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)

-

+ +

+ -

-

-