1. Stephan Krause
Director, QA Technology
AstraZeneca Biologics
PDA Annual Meeting
16-18 March 2015
Las Vegas, NV
Alert, Action, and Specification
Limits for Bioburden and
Endotoxin

2. Outline
• Control strategy
• CQA risk assessment process
• Specification setting process
• Examples for bioburden and endotoxin specification revisions
• Considerations for alert and actions levels
• QRM-developed and justified alert and action levels
The content and views expressed in this paper by the author are not
necessarily views of the organization he represents.
PDA Annual Meeting - Las Vegas 17March15

3. Risk Assessment(s) and Control Strategy Elements During Product
Development
FTIH POC BLAQTPP
Final CQAs &
Control Strategy Approval
Potential CQAs
Product & Process
Design
Life-Cycle
Management
POST-APPROVAL
CHANGES
PHASE 3PHASE 1/2Pre-IND
CQA
Patient Impact
Severity
Assessed
(Safety and
Efficacy)
Overall Risk Assessment
(ex., FMEA)
Final Assessment
Uncertainty
Detectability
Occurrence
Control
Strategy
Procedural
Control
Process
Validation
Lot Release
Testing
Raw Material
Control
Stability
Testing
Operational
Parameters
Risk(s)
Control(s)
Re-assessed
Re-assessed
In-Process
Testing
Characterization
Testing
Reproduced from Schenerman, M. et al. ,
Wiley Interscience. pp. 53-84, 2009. PDA Annual Meeting - Las Vegas 17March15

4. Risk Assessment Process During Product Development
Overall Risk Assessment
(ex., FMEA) Scoring
Severity Score
Probability
Score
Detectability
ScoreControl
Strategy
(p)CQA
(Prior to PV
Stage 2)
X
CQA
(at/after PV
Stage 2)
X
PDA Annual Meeting - Las Vegas 17March15

5. General Specification Setting Process
Acceptance
Criteria
Existing
Knowledge of
Mfg/Analytical
Capability
Historical Data
from this
Product and Mfg
Plant
Patient Impact
“Platform”
Knowledge from
Similar Product
and Process
Product-Specific
Clinical
Consideration
and/or Experience
Regulatory/
Industry Standard
PDA Annual Meeting - Las Vegas 17March15

6. Definitions (FDA Draft Guidance and PDA Bioburden/Biofilm TR)
Alert Limit- An established microbial or particulate level giving early warning of potential
drift from normal operating conditions and which trigger appropriate scrutiny and follow-
up to address the potential problem. Alert Limits are always lower than Action Limits.
Action Limit- An established microbial or particulate level which when exceeded should
trigger appropriate investigation and corrective action based on the investigation.
Acceptance Criterion/Specification – Failure to meet an established acceptance criterion
or specification renders the product unacceptable.
Bioburden- Total number of microorganisms associated with a specific item prior to
sterilization.
Colony Forming Unit (CFU)- A microbiological term which describes the formation of a
single macroscopic colony after the introduction of one (or more) microorganism(s) to
microbiological growth media. One colony forming unit is expressed as 1 CFU. (Alert
and Action Levels are reported as CFU/volume tested.)
Endotoxin- A pyrogenic product (e.g., lipopolysaccharide) present in the bacterial cell
wall. Endotoxin can lead to reactions in patients receiving injections ranging from fever
to death. (Alert/Action levels are reported as EU/mL or EU/mg protein).
Worst case- A set of conditions encompassing upper and lower processing limits and
circumstances, including those within standard operating procedures, which pose the
greatest chance of process or product failure (when compared to ideal conditions).
PDA Annual Meeting - Las Vegas 17March15

7. USP <85> Bacterial Endotoxins Test (BET by LAL – quantitative)
• BET assay description:
(Linear) standard curve: 0.005 – 5.0 EU/mL; results are measured in
EU/mL then converted to EU/mg (protein).
• Reporting of IPC and DS result:
< 0.005 EU/mL (= QL) x DF = reported result in EU/mL
EU/mL ÷ mg protein / mL = EU/mg protein
• Example for Product administered (1.0 mL with 100 mg/mL):
< 0.005 x 20 = 0.10 EU/mL ÷ 100 mg / mL = < 0.01 EU/mg (results are
“rounded up” to 1/100th EU/mg specification unit).
• Endotoxin Limit:
The endotoxin limit is 5.0 EU/kilogram, which represents the
approximate threshold pyrogen dose for humans and rabbits.
PDA Annual Meeting - Las Vegas 17March15

8. Examples for Clinical and Process Qualification (PV Stage 2) Drug
Substance Specifications for Bioburden and Endotoxin
Test /
Specification
IMP Phase 1-2
IMP Pivotal or
Phase 3
PQ Lots
(PV Stage 2)
Reported
Results
Example
Bioburden
NMT 10 CFU per
100 mL
NMT 1 CFU per
10 mL
NMT 1 CFU per
10 mL
0 CFU per
10 mL (1)
Endotoxin
(LAL)
NMT 1.75 EU/mg
protein
NMT 1.75 EU/mg
protein
NMT 0.50
EU/mg protein
0.01 EU/mg
protein
Bioburden: Specification for this critical safety quality attribute is compendia-based and an industry
standard. Specification can remain unchanged as it is already tightly controlled for early-stage clinical
studies. The use of a 10 mL sample volume was validated and justified with spiked DS samples.
(1) The 100 mL sample volume may be required, if a volume smaller than 100 mL cannot be validated
(equivalent) or upon request of a regulatory agency.
Endotoxin (LAL): A specification suggested in USP <85> for the Bacterial Endotoxins Test (by LAL; NMT
5.0 EU/kg body weight) can be used for early and late-stage clinical studies. Calculation: 5.0 EU/kg x 35
kg/100 mg = 1.75 EU/mg (not changed as 1.7549 EU/mg = 5.014 (5.0 EU/kg)). For PQ lots, the DS
specification is tightened, based on plant-specific manufacturing experience.
PDA Annual Meeting - Las Vegas 17March15

9. Example: DS/DP Endotoxin Specification and Alert/Action Level Options
• Due to limited data at/above the BET QL for DS, meaningful limits cannot be calculated.
• Instead, the specifications can be tightened, justified based on maximum patient exposure.
Examples:
A. Proposed Specification of ≤ 1.75 EU/mg remains unchanged from Phase 3 CTM.
B. The specifications are tightened from ≤ 1.75 EU/mg to ≤ 1.00 EU/mg.
C. The specifications are tightened from ≤ 1.75 EU/mg to ≤ 0.50 EU/mg.
D. The specifications are tightened from ≤ 1.75 EU/mg to ≤ 0.31 EU/mg.
For A-D: In addition to a specification level, action and alert levels can be set. Since no
endotoxin levels were detected at Late-Stage/Commercial Mfg plant , the following Action and
Alert Level options are proposed:
1. Alert Level = Quantitation Limit (0.005 EU/mL or (0.005 EU/mL x DF) / mg/mL => EU/mg)
2. Action Level = 5x QL (0.005 EU/mL or (0.025 EU/mL x DF) / mg/mL => EU/mg)
Action Level Example: (Worst case) for Clinical Product: 0.025 x 50/20 = 0.31 EU/mg (rounded)
Alert Level Example: (Worst case) for Clinical Product: 0.005 x 50/20 = 0.06 EU/mg (rounded)
PDA Annual Meeting - Las Vegas 17March15

10. Examples for DS/DP Endotoxin Specification Revision
Justifications
A. The specifications are tightened from ≤ 1.75 EU/mg to ≤ 1.00 EU/mg. The QL
for worst-case DF and protein concentration (50x and 20 mg/mL), 0.005 EU/mL x
50/20 = 0.0125 EU/mg (0.01 EU/mg), is about 100-fold lower than the proposed
specification. This proposed specification is tighter than a calculated maximum
specification based on the limit of 5.0 EU/kg of body weight (USP <85> BET), a
lowest patient weight of 35 kg, and a maximum dose of 20 mg. The highest
potential passing result of 1.0049 EU/mg for the proposed specification of 1.68
EU/mg equals 2.87 EU/kg.
B. The specifications are tightened from ≤ 1.75 EU/mg to ≤ 0.50 EU/mg. The QL
for worst-case DF and protein concentration (50x and 20 mg/mL), 0.005 EU/mL x
50/20 = 0.0125 EU/mg (0.01 EU/mg), is about 50-fold lower than the proposed
specification. This proposed specification is tighter than a calculated maximum
specification based on the limit of 5.0 EU/kg of body weight (USP <85> BET), a
lowest patient weight of 35 kg, and a maximum dose of 20 mg. The highest
potential passing result of 0.5049 EU/mg for the proposed specification of 0.50
EU/mg equals 1.44 EU/kg.
PDA Annual Meeting - Las Vegas 17March15

11. Possible IPC Alert/Action levels of Upstream/Downstream and DS
Specification Lifecycle Strategy - Endotoxin
DS Endotoxin Specification:
• For PQ and/or when extensive plant-specific experience exists, tighten DS specifications.
• Use statistically calculated limits and/or use justified limits (ex., clinical experience, maximum
patient exposure).
IPC Alert/Action Levels:
• For clinical and/or limited plant-specific historical experience, use QRM tools (ex., modified FMEA)
to establish risk-based IPC alert/action limits.
• Use three levels (high, medium, low) for each IPC sample based the QRM risk priority numbers.
• For commercial and/or when extensive plant-specific experience exists,
• Use calculated worse-case limits based on microbial proliferation opportunity. The primary
factors that affect the risk of microbial proliferation during hold times are the growth-
promoting properties of the in-process materials, initial bioburden level, and storage
conditions.
• Or, set statistically calculated limits (ex., based on plant-specific process capability.)
• Or, use a combination between the two as data may not be continuous (most results
reported as: < Alert Level (DL/QL) and/or therefore not normally distributed.
PDA Annual Meeting - Las Vegas 17March15

12. Possible IPC Alert/Action levels of Upstream/Downstream and DS
Specification Lifecycle Strategy - Bioburden
DS Bioburden Specification:
• Specification for this critical safety quality attribute is compendia-based and an industry standard.
• Specification can remain unchanged as it is already tightly controlled for early-stage clinical
studies.
• The use of less than 100 mL sample volume (ex., 10 mL) should be validated and justified with
spiked DS samples. Some regulatory agencies may require a 100 mL sample volume.
IPC Alert/Action Levels:
• For clinical and/or limited plant-specific historical experience, use QRM tools (ex., modified FMEA)
to establish risk-based IPC alert/action limits.
• Use three levels (high, medium, low) for each IPC sample based the QRM risk priority numbers.
• For commercial and/or when extensive plant-specific experience exists,
• Use calculated worse-case limits based on microbial proliferation opportunity. The primary
factors that affect the risk of microbial proliferation during hold times are the growth-promoting
properties of the in-process materials, initial bioburden level, and storage conditions.
• Or, set statistically calculated limits (ex., based on plant-specific process capability.)
• Or, use a combination between the two as data may not be continuous and/or normally
distributed. PDA Annual Meeting - Las Vegas 17March15

13. General Considerations for Bioburden and Endotoxin
IPC for Downstream
PDA Annual Meeting - Las Vegas 17March15
• IPC action and alert limits for bioburden and endotoxin are narrower
towards the end of the downstream process.
• Bioburden and endotoxin IPC limits are narrower at/after Virus Filtration.
• The alert limit is further tightened to ≤ 0 CFU/10 mL at the final step(s) so
that any presence of bioburden is investigated.
• Proliferation Opportunity (time, temperature, replicability – link to
endotoxin)
• Endotoxin limits for in-process intermediates are determined based on
the following factors:
• Quantitation Limit of 0.005 EU/mL
• Maximum valid dilution (MVD) – Dilution Factor used
• Expected clearance through the purification unit operations
• Proliferation Opportunity (time, temperature, replicability – link to
bioburden)

14. Score Rating Overall Risk Rating (for Alert/Action Levels, exluding Occurrence)
1-27 Low The potential impact is minimal or has been minimized through effective controls
36-108 Medium
The potential impact is somewhat significant. Effective controls are in the process of being
implemented or the existing controls are not comprehensive enough to fully mitigate the risk.
162-729 High
The impact is significant. The existing controls are not effective in mitigating the risk or no controls
are in place at all.
List of Mfg
Process Steps
(Examples)
(Potential) Failure
Mode Description
Unwanted Event
Result
S
(Severity -
Patient
and/or
Firm)
O
(Previous)
Occurance
D
(Detection
Probability)
P
(Proliferation
Opportunity)
C
(Control in Place
or Risk Mitigated)
RPN (Risk Priority
Number)
Downstream
(1, 3, 9)
1=low
9=high
(patient
impact)
(1-3)
1=low
2=medium
3=high
(1-3)
1=high
2=mediu
m 3=low
Hold Time
x Temp. x
Replicabilit
y (1,3,9)
1=low
9=high
1=yes 2=not
complete
3=no
Up to 27 = green
36-108 = yellow
162-729 = red
Modified QRM FMEA for Bioburden and Endotoxin IPC
Alert/Action Levels
PDA Annual Meeting - Las Vegas 17March15

15. Modified FMEA Example for Downstream Alert/Action Levels
PDA Annual Meeting - Las Vegas 17March15
List of Mfg
Process
Steps
(Examples)
(Potential)
Failure Mode
Description
Unwanted Event
Result
S
(Severity -
Patient
and/or
Firm)
O
(Previous)
Occurance
D
(Detection
Probability)
P
(Proliferatio
n
Opportunity)
C
(Control in
Place or Risk
Mitigated)
RPN (Risk Priority
Number)
Downstream
(1, 3, 9)
1=low
9=high
(patient
impact)
(1-3)
1=low
2=medium
3=high
(1-3)
1=high
2=medium
3=low
Hold Time x
Temp. x
Replicability
(1,3,9) 1=low
9=high
1=yes 2=not
complete
3=no
Up to 27 = green
36-108 = yellow
162-729 = red
Formulation
Buffer
Contaminated
(bioburden) buffer
can further
contaminate the
filtered, formulated
drug
substance/bulk.
Bioburden
contamination in
DS (above Action
Level).
Unacceptable
endotoxin levels
post-filtration.
9 [1] 2 3 3 162

16. Modified FMEA Example for Downstream Alert/Action Levels
Is Previous Occurrence a Factor Here ?
PDA Annual Meeting - Las Vegas 17March15
List of Mfg
Process
Steps
(Examples)
(Potential)
Failure Mode
Description
Unwanted Event
Result
S
(Severity -
Patient
and/or
Firm)
O
(Previous
Occurrence)
D
(Detection
Probability)
P
(Proliferation
Opportunity)
C
(Control in
Place or Risk
Mitigated)
RPN (Risk Priority
Number)
Downstream
(1, 3, 9)
1=low
9=high
(patient
impact)
(1-3)
1=low
2=medium
3=high
(1-3)
1=high
2=medium
3=low
Hold Time x
Temp. x
Replicability
(1,3,9) 1=low
9=high
1=yes
2=not complete
3=no
Up to 27 = green
36-108 = yellow
162-729 = red
Formulation
Buffer
Contaminated
(bioburden) buffer
can further
contaminate the
filtered, formulated
drug
substance/bulk.
Bioburden
contamination in
DS (above Action
Level).
Unacceptable
endotoxin levels
post-filtration.
9 [1] 2 3 3 162

17. Modified FMEA Example for Downstream Alert/Action Levels
Can Uncertainty be a Factor ?
PDA Annual Meeting - Las Vegas 17March15
List of Mfg
Process
Steps
(Examples)
(Potential)
Failure Mode
Description
Unwanted Event
Result
S
(Severity -
Patient
and/or
Firm)
U
(Uncertainty)
D
(Detection
Probability)
P
(Proliferation
Opportunity)
C
(Control in
Place or Risk
Mitigated)
RPN (Risk Priority
Number)
Downstream
(1, 3, 9)
1=low
9=high
(patient
impact)
(1-3)
1=low
2=medium
3=high
(1-3)
1=high
2=medium
3=low
Hold Time x
Temp. x
Replicability
(1,3,9) 1=low
9=high
1=yes
2=not complete
3=no
Up to 27 = green
36-108 = yellow
162-729 = red
Formulation
Buffer
Contaminated
(bioburden) buffer
can further
contaminate the
filtered, formulated
drug
substance/bulk.
Bioburden
contamination in
DS (above Action
Level).
Unacceptable
endotoxin levels
post-filtration.
9 [1] 2 3 3 162

18. Modified FMEA Example for Downstream Alert/Action Levels
PDA Annual Meeting - Las Vegas 17March15
Up to 27 = wide limits
36-108 = medium limits
162-729 = narrow limits
Scoring Description/Justification
Current Control(s)
and/or Possible
Risk Mitigation
Recommended
Action(s)
Sampling/Testing
Bioburden
RPN-based Action(s)
Alert/Action Level(s)
Endotoxin
RPN-based
Action(s)
Alert/Action Level(s)
(Potential) high bioburden and/or endotoxin
levelscould impact product safety. Product
released with high Endotoxin levels, causing
adverse events in patients. Higher S patient
multiplier (9) used in RPN calculation. Endotoxin
levels in Formulation buffer have been below
Alert Level (1). Detectability is limited (2) due to
sampling (sample may not be representative and
real-time testing not possible). Limited
proliferation possible (3) as processing
temerature and time in manufacturign is RT and
up to 10 hours. Replicability (doubling) in
formulation buffer is not prevented. Control is
limited to bioburden filtration and not endotoxin.
High levels of bioburden would also render the
formulation buffer unacceptable.
(RPN = S (patient) [x O] x D x P x C = 9 x 1 x 2
x 3 x 3 = 162
Formulation buffer is
to be tested and
rejected prior to use
if at/above Action
Level(s).
Sample formulation
buffer no later than xx
hours prior to use.
Store formulation
buffer at 2-8 C and no
longer tha xx hours at
RT.
Narrow Limit(s):
Alert = 1 CFU/10mL;
Action = 10 CFU/10mL
Narrow Limit(s):
Alert = 0.25 EU/mL;
Action = 1.00 EU/mL

19. Examples of PQ (PV Stage 2) Downstream In-Process Control
Alert and Action Limits for Bioburden and Endotoxin
Test / Alert/Action
Level
Mfg Process
Step/Material
Risk Priority
Number
Alert/Action Levels
Bioburden Formulation Buffer High
Alert: 1 CFU/10mL
Action: 10 CFU/10mL
Endotoxin (LAL) Formulation Buffer High
Alert: 0.25 EU/mL
Action: 1.00 EU/mL
Bioburden
Protein A Column Wash
Buffer
Medium
Alert: 3 CFU/10mL
Action: 30 CFU/10mL
Endotoxin (LAL)
Protein A Column Wash
Buffer
Medium
Alert: 0.25 EU/mL
Action: 2.0 EU/mL
Bioburden
Combined Protein A
Column Product Pool
Low
Alert: 10 CFU/10mL
Action: 100 CFU/10mL
Endotoxin (LAL)
Combined Protein A
Column Product Pool
Low
Alert: 1.0 EU/mL
Action: 5.0 EU/mL
PDA Annual Meeting - Las Vegas 17March15

20. Examples of PQ (PV Stage 2) Upstream In-Process Control
Alert and Action Limits for Bioburden and Endotoxin
Test / Alert/Action
Level
Mfg Process
Step/Material
Risk Priority
Number
Alert/Action Levels
Bioburden
Bioreactor Pre-Transfer
Seed Sample
High Alert/Action: 1 CFU/10mL
Endotoxin (LAL)
Bioreactor Pre-Transfer
Seed Sample
High
Alert: 0.25 EU/mL
Action: 1.00 EU/mL
Bioburden
Equilibration Buffer from
Harvest Tank
Medium
Alert: 1 CFU/10mL
Action: 10 CFU/10mL
Endotoxin (LAL)
Equilibration Buffer from
Harvest Tank
Medium
Alert: 0.25 EU/mL
Action: 2.0 EU/mL
Bioburden
Post-Harvest Conditioned
Medium
Low
Alert: 10 CFU/10mL
Action: 100 CFU/10mL
Endotoxin (LAL)
Post-Harvest Conditioned
Medium
Low
Alert: 0.25 EU/mL
Action: 5.0 EU/mL
PDA Annual Meeting - Las Vegas 17March15