Aplicação do QbD nos processos e produtos biotecnológicos
Luciana Mansolelli
24-Junho-2013
II SIMPÓSIO SINDUSFARMA / ANVISA / FIP-IPS
• Introduction - QbD
- Group B Streptococcus
- Prefilled syringe
• Approach for Product Understanding - Critical Quality Attributes
- Analytical Target Profile
• Approach for Process Understanding - Process Characterization & Critical Process Parameters
• Concluding remarks
2
Outline
3
QbD is “a systematic approach to development that begins with predefined
objectives and emphasizes product and process understanding and
process control, based on sound science and quality risk management in
order to ensure the quality of the product” - ICH Q8 (R2)
Product knowledge (quality criteria based on the demonstrated
safety/ efficacy)
Relationship between process parameters and critical quality
attributes/ performance
Product quality
ensured by risk-
based control
strategy for well
understood
product/process
Quality by Design
4
A “minimal”, empirical approach (as defined in ICH Q8 R2) for technical
development does not necessarily ensure a well known and controlled
product/ process, considering complexity of vaccines.
In other words, it is the complexity of vaccines that requires an
improved knowledge of product and process applying risk- based
approaches, according to Quality by Design.
Throughout this presentation we will use a case of QbD application to a
candidate vaccine against Group B Streptococcus (GBS), at drug
substance level, focusing on aspects potentially impacting the final
product quality. And also briefly discuss a prefilled syringe as a primary
packaging alternative, and discuss what kind of knowledge basis the
QbD approach brings to us.
QbD & vaccines
QTPP is a collection of Critical Quality Attributes (CQA)
Critical Quality Attributes (CQA) are:
• Physical, chemical, biological, etc. properties that should be within a desired range to ensure product quality
Typically resembles the specifications
• Dosage Form and Route of Administration
• Dosage Form Strengths
• Drug Release (Dissolution/Disintegration Time)
and/or Pharmacokinetic Profile
• Sterility, Purity, etc.
Quality Attributes
Selected salt form:
Salt form 4 row
Salt counter
ionStability DS
Stability with
Excipients
Polymorphism
(polymorphs,
hydrates, solvates)
Morphic
propertiesSolubility
Feasibility of
DS and DP
manufacturing
column 1 2 3 4 5 6 7
Salt form 1: Base a Low Low Low High Low High High
Salt form 2: Maleate b Low High Low Low Low High High
Salt form 3: Citrate c Low Low Low High Low High High
Salt form 4: HCl d Low Low Low High Low High High
Salt form 5: N.D. e Low Low Low Low Low Low Low
Guiding Criteria: Salt Selection
Insert file here.
“Low” indicates minimal risk to attribute evaluated “High” indicates risk needs to be further assessed
Risk Assessments
API Material Attributes (salt, polymorph...)
API Morphology (particle size…)
Formulation (API, excipients…)
Final Processing (mixing…)
Development Timeline
PreC Ph 1 PH 2b Ph 3
Risk Assessments
Fishbone
2 Raw Materials 4 Nibbler
3 Vibration Table1 Plant Factors
1.1 Temperature in
the plant
1.2 Humidity of the
milling air
3.1 Type
3.2 Frequency of
oscillation
3.3 Plane of oscillation
3.4 Form of oscillation
...
7 Filter
4.1 Type
4.2 Sieve size
4.3 Rotation speed
...
2.1 Purity of IP
2.2 PSD of IP
2.3 Flowability of IP
...
5 Feeder
5.1 Type of feeder
5.2 Type of screw
5.3 Rotation speed
...
6 Jet-Mill and Classifier
6.1 Type of jet-mill
6.2 Type of nozzle
6.3 Type of classifier
6.4 Feed rate of IP
...
7.1 Type
7.2 Pressure drop
7.3 Interval time of the
pressure impulse
...
8 Container Filling Unit
9 Blender 11 Sampling
10 Drum Filling Unit
8.1 Type of rotary gate feeder
8.2 Rotation speed of the rotary
gate feeder
...
9.1 Type
9.2 Rotation speed
9.3 Operation time
9.4 Interval for shifting
the rotation direction
...
10.1 Type filling unit
10.2 Type weighing machine
...
11.1 Procedure
11.2 Representativeness
...
CQAs (Ref. 2):
- PSD
- XRD-spectrum
- purity
(heavy metals)
- microbiological
tests
FMEA Table
DoE Design Space
high high
high
low
low
low
low
low
high
high high
low
low
high
high
high
high
low
high
high
low
low
low
low
H
A
B
C
D
E
F
G
I K
J
L Variable 1: (WA)
Variable 2: (GT)
Variable 3: (AF)
‘Out of Specification‘
‘Within Specification‘
Risk Assessments/DoEs/Design Space
Dynamic Process Data
Collection
Final Product CQAs
CPP9 – CQA5
-20
-10
0
10
20
-30 -20 -10 0 10 20 30
t[2]
t[1]
Batch Level Analysis of Granulation Process
R2X[1] = 0.5656 R2X[2] = 0.298283
Ellipse: Hotelling T2 (0.95)
S0010-B_85
S0011-A_85
S0011-B_85
S0012-A_85
S0012-B_85
S0013-A_85S0013_B_85
S0014_A_85
S0014_B_85
S0015-A_85
S0015_B_85
S0016-A_85
S0016-B_85
FMEA/
DoE
FMEA/
DoE
FMEA/
DoE
FMEA/
DoE
FMEA/
DoE
CPP2 – CQA2 CPP3 – CQA1,4
CPP6 – CQA5 CPP7 – CQA1
CPP4 – CQA4 CPP5 – CQA1,3 CPP8 – CQA2
FMEA/
DoE
Incoming Raw Material(s)
CPP1 – CQA3
Step 1
In process CQA
Step 2
In process CQA
Step 3
In process CQA
Step 4
In process CQA
Step 5
In process CQA
Control Strategy/Continual Improvement
10
GBS Vaccine: Purified capsular polysaccharides of serotypes Ia, Ib and III, each
conjugated to modified Corynebacterium diphtheriae toxoid (CRM197)
Indication: Prevention of invasive Group B streptococcus infection in infants via
immunization of pregnant women (>24 wks gestation) ; Phase 1.
CRM- GBS Ia
(simplified picture)
Coupling
Chemistry
(covalent
linkage)
+ Carrier
Protein
(CRM197)
Polysaccharide
(serotype specific
GBS antigen)
Polysaccharide-
Protein Conjugate
GBS vaccine
QbD: Overview of the assessment process Periodically refined based on gained knowledge
11
CQA
assessment
& ranges
What ?
Why ?
CPP/ KPP
assessment
Ensure proper
focus on the
process
parameters
impacting CQAs
(CPPs) as well as
performance
(KPPs)
Identify PPs
influencing
CQAs and PAs;
define CPP
ranges within
which product
quality is not
impacted
Determine
how well
we need to
control the
CPPs and
KPPs
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
In S
um
mary
Control
strategy
Define
risks
related to
meeting of
pre-
defined
quality
criteria
Based on
range
assessment,
Identify the
CPPs/ KPPs
which have to
be carefully
controlled to
ensure good
performance
and quality
Plan a set of
controls, that
ensures
process
performance
and product
quality
Overall
verification of
process/
product
monitoring, to
minimize risks
of failure
For each CQA,
evaluate risks
associated to
analytical
monitoring and
process; prioritize
based on criticality
score
Decide on
what
attributes
are critical
for safety
and efficacy
and define
their ranges
Analytical
Target
Profile
Challenge
analytical
methods to meet
the pre-defined
target for testing
of CQAs
Prioritize actions
to increase
knowledge on
criticality and on
ranges; build
strong rationales
for specifications
Define
minimum
and
optimum
requiremen
ts of
analytical
methods
Target
Product
Profile
(TPP)
Describe
product
requirements
for intended
use
Ensure that
product and
process
development
start from
patient’s
needs
Some quality attributes of GBS (DS) evaluated for criticality
n
O O
O H
O H
O H
O H O O
N H A c O
H O O
C O O -
H O A c H N
H O H 2 C
O H O
O H
O H O
O H H O
O H O
H H
O O
O H
O H
O H
O H O
O N H A c O
H O O
C O O -
H O A c H N
O H O
O H
O H O O
O H H O
O H O
H
O
N H
O H N a +
N a +
O O
O H
O H
O H
O H O O
N H A c O
H O O
C O O -
H O A c H N
O H O
O H
O H O
O H H O
O H O
H N a + O
O H
N H 2
12
Potential impact on efficacy
CRM 197 unfolding and structural modification
Antigen aggregation
Glycosylation Degree
Residual unconjugated CRM
Molecular dimensions
Sialic Acid retention in PS
N-acetyl groups in PS
Unconjugated PS
Polysaccharide Structural conformity
Oxidation degree
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
13
Quality
attribute
Efficacy Safety Overall
Score Impact Uncert Score Impact Uncert Score
Preservation of
terminal sialic
acid
25
Immune response
dependent on
terminal sialic acid
3
Non-
clinical
studies
75 2
Not expected
to impact
safety
4 Literature 8 75
Free CRM 2
Immune response
not exp to depend on
free CRM
4 Literature 8 2 Safety of CRM
is proven 1
Accepted by
community 2 8
Free
(unconjgated)
polysaccharide
25
Quality of
immunoresponse
could be dependent
on the amount of
conjugated
saccharide
4 Literature 100 2
Safety not
expected to be
impacted
4 Literature 8 100
General safety-
related
attributes
/ / / / / 25 Safety
impacted 1
Accepted by
community
25 25
Severity = Impact x Uncertainty
CQA risk assessment CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
14
Path to ensure proper control of potential CQAs
For each potential CQA:
Define the Analytical Target Profile and explore analytical approaches to ensure reliable testing
(release/ characterization/ in- process)
Confirm attribute criticality through nonclinical testing & eventually perform clinical evaluation of
CQA range
Periodically re-evaluate quality attributes criticality and ranges
Identify potential parameters impacting the CQA (potential CPPs)
Confirm process parameter criticality through screening DOEs
Evaluate normal operating ranges and proven acceptable ranges for confirmed CPPs
Define control strategy
This presentation will show how this path is being approached for free
polysaccharide in GBS glycoconjugates
15
Free polysaccharide
Minutes
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035PDA - 200nm
BulkRS2901(GGC_060)_TQ_100mMH3BO3_25mMSDS
BulkRS2901(GGC_060)TQ_100mMH3BO3_25mMSDS 10-22-2007 8-11-33 PM -Rep5.dat
“Free” PS
Addition of unconjugated “Free”
CRM
Glycoconjugate
Free polysaccharide is a residual of conjugation reaction.
For serotypes Ia and Ib, this component is not removed by any process step and it is
important to verify its impact on vaccine immunogenicity and to understand how
reliably it can be controlled (through analytical method and process).
Free polysaccharide is not a stability indicator as demonstrated by stability studies
16
Nonclinical studies (as defined in WHO TR 927, 2005- Annex 1) on CQAs
Added value
Increase product knowledge and support understanding of nonclinical test
sensitivity to molecular variation
Provide a basis for the rationale on the choice of attributes in
specifications (criticality confirmation- reduction of uncertainty score)
Notes
If correlation between nonclinical and clinical response is missing, these
studies are not used for setting specification ranges
In these cases, specification ranges can be supported by clinical studies
with batches reflecting process/ product variability
CQA confirmation CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
17
The purpose of the Analytical Target Profile (ATP) is to ensure that the test
methods are selected and developed based on quality attributes requirements,
including in- process and characterization; it can also reflect performance attributes
needs.
Quality
Attribute
DS Process steps for Free PS Assay Requirements
DS (glyco-
conjugate
purified bulk)
Conjugation Current
Assay Name
Assay
selectivity/
specificity
Sensitivity
(LOQ/LOD) Range Accuracy Precision Cycle time Throughput
Free
PS X
Capillary
Electro-
phoreis
Minimum/
Optimum:
no peak is
detected at
retention
time of PS
Minimum/
Optimum :
(typically
10µg/ml)
depending
on serotype
and sample
conc
Minimum/
Optimum:
(typically 10
- 200 µg/ml)
depending
on serotype
and conc
Minimum
spike
recovery
80-120%
Optimum
spike
recovery
90-110%
Minimum/
Optimum
statistical
criteria under
eval
Minimum
6 hrs
Optimum
1 hr
Minimum:
12
Optimum:
24
Free PS x RP + SE-
HPLC
Minimum/
Optimum:
no peak is
detected at
retention
time of PS
Minimum/
Optimum :
(typically
10µg/ml)
depending
on serotype
and sample
conc
Minimum/
Optimum:
(typically 10
- 1000
µg/ml)
depending
on serotype
and conc
Minimum
spike
recovery
80-120%
Optimum
spike
recovery
90-110%
Minimum/
Optimum
statistical
criteria under
eval
Minimum
2 h
Optimum
1 h
Minimum:
N/A
Optimum:
N/A
ATP- free polysaccharide CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
QbD: Overview of the assessment process Periodically refined based on gained knowledge
18
CQA
assessment
& ranges
What ?
Why ?
CPP/ KPP
assessment
Ensure proper
focus on the
process
parameters
impacting CQAs
(CPPs) as well as
performance
(KPPs)
Identify PPs
influencing
CQAs and PAs;
define CPP
ranges within
which product
quality is not
impacted
Determine
how well
we need to
control the
CPPs and
KPPs
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
In S
um
mary
Control
strategy
Define
risks
related to
meeting of
pre-
defined
quality
criteria
Based on
range
assessment,
Identify the
CPPs/ KPPs
which have to
be carefully
controlled to
ensure good
performance
and quality
Plan a set of
controls, that
ensures
process
performance
and product
quality
Overall
verification of
process/
product
monitoring, to
minimize risks
of failure
For each CQA,
evaluate risks
associated to
analytical
monitoring and
process; prioritize
based on criticality
score
Decide on
what
attributes
are critical
for safety
and efficacy
and define
their ranges
Analytical
Target
Profile
Challenge
analytical
methods to meet
the pre-defined
target for testing
of CQAs
Prioritize actions
to increase
knowledge on
criticality and on
ranges; build
strong rationales
for specifications
Define
minimum
and
optimum
requiremen
ts of
analytical
methods
Target
Product
Profile
(TPP)
Describe
product
requirements
for intended
use
Ensure that
product and
process
development
start from
patient’s
needs
Process understanding
Quality by Design & Vaccines: a Case Study| Technical Development, Novartis V&D 19
Conceptual steps for Process Characterization
SELECT POTENTIAL CRITICAL AND KEY PROCESS PARAMETERS
by risk assessment
PERFORM SCREENING STUDIES
by appropriate DoE to determine which among potential Critical and Key Process Parameters are
confirmed.
PERFORM A MAPPING STUDIES TO DEFINE DESIGN SPACE
Including all CPPs, by appropriate DoE studies to confirm tested ranges as PAR.
SET AND QUALIFY DOWN SCALE MODELS
For each relevant process step identified
Set-point : defined input value to be set during process.
Normal Operating range: fluctuation around set-point
due to equipment/operator control.
Acceptable range: range where the process still performs
adequately.
Process Characterization Objective:
Process Characterization Activities:
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
20
Free saccharide for serotype III is consistently removed during the routine process (well-controlled attribute) in the HA step
Process step impacting free saccharide attribute Conjugation reaction of serotype Ia and Ib
CRM197
Oxidation
Ultrafiltration
Conjugation
HA chromatography
Sterile filtration
Quenching/UF
Purified Polysaccharide
4 potential CPPs identified (see next slide)
21
Note: This screening DOE will be followed by robustness DOE to define NOR
and PAR of parameters impacting free PS % if the attribute will be confirmed
critical
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
• PS/ CRM ratio and PS oxidation are statistically significant for free
saccharide %
• PS/CRM ratio causes an effect on free saccharide % relevant in terms
of process
• It is important to evaluate statistically significant effects and their
practical process/ product relevance
• Further refinement of DOE interpretation is on-going
Preliminary Outputs Statistical evaluation
22
Control Strategy
Control strategy combines evaluations on testing and process performance to
ensure that the pre-defined product quality (CQAs) is consistently met.
• Nonclinical and clinical experiences combine to identify analytical and process
control parameters, and their appropriate specifications and operating ranges:
Quality attributes & quality boundaries of the vaccine should be ultimately defined based on
product characteristics (taking into account nonclinical studies, clinical trials outcome &
stability).
The ranges of variation of CPPs within which the quality of the vaccine is not impacted are
confirmed after Ph III, based on the results of process characterization and final clinical
outcome.
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
23
Towards control strategy for Free Saccharide
Current assessment for future control strategy:
1.QA criticality: non clinical evaluation of the attribute is ongoing; current
results would support reduction of criticality score with possible impact on
inclusion on specifications
2.Process impact: process parameters impacting free saccharide during
conjugation step are being identified by screening DOE; NOR and PAR
definition through robustness DOE will be performed if attribute criticality will be
confirmed
3.Testing strategy: tests on the bulk developed; method performances to be
confirmed through validation. Considered low risk as no sample pre-treatment
is needed before analysis by CE. In process control will be applied only for
process characterization
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
24
Prefilled syringes
There are many drivers and good reason to have vaccines in the prefilled
syringe presentation:
1. Vaccines are complex biological entities, therefore must be administered by
injection due to lack of significant advance in alternative delivery systems.
2. From a technical point of view, the precision of the dose and the risk of
manipulation and potential contamination.
Although the successful development of a vaccine product in prefilled syringe
requires careful evaluation of the interaction between the vaccine and the
primary packaging materials.
25
Prefilled syringes
A hollistic and integrated approach has been taken to focus on the linkage of
prefilled syringe in the following areas:
1. Target disease / health policy / user requirement
2. Formulation design
3. Prefilled syringe components
4. Manufacturing process (site and equipment)
5. Business / supply chain / procurement
6. Regulatory and Quality compliance requirements
These elements have to be considered throughout Product Design, Development,
Operation and Control.
26
Prefilled syringes
The glass syringe contains silicon oil for functionality purposes.
When protein interacts with silicon oil , potentially 2 phenomena that could lead
to particulates formation.
A hydrophobic interaction could enhance protein aggregation as a prerequisite
for visible particulates.
Also dispersed silicon oil droplets, although at low level, may be charged
dependent on the formulation conditoin.
This could lead to the induction of protein aggregation.
27
Concluding remarks
Benefits Points to consider
• Extent of QbD application is case-dependent.
• If a QbD element cannot be implemented, the associated risk can be clearly established
Structured approach consistently applicable to
any vaccine category
• Identification of potentially critical parameters ensures focus on the relevant process steps and proper monitoring/ characterization
Understanding of the relationship between process and quality
• Importance of structural characterization
• CQA risk assessments and quality- oriented process support prioritization of attributes to be experimentally tested for immunogenicity
Support rigorous rationales for specifications
• Consistency can be suitably defined and achieved when deep knowedge of product and process is built, with approaches like QbD.
Ensure that the pre-defined product quality is consistently met during
product life cycle
QTPP (Quality Target Product Profile)
• Route of administration (ex. Oral) • Pharmacokinetics (eg. Dissolution)
CQAs (Critical Quality Attributes)
• DS (eg. Particle size, moisture), DP (eg. Content uniformity, Dissolution)
Risk Management
• Eg. Risk Matrices, Fishbones, Failure Mode Effect Analysis, Peer Reviews
Design Space • Eg. Design of Experiments, Pareto analysis
Control Strategy • Eg. In-Process Controls, Process Analytical Technology
Continual Improvement • Flexibility within design space
Patient
Product
Process
Summary
29
Acknowledgement
• The work presented here was performed by Novartis
Vaccines & Diagnostics.
30
Questions
Back-ups
31
Health Authorities’ QbD Proposal:
“ICH Quality Guidelines …A more systematic approach to development… incorporation of prior knowledge... design of experiments… use of quality risk management…throughout the lifecycle of the product.
To Become:
Better (increased CpKs, continual improvement )
Faster (first time right, facilitates LEAN)
Cheaper (focused experiments)
With Regulatory Benefits:
Real Time Release &
Flexibility within a filed and approved Design Space
Purpose
EMEA QbD process
Design Space & Control Strategy
Summary
34
Opsono phagocytosis is used to determine the functional activity of vaccine-induced specific antibodies. Too variable to be used for release purpose.
Free polysaccharide criticality confirmation OPK (serotype Ia)
10 3902 9052 2733 8836 4847 6421
1352 5429 6429 5569 7444 2442
ELISA α-PS Ia (EU/mL) 2° protocol
Error bar represent mean*/- SD
Preliminary evaluation showed no impact on vaccine immunogenicity up to 50% of FS
(observed differences can be attributed to animal variability)
Inhibitory effect by increased dosages of total PS was not confirmed
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
Research, Serology
35
Non clinical testing current outcome- Free polysaccharide
IVRP doesn't discriminate completely between free and
conjugated PS and RP values increase proportionally with the
increasing of total saccharide in the DP.
Some confirmatory OPK experiments are needed before
deciding to downgrade free polysaccharide criticality.
As there is no proven correlation between ranges tested for
nonclinical testing, clinical evaluation of free polysaccharide
ranges could be planned, if the attribute’s criticality will not be
downgraded.
It is important to have suited analytical methods for the
investigated attributes
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
Conjugation Reaction Step Screening DoE Planned (serotype Ia/Ib)
Kinetic reactions followed in order to monitor the potential halt at desidered CQAs
Reactions not halt to evaluate all kinetic data
36
4 factors, 2 levels
Fractional design (Resolution IV)
24-1= 8 runs (+4 central points)
Maximum 3 runs/day
Blocks are different experimental sessions (different days)
All responses are evaluated during the same analytical session
SCR RUN
Block PS/CRM ratio PS concentration
Oxidation
degree%
Temperature °C
1 1 0.75 6.35 20 22
2 1 1.00 7.50 14 16
3 1 0.50 5.20 26 28
4 2 1.00 5.20 14 28
5 2 0.50 7.50 26 16
6 2 0.75 6.35 20 22
7 3 0.50 7.50 14 28
8 3 0.75 6.35 20 22
9 3 1.00 5.20 26 16
10 4 1.00 7.50 26 28
11 4 0.75 6.35 20 22
12 4 0.50 5.20 14 16
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
37
* Target not reached
time GD (w/w) %Poly > 300 Å % FS
SCR 1 15 1.50 25.1 11.6
SCR 2 37.5 1.79 23.9 17.7
SCR 3 8 1.29 26.7 6.4
SCR 4 36.5 1.74 20.5 18.6
SCR 5 11 1.32 25.9 8.0
SCR 6 16 1.47 25.3 11.9
SCR 7 11 n.a. 26.3 8.1
SCR 8 16 1.45 25.0 12.8
SCR 9 34.5 1.58 21.2 17.4
SCR 10 6 1.66 25.2 15.4
SCR 11 17 1.53 25.0 11.7
SCR 12 48 1.45 19.1 12.0
value min at target - 1.29
6.4
value max at target - 1.66 15.4
GBSIb PC SCR Conjugation Preliminary Outputs
Results of serotype Ib conjugation step screening
Note: Several of the explored conditions induced changes in free saccharide
percentage – statistical evaluation is on-going as well as data interpretation (see
next slides)
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
Conjugation step: an extract of cause- effect matrix
CQAs Free saccharide Glycosylation degree Conjugate dimension Total
PP
score Process Parameters
Rank Rationale Rank Rationale Rank Rationale
PS/CRM ratio in
reaction 7
Previos studies showed a potential effect of
stoichiometry on unconjugated PS 7
Previos studies showed a potential effect
of stoichiometry on glycosylation degree 7
Previos studies showed a potential effect
of stoichiometry on product dimension 21
PS concentration in
reaction 7
Previos studies showed a potential effect of
PS concentration on unconjugated PS
7
Previos studies showed a potential effect
of PS concentration on glycosylation
degree
7 Previos studies showed a potential effect
of PS concentration on product dimension 21
PS oxidation degree 7 Previous experiments performed with an
oxidation degree far from the target,
showed significant impact on unreacted PS 7
Previous experiments performed with an
oxidation degree far from the target,
showed significant impact on
glycosylation degree
7
Previous experiments performed with an
oxidation degree far from the target,
showed significant impact on conjugation
reaction and product dimension
21
Stirring rate of
product before
NaCNBH3 addition
1
No impact expected on product
characteristics (short duration, reaction has
not yet started)
1
No impact expected on product
characteristics (short duration, reaction
has not yet started)
1
No impact expected on product
characteristics short duration, reaction has
not yet started)
3
Temperature of
conjugation reaction 7
Temperature significantly impacts the
reaction kinetics ; effect of temperature on
unconjugated PS unknown 7
Temperature significantly impacts the
reaction kinetics ; effect of temperature
on glycosylation degree unknown
7
Temperature significantly impacts the
reaction kinetics ; effect of temperature on
product dimension unknown
21
Conjugation reaction
stop point 4
Variability in controlling the reaction stop
point should have a minimum impact on
the product
4 Variability in controlling the reaction stop
point should have a minimum impact on
the product 4
Variability in controlling the reaction stop
point should have a minimum impact on the
product
12
Stirring time of
product after WFI
addition
1 No impact on product expected. 1 No impact on product expected. 1 No impact on product expected. 3
Rank Input Process Parameters to Critical Quality Attributes
10 Relationship known
7 Relationship suspected or unknown
4 Slight relationship
1 No relationship
PP critical or potentially critical if at least one 7
or one 10 are present in the scoring
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
38
39
The purpose of the Analytical Target Profile (ATP) is to ensure that the test
methods are selected and developed based on quality attributes requirements,
including in- process and characterization; it can also reflect performance
attributes needs.
Quality
Attribute
DS Process steps for Free PS Assay Requirements
DS (glyco-
conjugate
purified bulk)
Conjugation Current
Assay Name
Assay
selectivity/
specificity
Sensitivity
(LOQ/LOD) Range Accuracy Precision Cycle time Throughput
Free
PS X
Capillary
Electro-
phoreis
Minimum/
Optimum:
no peak is
detected at
retention
time of PS
Minimum/
Optimum :
(typically
10µg/ml)
depending
on serotype
and sample
conc
Minimum/
Optimum:
(typically 10
- 200 µg/ml)
depending
on serotype
and conc
Minimum
spike
recovery
80-120%
Optimum
spike
recovery
90-110%
Minimum/
Optimum
statistical
criteria under
eval
Minimum
6 hrs
Optimum
1 hr
Minimum:
12
Optimum:
24
Free PS x RP + SE-
HPLC
Minimum/
Optimum:
no peak is
detected at
retention
time of PS
Minimum/
Optimum :
(typically
10µg/ml)
depending
on serotype
and sample
conc
Minimum/
Optimum:
(typically 10
- 1000
µg/ml)
depending
on serotype
and conc
Minimum
spike
recovery
80-120%
Optimum
spike
recovery
90-110%
Minimum/
Optimum
statistical
criteria under
eval
Minimum
2 h
Optimum
1 h
Minimum:
N/A
Optimum:
N/A
ATP- free polysaccharide CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
Process understanding PC Screening studies Conjugation step serotype Ia/Ib
40
Potential critical or key process parameters
TARGET
(stop reaction)
based on
conjugate
dimension
(poly% >300 A)
Outcome attributes Explored range
NOR Lower level Set point Upper level
PS/CRM (ratio w/w) 0.63- 0.90 0.5 0.75 1.0
Glycosylation Degree
PS concentration (mg/ml) 5.78- 6.92 5.2 6.35 7.5 Free saccharide%
PS oxidation degree (%) 13- 25 14 20 26
Reaction time Temperature (°C) 18- 26 16 22 28
CQA
assessment
& ranges
CPP/ KPP
assessment
Well-
controlled
CPPs/
KPPs
Combine
product and
process
understanding
Control
strategy
Analytical
Target
Profile
Target
Product
Profile
(TPP)
«Ad hoc» test was applied to monitor free saccharide and other
attributes during conjugation reaction
41
•GBS vaccines with different
concentrations of total PS have been
generated by spiking the glycoconjugate
(100% sample) with increasing
concentration of free saccharide (0-
75%) or conjugated saccharide (0-
75%).
•RP values increase proportionally with
the increasing of total saccharide in the
DP. Slightly better recognition of the
conjugated PS.
•IVRP doesn't discriminate completely
between free and conjugated PS.
Free polysaccharide criticality confirmation IVRP PS serotype Ia
% Total
saccharide % Free PS RP session 1 RP session 2 % Free PS RP session 1 RP session 2
100 0% 1,024 1,086 0% 1,024 1,086
105 5% 1,115 1,115 0% 1,136 1,099
111 10% 1,185 1,139 0% 1,215 1,204
118 15% 1,144 1,207 0% 1,273 1,317
133 25% 1,439 1,417 0% 1,436 nd
200 50% 1,931 1,953 0% 2,352 nd
400 75% 3,242 3,659 0% 4,456 nd
Free saccharide Conjugated saccharide
OPAGBS Ia
PBS/A
lum
CRM
-Ia,Ib
,III (
Tox lo
t)
CRM
-Ia,Ib
,III 0
% P
S free
CRM
-Ia,Ib
,III 2
5% P
S free
CRM
-Ia,Ib
,III 5
0% P
S free
CRM
-Ia,Ib
,III 7
5% P
S free
0
1000
2000
3000
4000
P value
Exact or approximate P value?
0.0286
Exact
0.0286
Exact0.0286
Exact P value summary *
mean = 1769
mean = 2981
mean = 1118
mean = 1089
mean = 539
OP
A t
iter
ELISA α-PS Ia (EU/mL)
10 4091 4426 2064 2583 1225
GBS Ia
Partial decrease trend observed in ELISA and OPK titers with increasing free-PS
Total PS doses changed with increasing free-PS
Investigation on the observed inhibitory effect due to two potential effects:
1. Presence of free PS
2. Increased amount of total PS in vaccine formulations
Free polysaccharide criticality confirmation
Slides provided by Research Serology 42
Serotype Ia - OPK and ELISA data
43
10 3902 9052 2733 8836 4847 6421
1352 5429 6429 5569 7444 2442
ELISA α-PS Ia (EU/mL)
GBS Ia
OPKA (515-Ia)
Preliminary evaluation showed no impact on vaccine immunogenicity and Ab functional activity due to
free PS (up to 50%):
observed differences in OPKA and ELISA titers among 0, 10 and 50% free PS
can be attributed to animal variability (see differences between paired cages)
A possible inhibitory effect by increased dosages of total PS was not confirmed
Additional variability evaluation by introducing a negative control in the protocol planned
Error bar represent mean*/- SD
Slides provided by Research Serology
Free-saccharide criticality confirmation Serotype Ia - OPK and ELISA data (protocol 2)
GBSIb Conjugation reaction An example of screening of serotype Ib
44
Reaction time (h)
GD %Poly > 300 Å FS. (% w/w)
1,6 30 15
1,3
0 6,3 100
1,5 2,92 7,6 60,0
12 nd 21,9 14,3
13 1,64 22,9 13,0
14 1,57 24 12,3
15 1,50 25,1 11,6
16 1,47 26,3 10,8
17 1,42 27,5 10,4
18 1,40 28,4 10,0
19 1,39 29,1 9,6
20 1,37 30,2 9,2
Screening 1
0
20
40
60
80
100
0 5 10 15 20 25
%
Time (h)
Free Saccharide
0
1
2
3
4
0 5 10 15 20 25
µgPS/µgCRM
Time (h)
Glycosylation Degree
0
10
20
30
40
50
0 5 10 15 20 25
%
Time (h)
% Poly > 300 Å
Reaction is halted at 25% Poly> 300A
45
Preliminary results on serotype Ib
Method Analytical Tools Detector Output
1 2D Chromatography
(C4+ TSK5000) UV (210nm)
Free Saccharide
amount/percentage/size
2 SEC (TSK6000)
UV (280nm) CRM conversion amount/percentage
Refractive
Index
Conjugate Size (for kinetic profile
expressed by % of molecules with Rn >
300 Ǻ)
Glycosylation
degree
Conjugation reaction can be monitored by combining two analitycal methods able to follow quality
attributes trends.
Kinetic profile of Conjugation reaction at standard conditions for serotype Ib:
In process control to monitor and to control conjugation reaction
FS % and GD diminished during reaction, conversely
average MW increased.
Reaction conducted at standard conditions reaches a
plateau of molecular attributes (i.e Free saccharide %
after 16 h) (back-up for kinetic profiles)
-
+ +
+ -
-
-