequential steps for dose level increase (ie, from 5 to 20 mg BID and from 20 to 


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50 mg BID; no increases beyond 50 mg BID will be allowed). The first dose of study treatment 
on Day 1 should be taken at the study site following all Day 1 assessments (with the exception of 
eDiary assessments) as depicted in the Schedule of Assessments (Section 10.13). 
Subjects will be assessed for safety and efficacy (as defined by Hb increase) every 4 weeks 
during Part 1, to determine if their dose should be increased, maintained at the current level, or 
decreased. At the Week 4 and Week 8 Visits, study treatment dose should be increased to the 
next dose level if the subject has met both of the following criteria: 
 The subject is tolerating the study treatment, and  
 The subject’s Hb concentration on the day of the visit based on local laboratory 
results is lower than 2.5 g/dL (1.55 mmol/L) below the upper limit of normal (ULN), 
as applies to men and women. 
Dose re-escalation or re-introduction should be avoided after the Week 8 Visit, but may be 
permitted, after discussion with the Independent Medical Monitor, or designee. 
At the Week 12 Visit, if the subject has tolerated the study treatment, the subject will remain at 
his/her current dose level. If the Investigator deems it necessary to reduce the study treatment for 
safety reasons, the subject’s dose may be reduced to 1 of the 2 available lower dose levels 
(ie, 5 mg BID, 20 mg BID). If the subject is already receiving 5 mg BID and/or cannot tolerate 
BID dosing, another regimen may be allowed after discussion with, and approval by, the 
Independent Medical Monitor, or designee. 
If questions arise about whether the dose level of a given subject should be increased, 
maintained, or decreased, the Independent Medical Monitor, or designee, should be contacted.  
At any time during the study, the Investigator can discontinue, interrupt, or reduce the 
subject’s dose of study treatment for reasons related to safety (as described in Section 9.5). 
Over the course of Part 1 of the study, subjects will attend visits on Day 1, Week 2, Week 4, 
Week 6, Week 8, Week 10, and Week 12. Details on the timing and type of assessments for 
Part 1 are outlined in the Schedule of Assessments (Section 10.13). 
7.1.4. 
Part 2: Fixed Dose Period  
Part 2 is the 12-week period following the Week 12 Visit through Week 24. 
Following Part 1, each subject will remain on his/her individually optimized dose and enter the 
Fixed Dose Period (Part 2). For the purposes of dosing during the Fixed Dose Period, the dose 
the subject is being administered at the Week 12 Visit will be considered the subject’s optimized 
dose and will be the dose the subject receives during Part 2. 
At any time during the study, the Investigator can discontinue, interrupt, or reduce the 
subject’s dose of study treatment for reasons related to safety (as described in Section 9.5).  
Dose re-escalation or re-introduction should be avoided after the Week 8 Visit (Part 1), but may 
be permitted, after discussion with the Independent Medical Monitor, or designee. 
Subjects will visit the study site every 4 weeks through Week 24 (ie, Week 16, Week 20, and 
Week 24). Each study visit will include efficacy and safety assessments. Assessments will be 
performed as outlined in the Schedule of Assessments (Section 10.13).  


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7.1.5. 
Study Discontinuation and Follow-up 
Subjects who discontinue the study prior to the Week 24 Visit should attend the End of Study 
Visit 28±4 days after the last study visit that the subject attended or 28±4 days after the last dose 
of study treatment (including the recommended dose taper), whichever is later. This visit will be 
identical to the Week 24 Visit with these exceptions: no study treatment will be dispensed at the 
End of Study Visit, and if the subject has not already done so, they should return their eDiary and 
remaining study treatment. These subjects do not need to complete the Follow-Up Visit. 
Subjects who continue study treatment through Week 24 should continue taking study treatment 
at least through the morning dose of the Week 24 Visit. All subjects who remain on study during 
Part 2 through the Week 24 Visit may be eligible for an open-label extension study, in which all 
subjects will receive AG-348. 
 Subjects who continue study treatment through the Week 24 Visit but do not continue 
on into an extension study, should undergo the recommended dose taper and then 
attend the Follow-up Visit 28±4 days after the last dose of study treatment (including 
the recommended dose taper). 
 Subjects who continue on into an extension study at the Week 24 Visit will not be 
required to attend the Follow-up Visit.  
 Subjects who initiate or are undergoing the recommended dose taper, and for whom 
participation in an extension study remains undetermined, should perform the taper as 
detailed in Section 9.5.3. Transition to an extension study should be discussed with 
the Independent Medical Monitor. 
All subjects who discontinue or interrupt study treatment during the study should undergo the 
recommended dose taper (see Section 9.5.3), unless an emergency situation justifies 
discontinuing or interrupting the study treatment abruptly. 
Guidelines for the follow-up of subjects with any AE at the subject’s completion of the study are 
described in Section 11. 
7.2. 
Number of Subjects 
Approximately 76 subjects are planned for enrollment. 
7.3. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
Circumstances that may warrant termination include, but are not limited to the following: 
 Determination of unexpected, significant, or unacceptable risk to subjects 
(eg, as determined by the Independent Data Monitoring Committee [IDMC]). 
 Plans to modify, suspend, or discontinue the development of the study treatment. 
 Decisions of competent authorities or Institutional Review Board (IRB)/Independent 
Ethics Committee (IEC) 


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 Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee.  


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8. 
SELECTION AND WITHDRAWAL OF SUBJECTS 
8.1. 
Inclusion Criteria 
For enrollment into this study, subjects must meet all of the following criteria during the 
Screening Period: 
1. Have provided signed written informed consent prior to performing any study procedure, 
including screening procedures. 
2. Be aged 18 years or older. 
3. Have documented clinical laboratory confirmation of PK deficiency, defined as 
documented presence of at least 2 mutant alleles in the PKLR gene, of which at least 1 is 
a missense mutation, as determined per the genotyping performed by the central 
genotyping laboratory. 
4. Have an Hb concentration less than or equal to 10.0 g/dL (6.21 mmol/L) regardless of 
gender (average of at least 2 Hb measurements [separated by a minimum of 7 days] 
during the Screening Period). 
5. Be considered not regularly transfused, defined as having had no more than 4 transfusion 
episodes in the 12-month period up to the first day of study treatment and no transfusions 
in the 3 months prior to the first day of study treatment.  
6. Have received at least 0.8 mg oral folic acid daily for at least 21 days prior to the first 
dose of study treatment, to be continued daily during study participation. 
7. Have adequate organ function, as defined by: 
a. Serum AST ≤2.5 × ULN (unless the increased AST is assessed by the Investigator as 
due to hemolysis and/or hepatic iron deposition) and ALT ≤2.5 × ULN (unless the 
increased ALT is assessed by the Investigator as due to hepatic iron deposition). 
b. Normal or elevated levels of serum bilirubin. In subjects with serum bilirubin >ULN, 
the elevation must not be associated with choledocholithiasis, cholecystitis, biliary 
obstruction, or hepatocellular disease. Elevated bilirubin attributed to hemolysis with 
or without Gilbert’s syndrome is not exclusionary. 
c. Estimated glomerular filtration rate (GFR) ≥60 mL/min/1.73 m2, measured GFR 
≥60 mL/min, or calculated creatinine clearance (CrCL; Cockcroft-Gault) 
≥60 mL/min. 
d. Absolute neutrophil count (ANC) ≥1.0 × 109/L (based on an average of at least 
2 measurements [separated by a minimum of 7 days] during the Screening Period). 
e. Platelet count ≥100 × 109/L in the absence of a spleen, or platelet count ≥50 × 109/L 
in the presence of a spleen and in the absence of any other cause of thrombocytopenia 
(based on an average of at least 2 measurements [separated by a minimum of 7 days] 
during the Screening Period). 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤1.25 × ULN, unless the subject is receiving therapeutic anticoagulants. 
8. For women of reproductive potential, have a negative serum pregnancy test during the 
Screening Period. Women of reproductive potential are defined as sexually mature 
women who have not undergone a hysterectomy, bilateral oophorectomy, or tubal 


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occlusion; or who have not been naturally postmenopausal (ie, who have not menstruated 
at all for at least the preceding 12 months prior to signing informed consent and have an 
elevated FSH level indicative of menopause during the Screening Period). 
9. For women of reproductive potential as well as men with partners who are women of 
reproductive potential, be abstinent as part of their usual lifestyle, or agree to use 2 forms 
of contraception, 1 of which must be considered highly effective, from the time of giving 
informed consent, during the study, and for 28 days following the last dose of study 
treatment for women and 90 days following the last dose of study treatment for men. A 
highly effective form of contraception is defined as combined (estrogen and progestin 
containing) hormonal contraceptives (oral, intravaginal, or transdermal) known to be 
associated with inhibition of ovulation; progestin-only hormonal contraceptives (oral, 
injectable, or implantable) known to be associated with inhibition of ovulation; 
intrauterine device; intrauterine hormone releasing system; bilateral tube occlusion; or 
vasectomized partner. The second form of contraception can include an acceptable barrier 
method, which includes male or female condoms with or without spermicide, and 
cervical cap, diaphragm, or sponge with spermicide. Women of reproductive potential 
using hormonal contraception as a highly effective form of contraception must also 
utilize an acceptable barrier method while enrolled in the study and for at least 28 days 
after their last dose of study treatment. 
10. Be willing to comply with all study procedures for the duration of the study. 
8.2. 
Exclusion Criteria 
Subjects who meet any of the following criteria during screening will not be enrolled in the 
study: 
1. Are homozygous for the R479H mutation or have 2 non-missense mutations, without the 
presence of another missense mutation, in the PKLR gene as determined per the 
genotyping performed by the central genotyping laboratory.  
2. Have a significant medical condition that confers an unacceptable risk to participating in 
the study, and/or that could confound the interpretation of the study data. Such significant 
medical conditions include, but are not limited to the following: 
a. Poorly controlled hypertension (defined as systolic blood pressure [BP] >150 mmHg 
or diastolic BP >90 mmHg) refractory to medical management.  
b. History of recent (within 6 months prior to signing informed consent) congestive 
heart failure; myocardial infarction or unstable angina pectoris; hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Cardiac dysrhythmias judged as clinically significant by the Investigator. 
d. Heart-rate corrected QT interval-Fridericia's method (QTcF) >450 msec (average of 
triplicate ECGs) with the exception of subjects with right or left bundle branch block. 
e. Clinically symptomatic cholelithiasis or cholecystitis. Prior cholecystectomy is not 
exclusionary. Subjects with symptomatic cholelithiasis or cholecystitis may be 
rescreened once the disorder has been treated and clinical symptoms have resolved. 
f. History of drug-induced cholestatic hepatitis. 


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g. Iron overload sufficiently severe to result in a clinical diagnosis by the Investigator of 
cardiac (eg, clinically significant impaired left ventricular ejection fraction), hepatic 
(eg, fibrosis, cirrhosis), or pancreatic (eg, diabetes) dysfunction. 
h. Have a diagnosis of any other congenital or acquired blood disorder or any other 
hemolytic process, except mild allo-immunization, as a consequence of transfusion 
therapy. Genetic findings that in isolation are predicted to be insufficient to explain 
the observed clinical phenotype may be allowed (eg, heterozygous status for certain 
recessive red blood cell disorders). 
i. Positive test for HBsAg or HCVAb with signs of active hepatitis B or C virus 
infection. If the subject is positive for HCVAb, a reverse transcriptase-polymerase 
chain reaction test will be conducted. Subjects with hepatitis C may be rescreened 
after receiving appropriate hepatitis C treatment. 
j. Positive test for HIV-1 or -2 Ab. 
k. Active infection requiring the use of parenteral antimicrobial agents or Grade ≥3 in 
severity (per NCI CTCAE [National Cancer Institute Common Terminology Criteria 
for Adverse Events) within 2 months prior to the first dose of study treatment. 
l. Diabetes mellitus judged to be under poor control by the Investigator or requiring 
>3 antidiabetic agents, including insulin (all insulins are considered 1 agent); use of 
insulin per se is not exclusionary.  
m. History of any primary malignancy, with the exception of: curatively treated 
nonmelanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
or other primary tumor treated with curative intent, no known active disease present, 
and no treatment administered during the last 3 years. 
n. Unstable extramedullary hematopoiesis that could pose a risk of imminent neurologic 
compromise. 
o. Current or recent history of psychiatric disorder that, in the opinion of the Investigator 
or Medical Monitor, or designee, could compromise the ability of the subject to 
cooperate with study visits and procedures. 
3. Have a splenectomy scheduled during the study treatment period or have undergone 
splenectomy within 12 months prior to signing informed consent. 
4. Are currently enrolled in another therapeutic clinical trial involving ongoing therapy with 
any investigational or marketed product or placebo. Prior and subsequent participation in 
the PK Deficiency NHS (NCT02053480) or PK Deficiency Registry is permitted, 
however, concurrent participation is not. Therefore, subjects enrolling in this current 
study will be expected to temporarily suspend participation in the NHS or Registry.  
5. Have exposure to any investigational drug, device, or procedure within 3 months prior to 
the first dose of study treatment. 
6. Have had any prior treatment with a pyruvate kinase activator. 
7. Have a prior bone marrow or stem cell transplant. 
8. Are currently pregnant or breastfeeding. 
9. Have a history of major surgery within 6 months of signing informed consent. Note that 
procedures such as laparoscopic gallbladder surgery are not considered major in this 
context. 


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10. Are currently receiving medications that are strong inhibitors of CYP3A4, strong 
inducers of CYP3A4, strong inhibitors of P-glycoprotein (P-gp), or digoxin (a P-gp 
sensitive substrate medication) that have not been stopped for a duration of at least 5 days 
or a timeframe equivalent to 5 half-lives (whichever is longer) prior to the first dose of 
study treatment. 
11. Are currently receiving hematopoietic stimulating agents (eg, erythropoietins, 
granulocyte colony stimulating factors, thrombopoietins) that have not been stopped for a 
duration of at least 28 days prior to the first dose of study treatment. 
12. Have a history of allergy to sulfonamides if characterized by acute hemolytic anemia, 
drug-induced liver injury, anaphylaxis, rash of erythema multiforme type or 
Stevens-Johnson syndrome, cholestatic hepatitis, or other serious clinical manifestations. 
13. Have a history of allergy to AG-348 or its excipients (microcrystalline cellulose, 
croscarmellose sodium, sodium stearyl fumarate, and mannitol).  
14. Are currently receiving anabolic steroids, including testosterone preparations, within 
28 days prior to the first dose of study treatment. 
8.3. 
Subject Withdrawal Criteria 
Subjects may withdraw from the study at any time for any reason. Subjects will be withdrawn 
from treatment and study-related procedures under the following conditions: 
 Withdrawal of consent 
 Development of an intercurrent medical condition that precludes further participation 
in the study 
 Subject requires use of a prohibited concomitant medication (Section 9.7.1) 
 Investigator decision 
 Persistent nonadherence to protocol requirements  
 Pregnancy 
 Lost to follow-up 
A subject who discontinues treatment will be encouraged to return for subsequent scheduled 
visits (or at a minimum the End of Study Visit) unless the subject withdraws consent. Should a 
subject decide to withdraw consent, all efforts will be made to complete and report the 
protocol-defined study observations up to the time of the subject’s withdrawal as completely as 
possible and to determine the reason for withdrawal. 
In the event a subject is withdrawn from the study treatment or the study, the Independent 
Medical Monitor, or designee, must be informed.  
When a subject withdraws from the study treatment or withdraws from the study, the primary 
reason for treatment discontinuation or study discontinuation must be recorded in the appropriate 
section of the electronic case report form (eCRF). 
Refer to Section 11 for details regarding the follow-up of AEs ongoing at the time a subject 
discontinues treatment.  


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8.4. 
Subject Replacement 
Subjects will not be replaced. 
8.5. 
End of Study 
End of Study is defined as the time at which all subjects have completed the study or are lost to 
follow-up. 


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9. 
TREATMENT OF SUBJECTS 
9.1. 
Description of Study Treatment 
9.1.1. 
Study Treatment 
The term “study treatment” is used throughout this protocol to define both the active (AG-348) 
drug and placebo. AG-348 will be supplied as 5, 20, and 50 mg strength tablets to be 
administered orally. Placebo will be supplied as matching tablets to be administered orally.  
Please see the IB for further details regarding AG-348. AG-348 is provided for investigational 
use only (considered an investigational medicinal product [IMP]) and is to be used only within 
the context of this study. All study treatment product will be supplied by the Sponsor. 
9.1.2. 
Study Treatment Packaging and Labeling 
AG-348 and matching placebo will be supplied as 7-day blister wallets and will be labeled 
appropriately as IMP for this study. The labeling for AG-348 and matching placebo will be 
identical. 
Subjects will be receiving 1 of 3 potential doses, each of which is supplied as a different sized 
tablet. Thus, to maintain blinding, each dose of study treatment will be supplied as 3 different 
sized tablets: 1 tablet will be the active drug and the other 2 tablets will be placebo for subjects 
who are randomized to active, and all 3 tablets will be placebo for subjects who are randomized 
to placebo. The packaging will not change if the subject undergoes the recommended dose taper. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.1.3. 
Study Treatment Storage 
AG-348 and matching placebo tablets must be stored according to the respective package label. 
All study treatment products must be stored in a secure, limited-access location and may be 
dispensed only by the Investigator or by a member of the staff specifically authorized by the 
Investigator.  
9.1.4. 
Study Treatment Administration 
AG-348 and matching placebo tablets are to be taken orally and swallowed whole with water. 
The tablets are not to be crushed, chewed, or dissolved in water. Doses of study treatment may 
be taken with or without food. Subjects will take 3 tablets, each of a different size, twice a day, 
approximately 12 hours apart (ie, 12 hours ±2 hours), regardless of randomization to active or 
placebo and dose level (ie, 6 tablets total each day). Subjects will be instructed to complete a 
dosing diary each day in the eDiary dispensed during screening. 
If a dose of study treatment is not taken 2 hours before or 2 hours after the scheduled dosing 
time, the dose should be skipped. If a dose of study treatment is skipped, the next dose should 
then be taken approximately 24 hours from the previous dose.  
Subjects should be advised not to discontinue or interrupt dosing without first speaking 
with the treating Investigator except in case of medical emergency; abrupt discontinuation 
or interruption of AG-348 may result in withdrawal hemolysis. If a subject needs to 


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discontinue or interrupt study treatment at any time during the study, guidance is 
provided in Section 9.5.  
9.1.5. 
Study Treatment Accountability 
Accountability for the study treatment at the clinical facility is the responsibility of the 
Investigator. The Investigator will ensure that the study treatment is used only in accordance 
with this protocol. Where allowed, the Investigator may choose to assign drug accountability 
responsibilities to a pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each subject, and return to the 
Sponsor, or the Sponsor’s designee (or disposal of the drug, if approved by the Sponsor). These 
records will adequately document that the subjects were provided the doses as specified in the 
protocol and should reconcile all study treatment received from the Sponsor. Accountability 
records will include dates, quantities, batch/serial numbers, expiration dates (if applicable), and 
subject numbers. The Site Monitor will review drug accountability at the site on a schedule 
agreed to by the Sponsor. 
Study treatment must not be used for any purpose other than the present study.  
All unused and used study treatment will be retained at the site until it is inventoried by the Site 
Monitor. All used, unused, or expired study treatment will be returned to the Sponsor, or the 
Sponsor’s designee or, if authorized, disposed of at the study site per the site’s Standard 
Operating Procedures and documented. 
Study treatment is expected to be dispensed to the subject at the study site. Under exceptional 
circumstances and with agreement of the Sponsor (or representative), study treatment can be 
provided at the subject’s home, if acceptable by practice and allowed by local regulations. 
9.1.6. 
Study Treatment Handling and Disposal 
All unused study treatment must be properly disposed of in compliance with local procedures 
and governing regulations. Documentation of the method of destruction should be maintained in 
the Investigator’s files. 
9.2. 
Randomization 
Eligible subjects will be randomized 1:1 to receive either AG-348 or matching placebo. The 
randomization will be stratified by the average of screening Hb concentrations 
(<8.5 vs ≥8.5 g/dL [5.28 mmol/L]) and the PKLR gene mutation category (missense/missense vs 
missense/non-missense). The randomization assignment will be double-blinded to minimize any 
potential assessment bias. In rare instances where PKLR gene mutation category cannot be made 
definitively (eg, if a subject harbors 3 mutant PKLR alleles), the subject will be assigned to the 
missense/non-missense category. 
An interactive response system (IXRS) will be used to assign subjects to treatment. The 
randomization code will be prepared and produced by a qualified randomization vendor. The 
Sponsor’s study biostatistician will review and approve the final dummy randomization. The 
final (production) unblinded randomization will be reviewed by the statistician from the qualified 
randomization vendor and transferred to the IXRS vendor directly. The Sponsor’s study team 


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will not have access to the final unblinded randomization until database lock and study 
unblinding. 
9.3. 
Blinding 
Blinding of treatment allocation to subjects, Investigators, and site personnel will be ensured by 
identical tablet appearance, and by identical labelling between placebo and active drug. 
The subject, Investigators, and site personnel will be blinded to the subject’s study treatment 
allocation until database lock, if that subject is not entering an extension study.  
If the subject expresses his/her intention to enter an extension study, the subject, Investigators, 
and site personnel will be unblinded to the subject’s study treatment allocation, but only after the 
subject completes the Week 24 assessments. 
The Sponsor study team will be blinded to study treatment allocation until the database has been 
locked. The subject, Investigators and site personnel, and Sponsor study team will not have 
access to the pharmacokinetic data until the database has been locked. 
9.3.1. 
Handling of Restricted Data 
The following data will be considered restricted data: RBC parameters, hemolysis parameters, 
and hormone data. The Investigators will have access to these restricted data, with the exception 
of the hormone data, for their own subjects. Although Hb is evaluated as the primary efficacy 
endpoint, it is also considered part of the safety evaluation. For this reason, it is not justifiable to 
restrict the Investigators to the Hb results of the subjects whose welfare they are responsible for.  
Subjects should not have access to their own restricted data, to reduce the potential for bias in the 
PRO measurements. 
The Sponsor study team will not have access to the restricted data. The Independent Medical 
Monitor, or designee, will have access to the restricted data to be able to provide guidance to the 
Investigators and perform periodic review of these data. Additional details are provided in the 
data blinding/unblinding plan. 
9.4. 
Unblinding 
In the event of a medical emergency or pregnancy in a female subject, or in the female sexual 
partner of a male subject, in which knowledge of the treatment allocation is critical to the 
subject’s management, the blind for that subject may be broken by the Investigator. Prior to 
unblinding, Investigators are encouraged to discuss a plan to break the blinding code with the 
Independent Medical Monitor, or designee. 
The Investigator will be able to access the interactive response technology to reveal the identity 
of the treatment for that subject. The Investigator must record the nature of the emergency that 
required the unblinding, along with the date and time of the unblinding on the proper source 
documentation, and notify the Independent Medical Monitor, or designee, of the unblinding. 
In the event that a subject’s treatment assignment is unblinded to the subject, Investigator, or the 
Sponsor study team (not including the Safety Officer who is not part of the Sponsor study team 
and not involved in the current study conduct), either accidentally or in the case of emergency 


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unblinding, the subject will be allowed to continue study treatment, but the data after unblinding 
will not be included in the efficacy analyses. 
9.5. 
Criteria for Dose Modification and Study Treatment Discontinuation 
or Interruption for Adverse Events or Excessive Hb Response 
It is important that, as much as possible, a subject does not abruptly discontinue or 
interrupt study treatment due to the risk of withdrawal hemolysis. The Investigator will 
closely monitor all subjects for safety. 
A subject’s study treatment dose may be reduced (to 5 mg BID or 20 mg BID), interrupted, 
and/or discontinued; the possible scenarios in which study treatment reduction, interruption, 
and/or discontinuation may be considered include the following: 
1. Excessive Hb response, with the subject’s Hb concentration higher than 
2 g/dL (1.24 mmol/L) below the ULN as applies to men and women 
2. Occurrence of study treatment related AEs (except for excessive Hb response) 
3. Planned study treatment discontinuation with the recommended dose taper (ie, subject 
withdraws consent for study treatment) 
9.5.1. 
Excessive Hb Response 
For a subject with an excessive Hb response as defined as higher than 2 g/dL (1.24 mmol/L) 
below the ULN up to and including the ULN, a dose decrease to the next lower dose level should 
be considered for the subject, without a need for a dose taper (ie, if the subject experiences an 
excessive Hb response at 50 mg BID, his/her dose will be decreased to 20 mg BID). The same 
rule applies to the decrease from 20 mg BID to 5 mg BID. 
For a subject with an excessive Hb response higher than the ULN, the event should be reported 
as an AE and the subject’s dose will be decreased to the next lower dose level, without a need for 
a dose taper (ie, if the subject experiences an excessive Hb response at 50 mg BID, his/her dose 
will be decreased to 20 mg BID). The same rule applies to the decrease from 20 mg BID to 5 mg 
BID. 
9.5.2. 
Occurrence of Study Treatment Related Adverse Events (Except for Excessive 
Hb Response) 
Dose modification which may be required due to study treatment related AEs (except for 
excessive Hb response) are described in Table 1. 


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Table 1: 
Dose Modification for Adverse Events Considered Related to Study 
Treatment (Except for Excessive Hb Response), Study AG348-C-006 
Related Adverse 
Event(s) Severity 
Dose Modification 
Grade 1 
None required. 
Grade 2 
None required. Contact the Independent Medical Monitor, or designee, to 
discuss specific cases that may need to be managed as Grade 3 events (see 
below). 
Grade 3 
After careful consideration of the relative risk of maintaining the subject on the 
study treatment versus the risk of withdrawal hemolysis when stopping 
treatment abruptly or reducing the dose, the Investigator should determine 
which of the following options is appropriate: 
 
Maintaining the current dose, or 
 
Performing the recommended dose taper, or 
 
Stopping the study treatment abruptly 
If the decision is made to maintain the current dose of study treatment, then no 
dose changes are required. At least once weekly monitoring should be 
performed until the event resolves to baseline or Grade 1 (whichever is lower). 
If the event persists, performing the recommended dose taper or stopping the 
study treatment abruptly should be considered. 
If the decision is made to perform the recommended dose taper or to stop the 
study treatment abruptly, the below instructions should be followed for 
re-introduction or re-escalation of the study treatment, respectively. 
In all cases, re-introduction and re-escalation of study treatment should be 
performed only after discussion with the Independent Medical Monitor, 
or designee. 
 
Restarting study treatment after dosing was stopped:  
 
Once the event resolves to baseline or Grade 1 (whichever is 
lower) and the decision is made to restart treatment, the study 
treatment should be re-introduced at the 5 mg BID dose level. 
If the event does not re-occur after at least 4 weeks on 5 mg 
BID (with at least once weekly monitoring), the dose may be 
increased from 5 mg BID to 20 mg BID. If the event does not 
re-occur after at least 4 weeks on 20 mg BID (with at least 
once weekly monitoring), the dose may be increased from 
20 mg BID to 50 mg BID. In both cases, dose escalation 
should follow the guidance in Section 7.1.3. 
 
Events resolving during the recommended dose taper (ie, the 
subject is still on study treatment):  
 
If during the recommended dose taper, the event resolves to 
baseline or Grade 1 (whichever is lower), the study treatment 
should be maintained at the dose at which the event resolved 


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Related Adverse 
Event(s) Severity 
Dose Modification 
for at least 4 weeks (with at least once weekly monitoring). If 
the event does not re-occur after at least 4 weeks, then the dose 
may be increased to the next highest BID dose (5 mg BID, 
20 mg BID, or 50 mg BID) with at least once weekly 
monitoring. If the event does not re-occur after at least 
4 weeks (with at least once weekly monitoring), and the 
subject is not already receiving 50 mg BID, then an increase to 
the next BID dose level should be considered. In both cases, 
dose escalation should follow the guidance in Section 7.1.3. 
 
Re-occurrence of the AE: 
 
If the AE re-occurs at any point during the above scenarios, 
the subject should undergo the recommended dose taper or 
stop study treatment abruptly, if necessitated by the risk of the 
AE. If the subject undergoes the recommended dose taper and 
the AE resolves during the taper, study treatment should be 
maintained at the next lowest BID dose below the dose at 
which the AE resolved. If the subject cannot tolerate BID 
dosing, another regimen may be allowed after discussion with, 
and approval by, the Independent Medical Monitor, or 
designee. If the AE does not resolve to baseline or Grade 1 
(whichever is lower) after the dose is decreased, a further 
decrease in the dose should be considered. If the AE still does 
not resolve, study treatment should be permanently 
discontinued. 
Grade 4 
After careful consideration of the relative risk of withdrawal hemolysis when 
stopping study treatment abruptly versus reducing the dose, the Investigator 
should determine which of the following options is appropriate: 
 
Performing the recommended dose taper or 
 
Stopping the study treatment abruptly 
If the event resolves, and the Investigator believes that re-introducing study 
treatment is justified, the Independent Medical Monitor, or designee, should be 
consulted before any further study treatment is administered. 
Abbreviations: AE = adverse event; BID = twice daily; Hb = hemoglobin. 
9.5.3. 
Study Treatment Discontinuation or Interruption With Recommended Dose 
Taper Regimen 
All subjects who discontinue or interrupt study treatment during the study should undergo the 
recommended dose taper regimen in Table 2. This regimen is based on the study treatment dose 
administered to the subject at the start of the taper and occurs in 1 or 2 sequential steps. Subjects 
who were unblinded and allocated to placebo do not need to undergo the recommended dose 
taper when discontinuing or interrupting study treatment. 


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Subjects undergoing the recommended dose taper should be monitored as clinically indicated for 
signs of withdrawal hemolysis and worsening of anemia. If the recommended dose taper is 
performed in order to permanently discontinue study treatment, subjects stop taking the study 
treatment after the taper has been completed. 
Table 2: 
Recommended Dose Taper Regimen, Study AG348-C-006 
Starting Dose 
(at the time of the dose taper) 
First Step 
×7 days 
Second Step 
×7 days 
5 mg BID 
5 mg QD 
--- 
20 mg BID 
20 mg QD 
5 mg QD 
50 mg BID 
50 mg QD 
20 mg QD  
Abbreviations: BID = twice daily; QD = once daily. 
9.6. 
Treatment Compliance 
Treatment compliance will be assessed by drug accountability (ie, number of tablets dispensed vs 
number returned). 
9.7. 
Prior and Concomitant Medications 
Prior medications are defined as those administered anytime within the 28 days prior to signing 
of the ICF until the first dose of study treatment and concomitant medications are defined as 
those administered from the point of first dose of study treatment through the subject’s 
completion of the study. All prior and concomitant medications must be recorded in the 
appropriate section of the source documentation and eCRF along with any dosage information, 
dates of administration, mode of administration, and reason for use. For non-drug therapies, 
please reference Section 9.8. 
9.7.1. 
Prohibited Medications  
Concomitant use of investigational drugs is not allowed while subjects are participating in this 
study. All subjects must discontinue any investigational drug no less than 3 months prior to the 
first dose of study treatment. 
In vitro studies using human liver microsomes and recombinant CYP enzymes have shown that 
AG-348 is primarily metabolized by CYP3A4 (>70%), with minor contributions from CYP2C9, 
CYP2C8, and CYP1A2. In addition, AG-348 has been shown to be a weak time-dependent 
CYP3A4 inhibitor and a potential inducer of CYP3A4 and CYP2B6 in vitro. In vitro transporter 
studies have shown that AG-348 is a substrate and inhibitor of P-gp.  
Also, AG-348 exhibits pH-dependent solubility. Therefore, proton-pump inhibitors and 
H2-receptor antagonists may decrease the absorption of AG-348.  
Based on these results, below is a list of concomitant therapy to be avoided and concomitant 
therapy requiring careful monitoring. 
The following are prohibited at all times during participation in this study: 
 Strong inhibitors of CYP3A4 (listed in the AG-348 IB) 


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 Products known to inhibit CYP3A4, such as grapefruit or grapefruit juice 
 Strong inducers of CYP3A4 (listed in the AG-348 IB) 
 Strong inhibitors of P-gp (listed in the AG-348 IB) 
 Digoxin, a P-gp transporter-sensitive substrate 
If a subject is taking any medication listed in the AG-348 IB and/or digoxin prior to enrolling in 
the study, the medication must be discontinued at least 5 days or a time frame equivalent to 5 
half-lives (whichever is longer) prior to Day 1 dosing. 
 Hematopoietic stimulating agents (eg, erythropoietins, granulocyte colony stimulating 
factors, thrombopoietins) must be discontinued no less than 28 days prior to the first 
dose of study treatment. B12 injections are permitted for subjects with a prior 
diagnosis of B12 deficiency syndromes. Subjects must be repleted to stability of the 
Hb and mean corpuscular volume [MCV] prior to enrollment in the study. 
 Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study treatment. 
The medications that fall under the categories mentioned below should be avoided and replaced 
with alternative treatments. If this is not possible, subjects receiving these medications should be 
carefully monitored. A general monitoring guideline for Investigators whose patients take 
medications that fall under the categories mentioned below is as follows: Investigators must 
monitor subjects for lack of efficacy of the prescribed medication or for side effects arising from 
the medication. If either a lack of efficacy of the prescribed medication or side effects suspected 
to be related to the prescribed medication are noticed, then the Investigator should make 
appropriate modifications to the dose of the prescribed medication or find alternatives to the 
prescribed medication. 
 Corticosteroids (sensitive substrates of CYP3A4 and weak CYP3A4 inducers) 
 Sensitive substrates of CYP3A4 (listed in the AG-348 IB) 
 Moderate inhibitors of CYP3A4 (listed in the AG-348 IB) 
 Sensitive substrates of CYP2B6 (listed in the AG-348 IB) 
 Proton-pump inhibitors and H2-receptor antagonists (listed in the AG-348 IB). 
Antacids, such as magnesium hydroxide and aluminum hydroxide, can be used with 
AG-348. 
AG-348, being a potential CYP3A4 inducer, has the potential to reduce the effectiveness of oral 
contraceptives. Therefore, women using oral contraceptives must also utilize a barrier method 
while enrolled in the study and until at least 28 days after their last dose of study treatment, as 
specified in Inclusion Criterion 9 (Section 8.1). 
9.7.2. 
Allowed Concomitant Medications 
Medications other than those specified above (Section 9.7.1) are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, 


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anti-infectives, and antipyretics as medically indicated and consistent with the guidance in 
Section 9.7.1.  
Subjects may continue iron chelation therapy with deferoxamine, deferasirox, or deferiprone. As 
iron overload is a long-term complication of PK deficiency, any initiation, completion, or change 
of iron chelation therapy will be of particular interest. Data about chelation therapy use will be 
carefully collected. 
Subjects must continue taking at least 0.8 mg oral folic acid daily for the duration of the study.  
9.8. 
Prior and Concomitant Non-Drug Therapies 
Prior non-drug therapies determined to be relevant for medical/surgical history and/or eligibility 
criteria, such as major surgeries within 6 months prior to signing the ICF (see Section 8.2, 
Exclusion Criterion 9) and splenectomy within 12 months prior to signing the ICF (see 
Section 8.2, Exclusion Criterion 3) should be recorded.  
Relevant concomitant non-drug therapies used to treat an AE should be collected from the 
signing of ICF until the subject completes the study.  
9.8.1. 
 Prior and Concomitant Transfusions 
Transfusions administered within 12 months prior to the first day of study treatment should be 
recorded to verify that the subject meets the inclusion criterion for “not regularly transfused” 
(see Section 8.1, Inclusion Criterion 5) 
For each transfusion administered from Day 1 until the end of the study, the date of transfusion 
and number of RBC units transfused will be recorded. 


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10. 
STUDY ASSESSMENTS 
The timing of all study assessments is indicated in the Schedule of Assessments (Section 10.13). 
10.1. 
Informed Consent 
A description of the study is to be presented to each potential subject and a signed and dated ICF 
is to be obtained before any study-specific procedures are performed. The ICF will contain a 
separate section regarding the option to use leftover biological samples for analysis of additional 
biomarkers; subjects may opt-in or decline; this will not affect their eligibility for the study. 
10.2. 
Study Eligibility 
A subject’s eligibility will be assessed and confirmed during the Screening Period. The 
Investigator will determine whether each subject meets all the inclusion criteria and none of the 
exclusion criteria. Eligibility of each subject will be confirmed by the Medical Monitor, or 
designee.  
If a subject is deemed ineligible for the study due to a transient condition (eg, prohibited 
concomitant medication, curable medical condition), the subject may be rescreened after the 
criterion that made the subject ineligible has resolved. Subjects who were ineligible according to 
a previous version of the protocol may be rescreened if the subject could be eligible according to 
the current version of the protocol. 
The following assessments will not need to be repeated at rescreening if they were performed 
correctly at the initial screening: demographics, medical/surgical history (unless new information 
needs to be added), liver MRI (unless done more than 3 months prior), DXA scan (unless done 
more than 3 months prior), and PKLR genotyping. 
10.3. 
Demographics 
Subject demographic data will include gender, year of birth, race, and ethnicity.  
Race and ethnicity will be collected to ensure that any race and ethnicity related specificities in 
the safety, pharmacokinetics, and/or efficacy of AG-348 can be captured and interpreted 
accurately.  
Collection of demographic data will be modified by country regulatory requirements, as 
appropriate.  
10.4. 
Medical/Surgical History 
All medical and surgical history deemed to be relevant per the Investigator (in particular, 
pertaining but not limited to the diagnosis of PK deficiency) and current medical conditions are 
to be recorded on the source documentation and included in the eCRF during screening.  
Prior history of splenectomy and/or cholecystectomy must be documented and included in the 
eCRF during screening for all subjects.  


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10.4.1. 
Prior Transfusions 
The dates of transfusions for the 12-month period prior to the first day of study treatment will be 
required for subject eligibility confirmation (Section 8.1, Inclusion Criterion 5). 
10.4.2. 
Iron Overload-Related History 
Historical use of chelation therapy for the 12 months prior to signing the ICF should be recorded, 
including type of iron chelation therapy, start date, stop date, and dose. Additionally, serum iron, 
ferritin, transferrin saturation, and LIC data should be recorded for the 12 months prior to signing 
the ICF. 
10.5. 
Pyruvate Kinase Deficiency Specific Assessments 
10.5.1. 
PKLR Genotyping 
The PKLR genotyping and genotype classification (ie, missense or non-missense) will be 
performed during screening by a central genotyping laboratory for confirmation of study 
eligibility.  
10.5.2. 
PK Enzyme Assay 
The PK enzyme assay will be performed on the blood sample collected during screening by a 
central laboratory. 
10.6. 
Assessment of Efficacy 
10.6.1. 
Hemoglobin Laboratory Assessment 
Hemoglobin concentrations will be collected to support efficacy (and safety) assessments. Blood 
will be drawn for hematology assessments as outlined in the Schedule of Assessments 
(Section 10.13).  
10.6.2. 
Additional Laboratory Assessments for Efficacy 
All the RBC parameters, haptoglobin, and LDH values will be collected to support additional 
efficacy assessments. Blood will be drawn for these assessments as outlined in the Schedule of 
Assessments (Section 10.13).  
10.7. 
Assessments of Safety 
10.7.1. 
Vital Signs 
Vital signs will be recorded and will include systolic and diastolic BP, heart rate, and body 
temperature.  
10.7.2. 
Physical Examination 
Complete PEs will be performed according to the Schedule of Assessments (Section 10.13) and 
additionally when clinically indicated, at the discretion of the Investigator. 
Height will be recorded only during screening.oh 


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10.7.3. 
Prior and Concomitant Medications 
Prior and concomitant medications will be captured as described in Section 9.7 and per the 
Schedule of Assessments (Section 10.13).  
10.7.4. 
Safety Laboratory Assessments 
Safety laboratory assessments are described in Section 10.8.1. 
10.7.5. 
Electrocardiogram 
The 12-lead ECGs should be performed following 5 minutes of recumbence and in triplicate, 
using the ECG machine provided by the central vendor and according to the vendor manual. The 
ECG at Week 12 will be performed prior to, but within the same window as, the pharmacokinetic 
sample collected 1 hour (±5 minutes) after the study treatment dose administration to align with 
the approximate Tmax (time to maximum [peak] concentration) of the study treatment. 
The ECGs will be read promptly by a qualified physician at the study site to detect any eligibility 
or safety issue. Only QTcF (not heart rate-corrected QT interval by Bazett’s method [QTcB]) 
will be used for determination of eligibility. In addition to the local read, the ECGs will be sent 
promptly to the central vendor for a data-analysis read. 
An ECG will be repeated if clinically significant abnormalities are observed, if artifacts are 
present, or if machine/equipment errors occur.  
10.7.6. 
Dual-Energy X-ray Absorptiometry Scans 
The DXA scans of the lumbar spine and proximal femur (trochanter and inter-trochanter, which 
comprise the total hip, and femoral neck) will be performed according to the instructions 
provided by the central vendor (see vendor’s manual for details). The DXA scans will be 
transmitted promptly to the central vendor for assessment of technical adequacy and may have to 
be repeated (before first dose for the screening DXA scan) if not technically adequate. The DXA 
scans will be read and interpreted by the central vendor. 
10.7.7. 
Menstrual Cycle Diary 
Menstruating female subjects will be required to fill out an electronic menstrual cycle diary for 
each menstrual period in order to detect any change in menstrual cycles. Subjects will record the 
start date, stop date, and any notable characteristics of each menstrual cycle in the eDiary 
provided at screening. 
10.8. 
Laboratory Assessments 
Laboratory assessment samples will be collected according to the Schedule of Assessments 
(Section 10.13). All blood samples are to be analyzed by the central laboratory. If results from 
the central laboratory are not available to support necessary clinical decision-making then results 
from a local laboratory may be used. Blood samples for the second Hb assessment during 
screening and at the Week 10 assessments may be collected outside of the study site and sent to 
the central laboratory by qualified personnel (eg, home health care nurse), if allowed by local 
regulations. 


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If Investigators believe that it is clinically indicated to obtain safety laboratory results from their 
own local laboratories on the day of the subject’s visit, they are free to exercise their discretion to 
do so.  
All clinically significant laboratory abnormalities noted on testing will be followed by repeat 
testing and further investigated according to the judgment of the Investigator.  
Safety laboratory assessments and other laboratory assessments are listed in Section 10.8.1 and 
Section 10.8.2, respectively.  
10.8.1. 
Safety Laboratory Assessments 
The following safety laboratory parameters will be measured: 
Hematology: 
Complete blood count (hematocrit [HCT], Hb, RBC count, percent 
reticulocyte, absolute reticulocyte count [if available], immature 
reticulocyte fraction [IRF-H and IRF-M+H] [if available], MCV, 
mean corpuscular Hb, mean corpuscular Hb concentration, red cell 
distribution width, nucleated RBC count, white blood cell count, 
ANC, absolute lymphocyte count, eosinophil count, basophil 
count, absolute monocyte count, and platelet count). 
Serum Chemistry: 
Sodium, potassium, chloride, calcium, magnesium, phosphorus, 
carbon dioxide or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen or urea, uric acid, and creatinine. At screening, 
estimated GFR, measured GFR, or calculated (Cockcroft-Gault) 
CrCL will be assessed. 
LFTs: 
Alkaline phosphatase, ALT, AST, total bilirubin, direct bilirubin, 
and indirect bilirubin 
Sex Steroid Testing: 
Testosterone (total and free), estrone, and estradiol.  
Fasting Lipids: 
Total cholesterol, high-density lipoprotein cholesterol, low-density 
lipoprotein cholesterol, and triglycerides measured by standard 
method and nuclear magnetic resonance 
Coagulation Studies: 
Fibrinogen, aPTT, and INR 
Dipstick Urinalysis: 
Protein, glucose, leukocytes, and blood  
Pregnancy Test (if 
applicable): 
Serum (hCG) or urine pregnancy tests (with the exception of the 
screening pregnancy test which must be serum) 
UGT1A1 Genotyping: 
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10.8.2. 
Other Laboratory Assessments 
A blood sample for serology, including HBsAg, HCVAb screen, and HIV-1 and HIV-2 Ab, will 
be collected from all subjects for eligibility criteria. 
The following other laboratory parameters will be assessed: 
 FSH (to confirm post-menopausal status) 
 Iron panel and related markers (iron, serum ferritin, total iron-binding capacity 
[TIBC], transferrin saturation, non-transferrin bound iron [NTBI], hepcidin, 
C-reactive protein [CRP], and other markers of iron metabolism). Remaining sample 
may be used for analyses of lipoproteins (only in subjects who have agreed to this 
optional analysis in the ICF). 
 Markers of erythropoietic activity: soluble transferrin receptor, erythropoietin (EPO), 
erythroferrone, and other markers of erythropoiesis 
 Assessments of complications of iron overload: thyroxine, thyroid-stimulating 
hormone, parathormone, fructosamine, and Vitamin D 
10.9. 
Other Assessments 
Liver iron concentration will be measured by MRI based on the measurement and imaging of 
proton transverse relaxation rates (R2). The MRI data collected at the site will be transferred to a 
central vendor for analysis. For further details, please refer to the vendor’s manual. 
10.10. 
Pharmacokinetic Assessments 
10.10.1. 
Blood Sample Collection 
On days of pharmacokinetic blood sample collection, the morning dose of study treatment must 
be administered at the study site. On days where a predose sample is required, the study 
treatment must be administered after the predose sample is taken. Plasma samples for 
pharmacokinetic analysis of AG-348 will be collected at the following time points (note: time 
points are listed in relation to the morning dose of study treatment): 
 Day 1 [predose (within 60 minutes prior to study treatment administration)] 
 Week 2 (1-2 hours and 3-4 hours post study treatment administration) 
 Week 6 (4-5 hours and 6-7 hours post study treatment administration) 
 Week 12 (full profile blood sampling)  
 predose (within 60 minutes prior to study treatment administration) 
 30 minutes (±5 minutes) post study treatment administration 
 1 hour (±5 minutes) post study treatment administration 
 2 hours (±5 minutes) post study treatment administration 
 4 hours (±30 minutes) post study treatment administration 
 8 hours (±30 minutes) post study treatment administration 


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 Week 16 [predose (within 60 minutes prior to study treatment administration)]  
The actual date and time of sample collection will be recorded in the source documents and 
eCRF. An explanation should be provided in the source documents for any missed or mishandled 
pharmacokinetic samples, as well as for any samples collected outside the time windows.  
10.10.2. 
Sample Analysis 
Pharmacokinetic samples will be analyzed for AG-348 using a validated liquid 
chromatography-tandem mass spectrometry method. Remaining samples may be used for 
analyses of AG-348 metabolism (only in subjects who have agreed to this optional analysis in 
the ICF). 
Plasma pharmacokinetic parameters will be computed, when data allow, using standard 
noncompartmental methods, based on observed plasma AG-348 concentrations and on actual 
sample collection times. These parameters will include, but may not be limited to, the following: 
 AUC0-last: The area under the plasma concentration × time curve from time 0 to the 
time of the last measurable concentration 
 Tlast: Time of last measureable concentration 
 Cmax: Maximum (peak) concentration  
 Tmax: Time to maximum (peak) concentration  
 λz: Apparent terminal elimination rate constant, calculated from a semi-log plot of the 
plasma concentration versus time curve  
 t½: Terminal half-life  
 CL/F: The apparent total plasma clearance (CLp) following oral (extravascular) 
dosing 
 Vz/F: The apparent volume of distribution during the terminal elimination phase 
following oral (extravascular) dosing 
10.11. 
Pharmacodynamic Assessments 
Pharmacodynamic samples to measure PKR protein levels in whole blood will be taken at the 
Day 1 Visit before administration of the first dose and predose at Week 16. Additional analysis 
of exploratory biomarkers (PKM protein levels and levels of intermediates in the metabolic 
pathways affected by PKR) to further the understanding of the mechanism of action of AG-348 
may be performed on leftover samples in subjects who have agreed to this optional analysis in 
the ICF. 
Subjects in the study, contingent on clinical site feasibility, will have blood taken at the Day 1 
Visit before administration of the first dose and predose at Week 12 for the PKR flux assay. The 
PKR flux assay measures the change in carbon flow from glucose through the PKR reaction to 
pyruvate and then to lactate after AG-348 treatment.  
On days of sample collection, the morning dose of study treatment must be administered at the 
study site following the predose collections. Predose samples for pharmacodynamic assessments 
should be collected within 60 minutes prior to study treatment.  


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10.12. 
Health-Related Quality of Life Assessments and CGIC 
Patient-reported outcome measures, specifically PKDD and PKDIA, will be evaluated during 
screening and throughout the study. Several other HRQoL measures have been included as 
validators (including relevant subscales) in this study to allow for in-trial validation of the PKDD 
and PKDIA. The exact methods and techniques to be used for PKDD and PKDIA calibration and 
scoring will be included in the formal PRO calibration and validation statistical analysis plan 
(SAP). The following measures will be used for validation of the PKDD and PKDIA:  
 EQ-5D-5L 
 The 12-item Short Form Health Survey, Version 2 (SF-12v2) 
 FACT-An – “Additional Concerns” 
 Patient Global Impression of Severity (PGIS) 
 Patient Global Impression of Change (PGIC) 
 Clinician Global Impression of Change (CGIC) 
Subjects will use the eDiary (provided during screening) to record responses to each of the 
HRQoL assessments (Section 10.12.1 through Section 10.12.7). The HRQoL assessments should 
be completed in the evening (between 5:00-11:00 PM) within the window for the relevant study 
visit as indicated in the Schedule of Assessments (Section 10.13). 
10.12.1. 
Pyruvate Kinase Deficiency-Specific HRQoL Assessments 
The PKDD is a 7-item PRO measure of the core signs and symptoms associated with PK 
deficiency in adults. Subjects rate their experience with symptoms of PK deficiency on the 
present day. The symptoms include those associated with tiredness, jaundice, bone pain, 
shortness of breath, and energy level.  
The PKDIA is a 12-item PRO measure of the common impacts of PK deficiency on activities of 
daily living. Subjects rate how PK deficiency has impacted aspects of daily living in the past 
7 days, including impacts on relationships; perceived appearance; work performance; and 
leisure, social, mental, and physical activities.  
These 2 tools were developed by the Sponsor to systematically assess and capture changes in 
symptom burden and impact on HRQoL.  
10.12.2. 
European Quality of Life Five-Dimensional Descriptive System 
The EQ-5D-5L is a standardized instrument for evaluating QoL over 5 dimensions: mobility, 
self-care, usual activities, pain, and mood. It is applicable over a broad range of health conditions 
and is used widely in clinical trials. 
10.12.3. 
Patient Global Impression of Severity 
The PGIS is a single-item questionnaire used to rate the subject’s impression of the severity of 
his/her condition. 


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10.12.4. 
12-Item Short Form Healthy Survey, Version 2 
The SF-12v2 is a widely used generic measure of health status and measures 8 concepts of 
health: physical functioning, role limitations due to physical health problems, bodily pain, 
general health, vitality (energy/fatigue), social functioning, role limitations due to emotional 
problems, and mental health (psychological distress and psychological well-being). 
10.12.5. 
Functional Assessment of Cancer Therapy Anemia – “Additional Concerns” 
The FACT-An is a questionnaire developed within the Functional Assessment of Chronic Illness 
Therapy (FACIT) system. The FACT-An – “Additional Concerns” is a 20-item QoL measure for 
assessing fatigue and anemia-related concerns.  
10.12.6. 
Patient Global Impression of Change 
The PGIC is a self-reported measure that reflects a subject’s perspective on his or her overall 
health status with respect to the study treatment. The assessment rates the subject’s perceived 
overall improvement while receiving study treatment.  
10.12.7. 
Clinician Global Impression of Change 
The CGIC is an assessment completed by the study Investigator to provide the clinician’s view 
of the subject’s overall improvement and health status prior to and after initiating study 
treatment.  
10.13. 
Schedule of Assessments 
The Schedule of Assessments is in Table 3.


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Table 3: 
Schedule of Assessments, Study AG348-C-006 
 
Screening 
Period 
Dose Optimization Period (Part 1) 
Fixed Dose Period (Part 2) 
Follow-up 
Visit5 
Visit: 
SCRN1 
D12 
W2 
W4 
W6 
W8 
W103 
W12 
W16 
W20 
W24/End 
of Study4 
28D after 
last dose5 
Study Day: 
D -42 to D -1 
1 
15 
29 
43 
57 
71 
85 
113 
141 
1694 
Visit Window: 
0  
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±4 D 
±4 D 
Procedures: 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Pyruvate kinase enzyme assay  
X 
 
 
 
 
 
 
 
 
 
 
 
PKLR genotyping 
X33 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 genotyping6 
 
X 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical/surgical history7  
X 
X 
 
 
 
 
 
 
 
 
 
 
Prior Medications8 
X 
X 
 
 
 
 
 
 
 
 
 
 
Eligibility confirmation9 
X 
 
 
 
 
 
 
 
 
 
 
 
Randomization9 
 
X 
 
 
 
 
 
 
 
 
 
 
Liver MRI to assess LIC10 
X 
 
 
 
 
 
 
 
 
 
X 
 
PE11 
X 
X 
 
 
 
 
 
X 
 
 
X 
 
Vital signs12 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
12-lead ECG13 
X 
X 
 
 
 
 
 
X 
 
 
X 
X 
DXA Scan10 
X 
 
 
 
 
 
 
 
 
 
X 
 
Dispense eDiary14 
X 
 
 
 
 
 
 
 
 
 
 
 
Menstrual Cycle Diary14 
X 
HRQoL Assessments14 
EQ-5D-5L 
X34 
 
 
 
 
 
 
X 
 
 
X 
 


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Screening 
Period 
Dose Optimization Period (Part 1) 
Fixed Dose Period (Part 2) 
Follow-up 
Visit5 
Visit: 
SCRN1 
D12 
W2 
W4 
W6 
W8 
W103 
W12 
W16 
W20 
W24/End 
of Study4 
28D after 
last dose5 
Study Day: 
D -42 to D -1 
1 
15 
29 
43 
57 
71 
85 
113 
141 
1694 
Visit Window: 
0  
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±4 D 
±4 D 
Procedures: 
 
 
 
 
 
 
 
 
 
 
 
 
SF-12v2 
X34 
 
 
 
 
 
 
X 
 
 
X 
 
PKDD14 
X–Daily–X 
X–Daily–X 
 
PKDIA 
X34 
 
 
X 
 
X 
 
X 
X 
X 
X 
 
FACT-An – “Additional 
Concerns” 
X34 
 
 
 
 
 
 
X 
 
 
X 
 
PGIS 
X34 
 
 
X 
 
X 
 
X 
 
 
X 
 
PGIC 
 
 
 
X 
 
X 
 
X 
 
 
X 
 
CGIC 
 
 
 
X 
 
X 
 
X 
 
 
X 
 
Dosing eDiary14 
 
X 
Return eDiary15 
 
 
 
 
 
 
 
 
 
 
X4 
X 
Clinical Laboratory Evaluations16 
HBsAg, HCVAb, HIV-1 and -2 
Ab17 
X 
 
 
 
 
 
 
 
 
 
 
 
FSH18 
X 
 
 
 
 
 
 
 
 
 
 
 
Hematology19 
X, X35 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin and LDH 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum chemistry20 
X 
X 
 
 
 
 
 
X 
 
 
X 
X 
LFTs21 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Coagulation studies22 
X 
X 
 
 
 
 
 
X 
 
 
X 
 
Urinalysis23 
X 
X 
 
 
 
 
 
X 
 
 
X 
 


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Screening 
Period 
Dose Optimization Period (Part 1) 
Fixed Dose Period (Part 2) 
Follow-up 
Visit5 
Visit: 
SCRN1 
D12 
W2 
W4 
W6 
W8 
W103 
W12 
W16 
W20 
W24/End 
of Study4 
28D after 
last dose5 
Study Day: 
D -42 to D -1 
1 
15 
29 
43 
57 
71 
85 
113 
141 
1694 
Visit Window: 
0  
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±4 D 
±4 D 
Procedures: 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test24 
X 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
Iron panel and related 
markers25 
X 
X 
 
 
 
 
 
X 
 
 
X 
 
Markers of erythropoietic 
activity25 
X 
X 
 
 
 
 
 
X 
 
 
X 
 
T4, TSH, PTH, Fructosamine, 
and Vitamin D 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Lipids26 
X 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
Sex Steroids27 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
PD Assessments28 
PKR protein  
 
X 
 
 
 
 
 
 
X 
 
 
 
PKR flux assay 
 
X 
 
 
 
 
 
X 
 
 
 
 
Dispense study treatment29 
 
X 
 
X 
 
X 
 
X 
X 
X 
X4 
 
Study treatment dosing 
 
X 
 
Return study treatment 
 
 
 
X 
 
X 
 
X 
X 
X 
X4 
X 
Pharmacokinetic Assessments 
Pharmacokinetic sparse blood 
sampling30 
 
X 
X 
 
X 
 
 
 
X 
 
 
 
Pharmacokinetic full-profile 
blood sampling31 
 
 
 
 
 
 
 
X 
 
 
 
 
SAEs/AEs/AESIs32 
X 


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Screening 
Period 
Dose Optimization Period (Part 1) 
Fixed Dose Period (Part 2) 
Follow-up 
Visit5 
Visit: 
SCRN1 
D12 
W2 
W4 
W6 
W8 
W103 
W12 
W16 
W20 
W24/End 
of Study4 
28D after 
last dose5 
Study Day: 
D -42 to D -1 
1 
15 
29 
43 
57 
71 
85 
113 
141 
1694 
Visit Window: 
0  
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±3 D 
±4 D 
±4 D 
Procedures: 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant 
medications/transfusions 
 
X 
Abbreviations: Ab = antibody; AE = adverse event; AESI = adverse event of special interest; ALC = absolute leukocyte count; ALP = alkaline phosphatase; ALT = alanine 
aminotransferase; ANC = absolute neutrophil count; aPTT = activated partial thromboplastin time; AST = aspartate aminotransferase; BP = blood pressure; BUN = blood urea 
nitrogen; CBC = complete blood count; CGIC = Clinical Global Impression of Change; CO2 = carbon dioxide; CrCL = creatinine clearance; CRP = C reactive protein; D = day; 
DXA = dual-energy x-ray absorptiometry; ECG = electrocardiogram; eDiary = electronic diary; EPO = erythropoietin; EQ-5D-5L = European quality of life five-dimensional 
descriptive system; FACT-An = Functional Assessment of Cancer Therapy-Anemia; FSH = follicle-stimulating hormone; GFR = glomerular filtration rate; Hb = hemoglobin; 
HBsAg = hepatitis B surface antigen; hCG = human chorionic gonadotropin; HCT = hematocrit; HCV = hepatitis C virus; HDL-C = high-density lipoprotein cholesterol; 
HIV = human immunodeficiency virus; HRQoL = health-related quality of life; ICF = informed consent form; INR = international normalized ratio; LDH = lactate dehydrogenase; 
LDL-C = low-density lipoprotein cholesterol; LFT = liver function test; LIC = liver iron concentration; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular 
hemoglobin concentration; MCV = mean corpuscular volume; MRI = magnetic resonance imaging; NRBC = nucleated red blood cells; NTBI = non-transferrin bound iron; 
PD = pharmacodynamics; PE = physical examination; PGIC = Patient Global Impression of Change; PGIS = Patient Global Impression of Severity; PKDD = Pyruvate Kinase 
Deficiency Diary; PKDIA = Pyruvate Kinase Deficiency Impact Assessment; PKR = red blood cell-specific form of pyruvate kinase; PTH = parathyroid hormone; RBC = red 
blood cell; RDW = red cell distribution width; RT-PCR = reverse transcriptase-polymerase chain reaction; SAE = serious adverse event; SCRN = Screening; SF-12v2 = 12-item 
Short Form Health Survey, version 2; T4 = thyroxine; TIBC = total iron-binding capacity; TSH = thyroid-stimulating hormone; UGT1A1 = uridine diphosphate glucuronosyl 
transferase 1A1; ULN = upper limit of normal; W = week; WBC = white blood cell. 
 
1 A subject’s Screening Period duration may be extended beyond 42 days upon the Medical Monitor’s, or designee’s, approval. 
2 The first dose of study treatment on Day 1 should be taken at the study site following all Day 1 assessments (with the exception of eDiary assessments; these assessments should 
be performed in the evening of Day 1). 
3 The Week 10 Visit will consist of laboratory assessments. 
4 Subjects who continue study treatment through Week 24 should continue taking study treatment at least through the morning dose of the Week 24 Visit. All subjects who remain 
on study during Part 2 through the Week 24 Visit may be eligible for an open-label extension study, in which all subjects will receive AG-348. Subjects who discontinue the 
study prior to the Week 24 Visit should attend the End of Study Visit 28±4 days after the last study visit that the subject attended or 28±4 days after the last dose of study 
treatment (including the recommended dose taper), whichever is later. Note, this may occur at any time during the study (Part 1 and Part 2). This visit will be identical to the 
Week 24 Visit with these exceptions: no study treatment will be dispensed at the End of Study Visit, and if the subject has not already done so, they should return their eDiary 
and remaining study treatment. These subjects do not need to complete the Follow-Up Visit. 
5 Subjects who continue study treatment through the Week 24 Visit but do not continue on into an extension study, should undergo the recommended dose taper and then attend the 
Follow-up Visit 28±4 days after the last dose of study treatment (including the recommended dose taper). Subjects who continue on into an extension study at the Week 24 Visit 
will not be required to attend the Follow-up Visit. Subjects who initiate or are undergoing the recommended dose taper, and for whom participation in an extension study remains 
undetermined, should perform the recommended dose taper as detailed in Section 9.5.3. Transition to an extension study should be discussed with the Independent Medical 
Monitor, or designee. 
6 UGT1A1 genotyping results are not required prior to randomization. 


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7 Transfusion history in the 12-month period prior to first day of study treatment should be reviewed for eligibility assessment. Subjects who received any transfusions within the 
3 months prior to the first day of study treatment are not eligible. Additionally, serum iron, ferritin, transferrin saturation, and LIC data should be recorded for the 12 months 
prior to signing the ICF. Historical use of chelation therapy for the 12 months prior to signing the ICF should be recorded, including type of iron chelation therapy, start date, 
stop date, and dose. 
8 Prior medications are defined as those administered anytime within the 28 days prior to signing of the ICF until the first dose of study treatment. 
9 The Investigator will determine whether each subject meets all the inclusion criteria and none of the exclusion criteria. Eligibility of each subject will be confirmed by the 
Medical Monitor, or designee, during screening. Randomization can occur up to 24 hours prior to Day 1. 
10 The DXA and liver MRI should be performed per the vendor manuals. If the screening DXA and/or MRI scan(s) are deemed to be of low quality by the central vendors, the 
scan(s) will be repeated before the first dose of study treatment is administered. 
11 A complete PE will be performed. Height will be collected during screening only. Additional PEs may be performed when clinically indicated, at the discretion of the 
Investigator. 
12 Vital signs of systolic and diastolic BP, heart rate, and body temperature will be collected. 
13 The 12-lead ECGs will be conducted using the equipment provided by the vendor and according to the vendor manual, after 5 minutes of recumbence and in triplicate. The 
ECGs will be read promptly by a qualified physician at the study site to detect any eligibility or safety issue. In addition to the local read, the ECGs will be sent promptly to the 
central vendor for a data-analysis read. The ECG at Week 12 will be performed prior to but within the same window as the pharmacokinetic sample collected 
1 hour (±5 minutes) after the study treatment dose administration to align with the approximate Tmax of the study treatment. 
14 All subjects will be given an eDiary to record responses to HRQoL assessments and dosing. In addition, menstruating female subjects will record their menstrual cycles (start 
date, stop date, and notable characteristics) in the eDiary; the menstrual diary can be completed at any time of day. The PKDD assessment will be collected daily throughout 
screening, Part 1, and Part 2. The HRQoL assessments should be completed in the evening (between 5:00-11:00 PM) within the window for the relevant study visit. The dosing 
diary should be completed every day in the evening (between 5:00-11:00 PM). 
15 Subjects should return their eDiary once they complete the study. Subjects who discontinue early and attend the End of Study Visit should return their eDiary at this visit. 
Subjects should return their eDiary at their last visit of this study. 
16 On Day 1 of Part 1, blood samples for all clinical laboratory assessments should be collected predose. All clinical laboratory evaluations should be analyzed centrally. If results 
from the central laboratory are not available, to support necessary clinical decision-making, then results from a local laboratory may be used. For a safety issue that may require 
local laboratory evaluations, refer to Section 10.8. 
17 If the subject is positive for HCVAb, an RT-PCR test will be conducted. The subject is ineligible to enroll if active hepatitis C is present. The subject may be rescreened after 
receiving appropriate hepatitis C treatment. 
18 The FSH assessment will be performed only at screening for female subjects for confirmation of postmenopausal status (ie, female subjects who have not menstruated at all for 
at least the preceding 12 months prior to signing informed consent). Samples should be drawn in the morning (does not need to be fasting). 
19 Hematology parameters (ie, CBC with differential) include HCT, Hb, RBC count, percent reticulocyte, absolute reticulocyte count (if available), immature reticulocyte fraction 
(IRF-H and IRF-M+H) (if available), MCV, MCH, MCHC, RDW, NRBC, WBC count, ANC, ALC, eosinophil count, basophil count, absolute monocyte count, and platelet 
count. 
20 Serum chemistry parameters include sodium, potassium, chloride, calcium, magnesium, phosphorus, CO2 or bicarbonate, albumin, total protein, glucose, BUN or urea, 
creatinine, and uric acid. At screening, estimated GFR, measured GFR, or calculated (Cockcroft-Gault) CrCL will be assessed.  
21 Liver function tests include ALP, ALT, AST, and total, direct, and indirect bilirubin. 
22 Coagulation studies include fibrinogen, aPTT, and INR. 
23 Urinalysis will be performed by a dipstick method and include assessments of protein, glucose, leukocytes, and blood. 
24 A serum (hCG) pregnancy test must be performed during screening. A urine or serum (hCG) pregnancy test must be performed and documented to be negative on Day 1, before 
administration of the first dose of study treatment. A urine or serum (hCG) pregnancy test must be repeated every 4 weeks at the indicated visits and must also be performed at 
any point throughout the study if pregnancy is clinically suspected. All pregnancy tests, other than the test performed at screening, should be performed locally. 
25 The iron panel and related markers include iron, serum ferritin, TIBC, transferrin saturation, NTBI, hepcidin, CRP, and other markers of iron metabolism. Erythropoietin, 
erythroferrone, soluble transferrin receptor, and other markers of erythropoiesis will be collected as markers of erythropoietic activity. Remaining sample may be used for 
analyses of lipoproteins (only in subjects who have agreed to this optional analysis in the ICF). 


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26 For lipid testing samples for total cholesterol, LDL-C, HDL-C, and triglyceride will be collected after an overnight fast (see central laboratory vendor manual for detail). If the 
subject reports that they did not adhere to an overnight fast, these samples should not be collected at the scheduled visit. Instead, these samples should be collected within 
2 weeks of the original time point at the next scheduled visit or at an unscheduled visit, after the subject has adhered to an overnight fast.  
27 Sexual steroid testing includes estrone, estradiol, and testosterone (total and free). Samples should be drawn in the morning (does not need to be fasting). 
28 Whole blood samples for PKR flux assay (contingent upon site feasibility) and samples for PKR protein are to be collected predose (within 60 minutes prior to study treatment 
administration). On days of sample collection, the morning dose of study treatment must be administered at the study site following the predose collections. 
29 Study treatment is expected to be dispensed to the subject at the study site. Under exceptional circumstances and with agreement of the Sponsor (or representative), study 
treatment can be provided at the subject’s home, if acceptable by practice and allowed by local regulations. Subjects who discontinue or interrupt study treatment should undergo 
the recommended dose taper (Section 9.5.3), unless an emergency situation justifies discontinuing or interrupting the study treatment abruptly; study treatment will be dispensed 
to these subjects until study treatment is completely stopped. Following full discontinuation of study treatment, it will no longer be dispensed at any subsequent study visits the 
subject may attend. 
30 Pharmacokinetic sparse blood sampling collection times are as follows: Day 1 (predose; within 60 minutes prior to study treatment administration), Week 2 (1-2 hours and 
3-4 hours post-study treatment administration), Week 6 (4-5 hours and 6-7 hours post-study treatment administration), and Week 16 (predose; within 60 minutes prior to study 
treatment administration). On days of pharmacokinetic blood sample collection, the morning dose of study treatment must be administered at the study site; on days where a 
predose sample is required, the study treatment must be administered after the predose sample is taken. 
31 Pharmacokinetic full profile blood sampling will be conducted in all subjects at the Week 12 Visit at the following time points: predose (within 60 minutes prior to study 
treatment administration) and 30 minutes (±5 minutes), 1 hour (±5 minutes), 2 hours (±5 minutes), 4 hours (±30 minutes), and 8 hours (±30 minutes) post study treatment 
administration. On days of pharmacokinetic blood sample collection, the morning dose of study treatment must be administered at the study site; on days where a predose sample 
is required, the study treatment must be administered after the predose sample is taken. 
32 The Investigator should ask the subject for information regarding sleep patterns, signs, and symptoms associated with insomnia. 
33 For eligibility confirmation, the PKR genotyping must be analyzed by the central laboratory; therefore, this must be drawn early in the Screening Period to ensure results are 
back in time to allow the Investigator to assess eligibility. 
34 PKDIA, FACT-An, SF12v2, EQ-5D-5L, and PGIS should all be completed on the first day of screening (+1 day) and then at weekly intervals (±1 day) until Part 1 Day 1.  
35 During screening, at least 2 hematology samples must be collected at least 7 days apart.


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11. 
ADVERSE EVENTS 
11.1. 
Reporting Period for Adverse Events and Serious Adverse Events 
Monitoring of AEs, including frequency, severity, and characterization of SAEs, AESIs, and 
AEs leading to discontinuation will be conducted throughout the study. Adverse events and 
SAEs will be recorded in the source documentation and eCRF from the time of the signing of 
ICF through the subject’s completion of study or withdrawal of consent, whichever occurs first.  
All AEs will be monitored until resolution of the AE to baseline, the AE is considered stable 
within the context of the trial, the subject is lost to follow-up, or until 28 days after the last dose 
of study treatment unless the subject is enrolled in an extension study, in which case ongoing 
AEs will be reported in the extension study database following consenting of the subject.  
All SAEs will be followed until final outcome of the SAE is known, the subject is lost to 
follow-up, or the subject is enrolled in an extension study, in which case the SAE follow-up 
information will be reported in the extension study database. Any SAEs that are assessed as 
related to study treatment that occur ≥28 days post-treatment are to be reported to the Sponsor 
directly by the Investigator. 
Adverse events will be evaluated by the Investigator and recorded as per Section 11.3. Any AEs 
already documented at a previous assessment and designated as ongoing will be reviewed at 
subsequent visits or assessment time points as necessary. If these AEs have resolved, this will be 
documented. 
All AEs will be graded using the CTCAE v4.03 grading system. 
11.2. 
Definition of Adverse Events 
11.2.1. 
Adverse Event 
A clinical AE is any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product and which does not necessarily have to have a causal 
relationship with the study treatment. An AE can, therefore, be any unfavorable and unintended 
sign (including an abnormal laboratory finding, for example), symptom, or disease temporally 
associated with the use of a medicinal product, whether or not considered related to the 
medicinal product. 
Pre-existing conditions that worsen during a study are to be reported as AEs. Withdrawal 
hemolysis is to be reported as a study treatment-related AE. 
11.2.2. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
 Death 


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 Life-threatening (meaning that the subject was at immediate risk of death from the 
reaction as it occurred; but it does not include a reaction that hypothetically might 
have caused death had it occurred in a more severe form) 
 Inpatient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the subject was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (eg, surgery performed earlier than planned). 
 A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
 Congenital anomaly/birth defect 
 Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization, but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the subject and 
may require medical or surgical intervention to prevent 1 of the outcomes listed in the 
definitions for SAEs. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood 
dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse. 
11.2.3. 
Adverse Events Occurring Secondary to Other Events 
In general, AEs occurring secondary to other events (eg, cascade events or clinical sequelae) 
should be identified by their primary cause, with the exception of severe or serious secondary 
events. However, medically significant AEs occurring secondary to an initiating event that are 
separated in time should be recorded as independent events on the AE eCRF. Please refer to the 
eCRF completion guidance for examples of how to record events occurring secondary to other 
events. 
All AEs should be recorded separately on the Adverse Event eCRF if it is unclear as to whether 
the events are associated. 
11.2.4. 
Pre-existing Medical Conditions 
A pre-existing medical condition is one that is present during screening for this study. Such 
conditions should be recorded on the Medical History eCRF. 
A pre-existing medical condition should be recorded as an AE only if the frequency, severity, or 
character of the condition worsens during the study. When recording such events on the Adverse 
Event eCRF, it is important to convey the concept that the pre-existing condition has changed by 
including applicable descriptors (eg, “more frequent headaches”). 
11.2.5. 
Abnormal Laboratory Values 
Abnormal laboratory tests should be repeated as soon as possible for confirmation. Not every 
laboratory abnormality qualifies as an AE. A laboratory test result should be reported as an AE if 
it meets any of the following criteria: 


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 Accompanied by clinical symptoms 
 Results in a change in study treatment (eg, dosage modification, treatment 
interruption, or treatment discontinuation) 
 Results in a medical intervention (eg, potassium supplementation for hypokalemia) or 
a change in concomitant therapy 
 Clinically significant in the Investigator’s judgment 
It is the Investigator’s responsibility to review all laboratory findings. Medical and scientific 
judgment should be exercised in deciding whether an isolated laboratory abnormality should be 
classified as an AE. 
If a clinically significant laboratory abnormality is a sign of a disease or syndrome, only the 
diagnosis should be recorded on the Adverse Event eCRF. 
If a clinically significant laboratory abnormality is not a sign of a disease or syndrome, the 
abnormality itself should be recorded on the Adverse Event eCRF, along with a descriptor 
indicating if the test result is above or below the normal range. 
11.2.6. 
Adverse Events of Special Interest 
An AESI can be serious or nonserious. Ongoing monitoring and rapid communication (within 
24 hours) by the Investigator to the Sponsor is required to allow for further characterization and 
reporting to regulatory authorities. 
11.2.6.1. Transaminase Increase 
Transaminase increase is an AESI for AG-348. In the event of a transaminase increase of 
>2.5 × baseline (defined as the mean of the screening and Day 1 values) or an increase in AST or 
ALT to Grade ≥2 in severity, whichever is lower. The study site should report this occurrence to 
the Sponsor, using the AESI page in the eCRF, within 24 hours of their first knowledge of the 
event.  
An LFT panel should then be performed weekly until the transaminases have decreased to 
<2.5 × baseline (defined as the mean of the screening and Day 1 values). Additionally, the 
following tests should be performed to gain further information on the possible cause of the 
transaminase increase: 
1. Rule out biliary obstruction by liver imaging (liver CT scan, liver MRI, liver ultrasound, 
or magnetic resonance cholangiopancreatography, as clinically indicated).  
2. Viral screen for Epstein-Barr virus (EBV), cytomegalovirus (CMV) Abs, Hepatitis A Ab, 
HBsAg, HCVAb (with an RT-PCR test performed if HCVAb is positive), HIV-1Ab, and 
HIV-2Ab. 
3. Autoimmune hepatitis panel consisting of the following: serum antinuclear antibody, 
antismooth muscle antibody, liver-kidney microsomal type 1, antibody to soluble liver 
antigen, and antimitochondrial antibody when transaminase increase meets the criteria of 
AESI and repeated 4 weeks later, if the results were negative in the first time.  


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The Investigator should refer to Section 9.5 to determine if a dose adjustment is needed. If the 
Investigator is not sure whether or not a dose adjustment is needed, they should consult with the 
Independent Medical Monitor, or designee. 
11.3. 
Procedures for Reporting Adverse Events, Adverse Events of Special 
Interest, and Serious Adverse Events 
Each subject must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of nonleading questions (eg, “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from subjects. 
The Investigator should ask the subject for information regarding sleep patterns, signs, and 
symptoms associated with insomnia. 
All AEs (serious and nonserious) spontaneously reported by the subject and/or in response to an 
open question from study personnel or revealed by observation, PE, or other diagnostic 
procedures will be recorded on the appropriate page of the eCRF. Any clinically relevant 
deterioration in laboratory assessments or other clinical finding is considered an AE and must be 
recorded on the appropriate pages of the eCRF. When possible, signs and symptoms indicating a 
common underlying pathology should be noted as 1 comprehensive event. 
Any deaths and any AEs assessed as life-threatening are to be reported immediately. All SAEs 
are to be reported within 24 hours from the point in time when the Investigator becomes aware of 
the SAE on the appropriate page of the eCRF. All SAEs must be reported whether or not they are 
considered causally related to the study treatment. 
In the event that the electronic data capture (EDC) system is unavailable, a paper SAE and fax 
coversheet should be completed and faxed/emailed to the Sponsor within no more than 24 hours 
after learning of the event using the contact details provided to Investigators in the Serious 
Adverse Event Report Form Completion Guidelines. 
Excessive Hb responses should only be reported as an AE if they meet the criteria for Hb 
increased per CTCAE (ie, Hb concentration is higher than the subject’s ULN, as applies to men 
and women). Any reports of excessive Hb response should be graded using the CTCAE grading 
system. 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local IRB/IEC will be promptly notified based on local regulations 
where required by the IRB/IEC of all serious, unexpected adverse drug reactions involving risk 
to human subjects. 
11.3.1. 
Intensity 
The intensity of all AEs will be graded according to the NCI CTCAE. It is important to 
distinguish between SAEs and AEs with a severe intensity. An AE of severe intensity may not be 
considered serious. Severity is a measure of intensity, whereas seriousness is defined by the 
criteria in Section 11.2.2. For example, a severe headache without any further findings would not 
be considered an SAE. Alternatively, a mild presentation of a serious event, such as a myocardial 


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infarction assessed as mild by a cardiologist, that leads to hospitalization would be considered an 
SAE. 
Severity of all AEs, including clinically significant treatment-emergent laboratory abnormalities, 
will be graded according to the NCI CTCAE v4.03. Adverse events not listed by the CTCAE 
will be graded as follows: 
 Mild (Grade 1): The event is noticeable to the subject but does not interfere with 
routine activity. 
 Moderate (Grade 2): The event interferes with routine activity but responds to 
symptomatic therapy or rest. 
 Severe (Grade 3): The event significantly limits the subject’s ability to perform 
routine activities despite symptomatic therapy. 
 Life-threatening (Grade 4): An event in which the subject was at risk of death at the 
time of the event. 
 Fatal (Grade 5): An event that results in the death of the subject. 
11.3.2. 
Relationship to Study Treatment 
Relationship to study treatment administration will be determined by the Investigator according 
to the following criteria: 
 Not Related: AEs will be considered related, unless they fulfill the criteria as 
specified below:  
 Evidence exists that the AE has an etiology other than the study treatment 
(eg, pre-existing medical condition, underlying disease, intercurrent illness, 
concomitant medication); and/or  
 The AE has no plausible temporal relationship to the administration of the study 
treatment (eg, cancer diagnosed 2 days after the first dose of study treatment). 
 Related: AEs will be considered related if they fulfill the criteria as specified below: 
 There is a plausible temporal relationship between the onset of the AE and 
administration of the study treatment, and the AE cannot be readily explained by 
the subject’s clinical state, intercurrent illness, or concomitant therapies; and/or 
 The AE follows a known pattern of response to the study treatment; and/or 
 The AE abates or resolves upon discontinuation of the study treatment or dose 
reduction and, if applicable, reappears upon re-challenge. 
11.4. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(eg, spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female subject that occurs during this study or within 28 days following the last 
dose of study treatment must be reported to the Independent Medical Monitor, or designee, 
within 24 hours of being notified of the pregnancy. Any pregnancy in a female sexual partner of 


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a participating male subject that occurs during this study or within 90 days following the last 
dose of study treatment, must be reported to the Independent Medical Monitor, or designee, 
within 24 hours of being notified of the pregnancy, if acceptable by practice and allowed by local 
regulations. If the pregnancy in a female sexual partner of a participating male subject occurs 
after the male subject has completed the study, the pregnancy must be reported to the 
Independent Medical Monitor, or designee, directly by the Investigator. 
The Investigator must follow up and document the course and outcome of all pregnancies even if 
the subject was discontinued from the study or if the study has finished. The female subject or 
female sexual partner of a male subject should receive any necessary counseling regarding the 
risks of continuing the pregnancy and the possible effects on the fetus. Monitoring should 
continue until conclusion of the pregnancy. 
All outcomes of pregnancy in a female study participant or consented female sexual partner of a 
male participant must be reported by the Investigator to the Sponsor or Sponsor’s designee on a 
Pregnancy Outcome Report form within 28 days after he/she has gained knowledge of the 
delivery or elective abortion. 
Any SAE that occurs during pregnancy in a female study participant or consented female sexual 
partner of a male participant must be recorded on the SAE report form (eg, maternal serious 
complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, neonatal death, 
congenital anomaly, or birth defect) and reported within 24 hours in accordance with the 
procedure for reporting SAEs. 
Women of reproductive potential as well as men with partners who are women of reproductive 
potential, must agree to be abstinent as part of their usual lifestyle, or agree to use 2 forms of 
contraception, 1 of which must be considered highly effective, from the time of giving informed 
consent, during the study, and for 28 days following the last dose of study treatment for women 
and 90 days following the last dose of study treatment for men. Periodic abstinence (eg, calendar, 
symptothermal, and post-ovulation methods) and withdrawal are not acceptable methods of 
contraception. 


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12. 
STATISTICAL METHODS 
The study data will be analyzed and reported when all subjects have completed their Week 24 
Visit or Follow-up Visit (if applicable) or have discontinued the study. Final analysis of all data, 
including efficacy and safety, will be performed by the Sponsor, or designee. A detailed analysis 
plan for the analysis of efficacy and safety data will be presented in a SAP, which will be 
finalized before the database lock and study unblinding. 
12.1. 
Analysis Sets 
Three analysis sets will be defined for evaluation of the study endpoints: Full Analysis Set 
(FAS), Per Protocol Set (PPS), and Safety Analysis Set (SAS). 
12.1.1. 
Full Analysis Set 
The FAS is defined as all subjects who are randomized. Subjects will be analyzed according to 
the treatment they were randomized to, regardless of any errors in dosing. The FAS will be the 
primary analysis set for the efficacy endpoints.  
12.1.2. 
Per Protocol Set 
The PPS is defined as all subjects who are randomized and dosed and have Hb assessments at 
Weeks 16, 20, and 24 during the Fixed Dose Period. Subjects will be analyzed according to the 
treatment to which they were randomized, regardless of any errors in dosing. The PPS will be 
used for a sensitivity analysis for the primary endpoint and the key secondary endpoint as 
detailed in Section 12.2.4.2.  
12.1.3. 
Safety Analysis Set 
The SAS includes all subjects who received at least 1 dose of study treatment. Subjects will be 
analyzed according to the actual treatment they received (eg, if a subject received any active 
treatment, the subject will be grouped to the active arm). The SAS will be the primary analysis 
set for the safety analyses. 
12.2. 
Statistical Analysis 
This section presents a summary of the planned analyses of efficacy and safety for this study. 
Additional supportive and exploratory analyses will be specified in the final SAP, which will be 
finalized before the database lock and study unblinding.  
12.2.1. 
General Methods 
All individual subject data for all subjects will be presented in data listings. 
Continuous variables will be summarized using the following descriptive summary statistics: 
the number of subjects (n), mean, standard deviation (SD), median, minimum value, and 
maximum value.  
Categorical variables will be summarized using counts and percentages.  


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Baseline value: Unless otherwise specified, the baseline value will be defined as the most recent 
non-missing measurement (scheduled or unscheduled) collected before the initial administration 
of the study treatment.  
 The individual subject’s baseline Hb concentration is defined as the average of all 
available Hb concentrations from the central laboratory for that subject during the 
Screening Period up to the first dose of study treatment. 
 For transaminase (AST and ALT) assessments, the baseline will be defined as the 
average of the Screening Period and Day 1 values.  
Change (Absolute Change) from baseline: will be calculated as postbaseline value – baseline 
value. 
Relative change from baseline: will be calculated and presented in percentage as 
100 × (postbaseline value – baseline value)/baseline value. 
Treatment-emergent period: is defined as the time from the first dose of study treatment to 
28 days after the last dose of study treatment. 
12.2.2. 
Background Characteristics 
Subject disposition, demographic and baseline characteristics, prior and concomitant 
medications, study treatment exposure, and other background characteristics will be summarized. 
All subject data will be presented in subject data listings. All summaries will be based on the 
FAS unless otherwise specified in the SAP.  
12.2.2.1. Subject Disposition 
The number and percentage of subjects in different analysis sets below will be summarized, with 
the percentage calculated using the number of all subjects.  
 Subjects randomized or dosed 
 Randomized (FAS) 
 Randomized but not dosed 
 Randomized and dosed  
 PPS 
 Dosed (SAS) 
The number and percentage of subjects in the following disposition categories will also be 
summarized. The percentage will be calculated based on the FAS. 
 Completed treatment 
 Prematurely discontinued treatment and the reasons for treatment discontinuations 
 Completed study 
 Prematurely discontinued the study and the reasons for study discontinuations 


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12.2.2.2. Demographics and Baseline Characteristics 
Demographics, background (eg, medical history), and baseline characteristics will be 
summarized and presented for the FAS. 
12.2.2.3. Prior and Concomitant Medications 
Medications used in this study will be coded by using the World Health Organization Drug 
Dictionary Enhanced and categorized as the following:  
 Prior medication: any medication that started before initial dosing of study 
treatment, regardless of when it ended 
 Concomitant medication: medication continued or newly received during the 
treatment-emergent period 
 Post-treatment medication: medication continued or newly received after the 
treatment-emergent period 
A given medication can be classified as a prior medication, a concomitant medication, or a 
post-treatment medication; both prior and concomitant; both concomitant and post-treatment; or 
prior, concomitant, and post-treatment. If a medication has a missing or partially missing 
start/end date or time and it cannot be determined whether it was taken before, during, or after 
the treatment-emergent period, it will be considered as prior, concomitant, and post-treatment 
medications. 
Prior medications and concomitant medications will be summarized descriptively based on the 
FAS. Prior medications, concomitant medications and post-treatment medications will all be 
listed for each subject.  
12.2.3. 
Study Treatment Exposure and Compliance 
12.2.3.1. Study Treatment Exposure 
Duration of study treatment exposure is defined as follows: last dose date – first dose 
date + 1 day, regardless of any interruptions in dosing. If the last dose date of study treatment is 
missing, the subject’s treatment discontinuation or treatment completion date will be used for 
analysis purposes. 
Duration of study treatment exposure will be summarized descriptively as a quantitative variable 
(number, mean, SD, median, minimum, and maximum). 
Exposure summaries will be based on the FAS. 
12.2.3.2. Study Treatment Compliance 
Study treatment compliance will be assessed by percentage of days on treatment and percentage 
of tablets taken.  
The percentage of days on treatment will be calculated as follows:  
100 × (1 – [total number of days on study treatment] / [duration of full study treatment 
exposure + total number of days study treatment interrupted after last dose, if any]). 


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The total number of days of study treatment interruption is defined as the sum of (the number of 
days of each study treatment interruption), where number of days of each study 
treatment interruption is defined as the interruption end date − the 
corresponding interruption start date + 1. 
Percent of tablets taken will be calculated as follows:  
 100 × (Total number of tablets administered) / (Expected number of tablets taken 
during the study) 
Both percentage of days on treatment and percentage of tablets taken will be summarized 
descriptively as quantitative variables (number, mean, SD, median, minimum, and maximum). 
The number and percentage of subjects whose compliance is <80% or ≥80% will be 
summarized. 
Study treatment compliance will be based on the FAS.  
12.2.4. Efficacy Analysis 
Unless specified otherwise, only central laboratory results will be considered in the Hb related 
efficacy analyses. Hemoglobin results collected within 2 months (61 days) after an RBC 
transfusion will be excluded. 
12.2.4.1. Analysis of Primary Endpoint 
In the primary analysis of the primary efficacy endpoint, subjects' HR status will be analyzed 
using a logistic regression model. The model will include the HR status (Yes vs No) as the 
dependent variable and treatment as the independent variable, adjusting for the stratification 
factors including the average of screening Hb concentrations (<8.5 vs ≥8.5 g/dL) and the PKLR 
gene mutation category (missense/missense vs missense/non-missense). The primary analysis 
will be based on the FAS. In the primary analysis, subjects who discontinue the study before 
having at least 2 Hb laboratory assessments during the Fixed Dose Period will be considered not 
achieving an HR. The primary result obtained from the logistic model will be the estimated odds 
ratio between the active arm vs the placebo arm, along with the 95% confidence interval (CI) and 
the 2-sided P value. If the 2-sided P value is less than the required alpha (0.05), then the null 
hypothesis will be rejected and a statistically significant difference will be claimed for the active 
arm (ie, AG-348). When the logistic model fails to converge or its maximum likelihood estimate 
does not exist due to quasi-complete separation, the nonparametric method 
(ie, Cochran-Mantel-Haenszel test [CMH]) will be used. 
A sensitivity analysis based on the PPS will be conducted to evaluate the impact of missing 
assessments due to early study discontinuation. The number and percentage of subjects with HR 
will also be summarized by treatment arm. 
Additional sensitivity and supportive analyses will be specified in the SAP.  
12.2.4.2. Analysis of Key Secondary Endpoint 
The average changes from baseline in Hb concentrations at Week 16, 20, and 24 will be analyzed 
and compared between the active arm and the placebo arm by the linear Mixed-Effect Model 
Repeat Measurement (MMRM) method. The model will include the change in Hb at each visit as 


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the dependent variable; treatment, visit, and treatment-by-visit interaction as fixed effects; and 
subject as a random effect with adjustment for the stratification factors including the average of 
screening Hb concentrations (<8.5 vs ≥ 8.5 g/dL) and the PKLR gene mutation category 
(missense/missense vs missense/non-missense).  
The primary result obtained from the model will be the average treatment effect at Weeks 16, 20, 
and 24 based on the least square estimate (LS Means) from MMRM. The estimated LS Means, a 
95% CI, and a 2-sided P value will be provided. Furthermore, the LS Means and the treatment 
effect at each postbaseline visit, obtained from the model, will also be provided. 
12.2.4.3. Analysis of Other Secondary Efficacy Endpoints 
Other Hb related secondary endpoints: 
The maximum change in Hb will be summarized. The difference in the maximum change in Hb 
between the active vs the placebo arm, along with the 95% CI based on normal approximation, 
will be provided. The maximum change in Hb concentrations will be compared between the 
active arm and the placebo arm based on analysis of covariance (ANCOVA) controlling for the 
stratification factors, including the average of screening Hb concentrations and the PKLR gene 
mutation category.  
The time to first HR will be analyzed using the Kaplan-Meier method. The estimated median, 
25th percentile, and 75th percentile will be provided. The time to first HR will be compared 
between treatment arms based on a log rank test stratified by the stratification factors, including 
the average of screening Hb concentrations and the PKLR gene mutation category at a 
0.05 significance level. 
Other markers of hemolysis, hematopoietic activity, and indicators of clinical activity: 
The changes from baseline at each visit over time in markers of hemolysis, hematopoietic 
activity, and indicators of clinical activities will be summarized. Additional modeling based on 
the MMRM method, similar to the one specified for the key secondary endpoint in 
Section 12.2.4.2, may be conducted and details will be provided in the SAP.  
Patient-Reported Outcomes: PKDD and PKDIA 
PKDD and PKDIA are currently being developed by the Sponsor, and the data from the current 
study will be used for their validation. The analyses used to validate these 2 instruments will be 
based on the final pooled blinded data conducted by the psychometric vendor. The validation 
details will be provided in a separate PRO validation analysis plan. Once the validation of these 
PROs are complete and an algorithm used to calculate their scores are finalized, the analyses of 
these 2 PROs will be conducted accordingly based on unblinded data.  
In the final analysis of the PROs based on validated and finalized instrument, summary statistics 
and comparison between treatment arms will be provided as considered appropriate for the data. 
Additional modeling may be further conducted. Details will be provided in the SAP.  
12.2.4.4. Multiplicity Adjustment 
A hierarchy testing will be implemented for the primary endpoint and the key secondary 
endpoints. Only when the primary endpoint is significant at a 2-sided 0.05 significance level, the 
key secondary endpoint will be further tested.  


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For the other secondary efficacy endpoints, the nominal P values will be reported without further 
adjustment for multiplicity. However, these other efficacy endpoints are listed according to their 
clinical importance and may be considered when interpreting their nominal P values.  
12.2.5. 
Safety Analysis 
The overall safety profile of the study treatment will be assessed in terms of the following safety 
and tolerability endpoints: 
 AEs 
 Clinical laboratory values  
 ECGs (standard 12-lead) 
 Vital signs 
 PE findings 
 DXA scans 
Safety endpoints will be analyzed based on the SAS. Only descriptive analysis of safety will be 
performed (ie, no formal testing will be performed). 
12.2.5.1. Adverse Events 
For analysis purposes, AEs will be classified as pretreatment AEs, treatment-emergent adverse 
events (TEAEs), or post-treatment AEs.  
 Pretreatment AE: any AE that started before initial dosing of study treatment. 
 TEAE: any AE that increased in severity or that was newly developed during the 
treatment-emergent period. 
 Post-treatment AE: any AE that increased in severity or that was newly developed 
after the end of the treatment-emergent period. 
For AEs with missing or partial start dates, if there is no clear evidence that the AE started before 
or after study treatment, then the AE will be classified as a TEAE. 
Adverse event summary tables will be presented for TEAEs only and will include the following: 
 All TEAEs  
 TEAEs by relationship  
 TEAEs by maximal severity 
 TEAEs leading to treatment discontinuation 
 TEAEs leading to treatment interruption 
 Serious TEAEs 
 AESIs 
 TEAEs leading to death 


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Summaries will be presented by Medical Dictionary for Regulatory Activities (MedDRA) 
system organ class and preferred term using frequency counts and percentages (ie, number and 
percentage of subjects with an event as well as total number of events). When summarizing the 
number and percentage of subjects with an event, subjects with multiple occurrences of the same 
AE or a continuing AE will be counted once, only the maximum severity level will be presented 
in the severity summaries, and only the worst/highest relationship level will be presented in the 
relationship summaries. The number of TEAEs, when each event is considered unique, will also 
be provided.  
In addition, listings containing individual subject data for all TEAEs leading to treatment 
discontinuation, TEAEs leading to treatment interruption, serious AEs, AESIs, and death will be 
provided separately. All AEs, including pre- and post-treatment AEs, will be presented in 
individual subject data listings. 
12.2.5.2. Clinical Laboratory Assessments 
For laboratory measurements, the raw values and change from the baseline values of the 
continuous hematology and chemistry results will be summarized at each scheduled time point.  
The number and percentage of subjects with transaminase increases of >2.5× baseline or 
increases to Grade ≥2 (AESI of elevated transaminase as defined in Section 11.2.6.1), will be 
summarized. In addition, the number and percentage of subjects who have had elevated 
AST/ALT (>3× ULN, >5× ULN, and >8× ULN) will be summarized. 
Results of urinalysis and the serum and urine pregnancy tests will be listed in individual subject 
data listings only. In addition, a listing containing individual subject hematology, chemistry, and 
coagulation values outside the reference ranges will be provided. This listing will include data 
from scheduled and unscheduled time points. 
12.2.5.3. Electrocardiogram 
For ECG measurements, a summary of raw values and change from baseline values will be 
provided at each scheduled time point for the following standard digital ECG measurements: PR, 
QRS, QT, and QT corrected for heart rate intervals (QTcB and QTcF), QRS duration, and heart 
rate. 
The number and percentage of subjects will be summarized by maximum QTcB and QTcF 
intervals, categorized as ≤450 msec, >450 to ≤480 msec, >480 to ≤500 msec, and >500 msec, as 
well as maximum change from baseline, categorized as <30 msec, >30 to ≤60 msec, and 
>60 msec.  
12.2.5.4. Vital Signs 
For vital signs measurements, the raw values and change from baseline values will be 
summarized at each scheduled time point: systolic BP and diastolic BP (mmHg), body 
temperature (C), and heart rate (beats per minute).  
12.2.5.5. Physical Examination 
Physical examination findings will be presented as a data listing only. 


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12.2.5.6. Dual-Energy X-Ray Absorption Scans 
For DXA scans, the raw values of bone mineral density, T- and Z-scores, and their change from 
baseline values will be summarized for each scanned area and at each scheduled time point.  
12.3. 
Interim and Independent Data Monitoring Committee Analyses 
12.3.1. 
Interim Analysis 
No interim analysis is planned. 
12.3.2. 
Independent Data Monitoring Committee Analysis 
At least 1 IDMC meeting is planned for this study. A statistician independent from the Sponsor 
clinical team will prepare the analysis for the IDMC. Details regarding the IDMC meeting 
schedules and scope of the analysis will be specified in the IDMC charter. 
12.4. 
 Sample Size and Power 
12.4.1. 
Sample Size for the Primary Endpoint 
Assuming a response rate of 35% in the active arm and 5% in the placebo arm, 76 subjects 
(38 per arm) are needed to have 90% power to detect a treatment effect in HR rate based on a 
2-sided Fisher’s Exact test with 0.05 significance level. As detailed in Section 12.2.4.1, in the 
primary analysis of the primary efficacy endpoint, a logistic regression model adjusting for the 
stratification factors, including baseline Hb and mutation type, will be used to model HR rate 
between the active arm and the placebo arm. Based on the logistic model, the odds ratio, along 
with the 95% CI and 2-sided P value will be provided. If the 2-sided P value is less than the 
required alpha (0.05), then the null hypothesis will be rejected and statistical significant 
difference will be claimed for the active arm (ie, AG-348). 
The sample power calculation was based on EAST© Cytel Inc. Version 6.4. 
12.4.2. 
Power for the Key Secondary Endpoint 
Assuming a standard deviation of 1.5, a sample size of 76 subjects (38 per arm) has 80% power 
to detect a difference of 1.4 in the average change in Hb at Weeks 16, 20, and 24, based on the 
simple 2-sample t-test. As specified in Section 12.2.4.2, the final analysis of the key secondary 
endpoint will be based on MMRM. The average treatment effect at Weeks 16, 20, and 24 will be 
compared between active and placebo arms based on the LS Means from MMRM at a 2-sided 
0.05 significance level. A hierarchy testing will be implemented for the primary and the key 
secondary endpoint.  


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13.
ADMINISTRATIVE REQUIREMENTS
13.1. 
Good Clinical Practices 
The study will be conducted in accordance with the International Council for Harmonisation 
(ICH) for Good Clinical Practice (GCP) and the appropriate regulatory requirement(s). The 
Investigator will be thoroughly familiar with the appropriate use of the study treatment as 
described in the protocol and the IB. Essential clinical documents will be maintained to 
demonstrate the validity of the study and the integrity of the data collected. Master files should 
be established at the beginning of the study, maintained for the duration of the study, and 
retained according to the appropriate regulations.  
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki. 
The Investigator must obtain IRB/IEC approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any subject into the 
study. The IRB/IEC will review all appropriate study documentation in order to safeguard the 
rights, safety, and well-being of the subjects. The study will only be conducted at sites where 
IRB/IEC approval has been obtained. The protocol, IB, ICF, advertisements (if applicable), 
written information given to the subjects, safety updates, annual progress reports, and any 
revisions to these documents will be provided to the IRB/IEC.  
The IRB/IEC is to be notified of any amendment to the protocol in accordance with local 
requirements. Progress reports and notifications of serious unexpected adverse drug reactions are 
to be provided to the IRB/IEC according to local regulations and guidelines. 
13.3. 
Subject Information and Informed Consent 
The Investigator or trained designee will ensure that the subject is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. 
Subjects must also be notified that they are free to discontinue from the study at any time. The 
subject should be given the opportunity to ask questions and allowed time to consider the 
information provided. 
After the study has been fully explained, written informed consent will be obtained from the 
subject prior to study participation.  
The subject’s signed and dated informed consent must be obtained before conducting any 
study-related procedures. The Investigator must maintain the original, signed consent form. A 
copy of the signed form must be given to the subject. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
13.4. 
Subject Confidentiality 
In order to maintain subject privacy, all source documents, study treatment accountability 
records, study reports and communications will identify the subject by the assigned subject 


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number. The Investigator will grant monitor(s) and auditor(s) from the Sponsor or the Sponsor’s 
designee and regulatory authority(ies) access to the subject’s original medical records for 
verification of data gathered on the source documents and to audit the data collection process. 
The subject’s confidentiality will be maintained and will not be made publicly available to the 
extent permitted by the applicable laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB/IEC approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
subjects. The IRB/IEC may provide, if applicable, where regulatory authority(ies) permit, 
expedited review and approval/favorable opinion for minor change(s) in ongoing studies that 
have the approval/favorable opinion of the IRB/IEC. The Sponsor, or designee, will submit all 
protocol modifications to the regulatory authority(ies) in accordance with the governing 
regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
subjects, the Investigator will contact the Medical Monitor (or Medical Director), if 
circumstances permit, to discuss the planned course of action. Any departures from the protocol 
must be fully documented in the source documents/database. 
13.6. 
Data Management 
All data for the subjects recruited for the trial will be entered onto the eCRFs via an EDC system 
provided by the Sponsor, or designee. Only authorized staff may enter data onto the eCRFs. If an 
entry error is made, the corrections to the eCRFs will be made according to eCRF guidelines by 
an authorized member of the site staff. 
Electronic case report forms will be checked for correctness against source document data by the 
Sponsor’s monitor. If any entries into the eCRF are incorrect or incomplete, the monitor will ask 
the Investigator or the study site staff to make appropriate corrections, and the corrected eCRF 
will again be reviewed for completeness and consistency. Any discrepancies will be noted in the 
eCRF system by means of electronic data queries. Authorized site staff will be asked to respond 
to all electronic queries according to the eCRF guidelines. 
13.7. 
Source Documentation and Electronic Case Report Form 
Completion 
Source documents will be completed for each study subject. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the subject’s source 
document/eCRF. The source document should indicate the subject’s participation in the study 
and should document the dates and details of study procedures, AEs, and subject status. 
The Investigator, or designated representative, should complete the source document as soon as 
possible after information is collected for a subject’s examination, treatment, or any other study 
procedure. Any outstanding entries must be completed after the final examination. An 
explanation should be given for all missing data. 


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The Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or the Sponsor’s designee. Monitoring will be 
performed by personal visits from a representative of the Sponsor (Site Monitor) and will include 
on-site review of the source documents for completeness and clarity, cross-checking with source 
documents, and clarification of administrative matters will be performed. The review of medical 
records will be performed in a manner to ensure that subject confidentiality is maintained. 
The Site Monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study treatment and other study materials should be destroyed or returned to the 
Sponsor, or designee, after the study has been completed, as directed by the Sponsor. 
Regulatory authorities, the IRB/IEC, and/or the Sponsor’s clinical quality assurance group, or 
designee, may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities.  
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards. 
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor, or designee, to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information only to 
conduct the study and not to use it for any other purpose without explicit consent from the 
Sponsor. 
It is understood that there is an obligation on the Investigator’s part to provide the Sponsor with 
the complete data obtained during the study. Such information will be used in the clinical 
development of AG-348 and may be disclosed to regulatory authorities, other Investigators, 
corporate partners, or consultants, as required. 




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