The QbD Approach to Development of the

New LFB Immune Globulin Intravenous

Product

P.Paolantonacci, B.Claudel, L.Siret, M.OllivierLFB Biotechnologie, France

Product Overview

█ Code Name : I10

█ Ready to use liquid Immune Globulin(100 mg/ml) for intravenous administration (IVIg)

█ Composition: Glycine (18.8 mg) ; Polysorbate 80 (0.05 mg) / WFI (1 ml qsf)

█ Low pH: 4.8 (prevention of aggregation)

█ High Purity (98% IgG)

█ controlled anti-A and anti-B hemagglutinin content

█ low IgA content

█ established elimination of procoagulant factors

█ Designed to comply with Eur.Ph. (0918) and CFR requirements

█ Pharmaceutical development according to QbD approach

2

Overall QbD Strategy

3

█ I10 is a conventional Blood Product with an almost continuous process fromcryosupernatant to DP with no formal DS stage before F&F operations, # biotechproducts

█ the large majority of QC testing is carried out at the DP stage, almost no QC at DS stage.

█ I10 should comply with compendial requirements for IVIg products, inducing a limitedfreedom for Drug Product Control Strategy

█ I10 QbD approach was a mix between traditional and enhanced approaches.

QbD Stage DS DP

QTPP Patient Oriented & Regulations

Product Risk Analysis Scientific Understanding and Evaluation

Process Risk Analysis Enhanced Enhanced

Process Risk Mitigation Enhanced Traditional

Control Strategy Enhanced Traditional

4

QbD Process for I10

Stage 1 Product DesignPatient oriented and Regulations

QTPP

Stage 2 Product Risk AnalysisPreliminary Hazard Analysis (Severity/Probability)

CQAs

Stage 3 Process Risk AnalysisFMECA

Critical StepsPotential CPPs

Stage 4 Process Risk Mitigation- DoE robustness- Process Verification

CPPsPARs

Stage 5 Control StrategyCombination of process/product controls

Specifications

Product/Process Design

5

Reduction of potential adventitious and non-adventitious

agents

Removal of activated coagulation factors to reduce the risk of

thrombogenic adverse events,

Removal of Anti-A and Anti-B haemagglutinins to avoid adverse events on patients of AB, A & B

blood groups,

Reduction of aggregates to avoid adverse events through complement activation,

Removal of IgA to avoid immune responses in patients deficient in

IgA,

I10 Purification Process

Cryo Separation

Ethanol Fractionation

Caprylic Fractionation

Depth Filtration

UF1

SD Treatment

AEX XtO

Affinity XtO

Filtration

Nanofiltration

UF2

Formulation

Clearance of procoagulant factors

Depletion of anti-A and anti-B haemagglutinins

Reduction of polymers and aggregates

Clearance of IgA and IgM

Inactivation of enveloped viruses

Retention of small viruses

Major QTPP Objectives Step Objective

I10 vs Mab Critical Quality Attributes

6

I10 MAb

Product Variants

Aggregation, ACA activity Aggregation

Fragmentation,Polyvalent Ig of plasma origin exhibits native humanvariants

Fragmentation, glycation, C-Terminal Lysine,Glycosylation, Deamidation, Oxydation, Disulfide bonds, Thioether Link

Purity

Plasma related protein with physiological activity : IgA, Thrombogenic factors, Prekallikrein Activator, Anti-A & Anti-B haemagglutinins, ...

Cell-Culture related: Selective Agent, cell culture medium components, DNA, HCP.

DSP related: Leachables, Purification Buffer Components (including SD reagents)

DSP related: Protein A, leachables, Purification BufferComponents

Microbiological Purity Microbiological Purity

Viral Purity, Prions Viral Purity

Drug Product Attributes

Foreign Particles, Clarity, Color Foreign Particles, Clarity, Color

pH, Osmolality pH, Osmolality

Quantity of Active Substance per vial Product Concentration, Volume

Multiple Potency Assays (several modes of action) Potency

Initial Process Risk Analysis

7

Impact of Manufacturing steps on CQAs

12 CriticalSteps

Main contributive step

DP StorageCappingAseptic filling � �

Finale Sterilising Filtration � � � � � �

Drug Substance Storage �

Formulation � � � � � � �

Ultrafiltration-Diafiltration 2 � � � � � � �

Nanofiltration 20 nm � � � � � � �

Filtration Cuno 90 � � � � �

Affinity chromatography � � � � �

Anion-exchange chromatography � � � � � � � � � �

SD treatment � � � �

Ultrafiltration-Diafiltration 1 � � � � � �

Activated carbon depth filtration � � � � � �

Caprylic acid Depth filtration � � � � � � � � � �

Caprylic acid precipitation � � � � � � � �

Precipitate solubilisation � � � �

Freezing of the precipitate � �

Ethanolic depth filtration � � � � � � � � �

Ethanol precipitation � � � � � � �

Depth filtration � �

Centrifugation �

Cryoprecipitation � �

Pre-thawing to thawing �

Plasma � � � � �

Apy

roge

nici

ty

Thr

ombo

géni

c A

ctiv

ity

Ant

i-com

plem

enta

ry A

ctiv

ity

IgA

Pol

ymer

s / A

ggre

gate

s

Qua

ntity

of a

ctiv

e su

bsta

nce

per

vial

Pur

ity

Leac

habl

es (

Pro

cess

rel

ated

)

Leac

habl

es (

Con

tain

er-c

losu

re

syst

em)

For

eign

mat

ters

/ V

isib

le p

artic

ule

Ste

rility

Gly

cine

Osm

olal

ity

Viru

ses

Prio

n

Bio

logi

cal A

ctiv

ity

Pre

kalli

krei

n A

ctiv

ator

Ant

i-A &

ant

i-B h

aem

aggl

utin

ins

TnB

P &

Oct

oxin

ol 1

0

Process Risk Mitigation Plan

8

Step ID Critical Steps Contributive studies

IIIPrecipitate solubilisation and Caprylic acid precipitation

Robustness studyCaprylic acid depth filtration

IV Activated carbon depth filtration

VI SD treatment Viral validation study

VII Anion-exchange chromatographyRobustness study

Ageing study

VIII Affinity chromatographyRobustness study

Ageing study

IX Filtration Robustness study

X 20nm Nanofiltration Viral validation study

XI Ultrafiltration 2 Robustness study

XII Formulation Validation batches

1 Final sterile filtration 0.22µm Filter validation study

2 Aseptic filling Filling validation study

NA DP storage Stability study

5 DoE Robustness Studies conducted at qualified small scale

Robustness Studies: DoE Methodology

Identification of the CPP and non-CPP (within the tested domain) For each parameter � Identification of the PAR values

Statistical analysis of the data (Design-Expert v.8.01 – Statease)

Robustness studies performed at qualified small scale

Experimental plan according to DoE

For each potential CPP:

Definition of the experimental domain

For each step:

Selection of the operating parameters to be tested

Robustness studies ���� for the critical steps

9

Robustness DoE Study: Anion Exchange Chromatography

Process parameter CQA ImpactedTesting domain

Residence time (min)

IgATnBP

Octoxinol 100 - 100

Column load (g/L)Binding conditions:

- pH- Conductivity (µS/cm)

Eluting conditions:- pH- Conductivity (µS/cm)

Gel bed height (cm)

10

Process parameter Critical Target valuesTested domain

PAR values

Residence time (min)

No 20 0 - 100 0 – 100

Column load (g/L) Yes 50 0 - 100 40 – 60Binding conditions:pH Conductivity (µS/cm)

NoNo

40 – 80900 – 1100

0 - 1000 - 100

0 – 1000 - 100

Eluting conditions:pH Conductivity (µS/cm)

YesYes

38 - 6233 – 66

0 - 1000 - 100

38 - 620 – 66

Gel bed height (cm) No 0 - 100 0 - 100 0 - 100

Design

33 experiments

Results

11

Process robustness studies - Results

█ Anion exchange chromatography Robustness study - Co nclusions

CQA: IgA Level

Identified CPPs: Column Loading, Eluting buffer (pH and Conductivity)

Statistical analysis: Quadratic model with interaction

Normal Operating range (NOR) and Proven Acceptable Range (PAR)

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

70.00

75.00

5.80

6.00

6.20

6.40

6.60

0.00

0.10

0.20

0.30

0.40

A: Charge Prot

B: pH élution 25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

70.00

75.00

5.80

6.00

6.20

6.40

6.60

0.00

0.10

0.20

0.30

0.40

Tau

x Ig

A

A: Charge Prot

B: pH élution

Taux

d’Ig

A

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

70.00

75.00

5.80

6.00

6.20

6.40

6.60

0.00

0.10

0.20

0.30

0.40

A: Charge Prot

B: pH élution Charge de la colonnepH d’élution

NOR PAR

Increasing pH Increasing loading

Increasing Conductivity

IgA

Leve

l

12

Process robustness studies - Results

█ Anion exchange chromatography Robustness study - Conclu sionsAttribute: IgM Level

Proven Acceptable Range (PAR)

5.80

6.00

6.20

6.40

6.60

25.00

37.50

50.00

62.50

75.00

0.004

0.013

0.023

0.032

0.041

0.051

0.060

Tau

x Ig

M

C: pH élution B: Charge Prot pH

of Elution bufferColumn load

IgM

leve

l

Elution bufferconductivity

Min

Max

� no model for the whole domain (too low variations)(high column load, low eluting pH and Low/High eluting Conductivity)

Low variations

Very high variations

Robustness Studies: Conclusion

13

Step Critical process parameters PAR

Caprylic acid fractionation and depth filtration

Acetate concentration (mMol)pH before precipitationRatio of caprylic acid to protein (g/g)Duration of the addition of the caprylic acid (min) Duration of the maturation with caprylic acid (min)Pressure applied to the filter press (bar)Filter press rinse volume (kg/L)

25 - 7533 - 6625 - 50

33 - 10020 - 460 - 10065 - 100

Activated carbon depth filtration

Filter relative surface area (cm2/L)Residence time (Flow rate) on the filter (L/h/m2)

10 -1000 - 83

Anion-exchange chromatography

Protein load (g/L)pH of the elution bufferConductivity of the elution buffer (µS/cm)

40 - 6038 - 620 - 66

Affinity chromatography

Residence time (min)Protein load (kg/L)Product pH

30 -1000 - 10038 - 100

FiltrationVolume load (L/m2)Product pH

0 - 9329 – 88

Ultrafiltration 2 None NA

Identification of CPPs and establishment of PARs

For non CPPs, PAR = tested domain

Process Verification

14

Verification of PARs at Industrial Scale, Different Strategies possible according to Industrial Constraints

Step Critical process parameters PAR Strategy

Caprylic acid fractionation and depth filtration

Acetate concentration (mMol)pH before precipitationRatio of caprylic acid to protein (g/g)Duration of the addition of the caprylic acid (min) Duration of the maturation with caprylic acid (min)Pressure applied to the filter press (bar)Filter press rinse volume (kg/L)

25 - 7533 - 6625 - 50

33 - 10020 - 460 - 10065 - 100

Target: 50Target: 50Target: 37

Range: 33 to 100Worst case: 20

Extend PAR: 110Target: 70

Activated carbon depth filtration

Filter relative surface area (cm2/L)Residence time (Flow rate) on the filter (L/h/m2)

10 -1000 - 83

Target: 70Target: 32

Anion-exchange chromatography

Protein load (g/L)pH of the elution bufferConductivity of the elution buffer (µS/cm)

40 - 6038 - 620 - 66

Extend PAR: 70Target: 50Target: 38

Affinity chromatography

Residence time (min)Protein load (kg/L)Product pH

30 -1000 - 10038 - 100

Target: 50Target: 36Target: 56

FiltrationVolume load (L/m2)Product pH

0 - 9329 – 88

Target: 15Target: 63

Process Risk Mitigation: Conclusion

15

Step ID Criticity Step Initial risk Mitigated risk

IIN Ethanol precipitation Moderate Moderate

N Ethanolic depth filtration Moderate Moderate

IIIY

Precipitate solubilisation and Caprylic acid precipitation

High Moderate

Y Caprylic acid depth filtration Moderate Acceptable

IV Y Activated carbon depth filtration Moderate Acceptable

V N Ultrafiltration 1 Moderate Moderate

VI Y SD treatment High Acceptable

VII Y Anion-exchange chromatography Moderate Acceptable

VIII Y Affinity chromatography Moderate Acceptable

IX Y Filtration Moderate Acceptable

X Y 20nm Nanofiltration High Acceptable

XI Y Ultrafiltration 2 Moderate Acceptable

XII Y Formulation High Acceptable

1 Y Final sterile filtration Moderate Acceptable

2 Y Aseptic filling High Acceptable

NA Y DP storage Moderate Acceptable

Control Strategy: positive outcome

16

Manufacturing Process ControlsProcess Step Step ID Objective Manufacturing Process Design

Manufacturing Process Validation

In-Process Controls

SD Treatment VIIntroduction of SD reagents in the manufacturing process

Quantity of SD introduced verified routinely

Anion Exchange Chromatography

VII Removal of SD reagents

Chromatographic step intended to remove SD reagents (TnBP and Octoxinol 10 are not retained on

anionic support)

Robustness Study identified one CPP and

established PARsControl of CPP: - protein load

Removal of SD reagent demonstrated as part of

Process validation

Final Ultrafiltration XI Removal of SD reagentsUF step designed to remove

chemicals

Robustness Study did not identify any CPP

Removal of SD reagent demonstrated as part of

Process validation

Product controlStage ID Routine testing Characterisation Stability Monitoring Method Status

Before and after Chromatography

VII

NoTnBP and Octoxinol 10

concentrations analysed on validation batches

No ValidatedBefore and after UF 11

Drug product

For the majority of CQAs, the Control Strategy demonstrated full control by the process.

Validation of two clearance steps justifies removal from routine testing

Control Strategy: limitations

17

Manufacturing Process ControlsProcess Step Step ID Objective Manufacturing Process Design

Manufacturing ProcessValidation

In-Process Controls

Affinity Chromatography VIII

Removal of anti-A and anti-B haemagglutinins by specific affinity capture

Specific step introduced in the manufacturing process to remove Anti-A & Anti-B

hemagglutinins

Robustness study identify three CPP and established

PARs Control of CPP: - residence time, - protein load, - pH

Efficency of process step to remove Anti-A & Anti-B

hemagglutininsdemonstrated as part of

Process Validation

Guarrantee affinity gel performance

Specific affinity gels (HypercelISOA and ISOB) developed to

remove Anti-A & Anti-B hemagglutinins

Aging studies showed that the gels can be used up to 100 cycles with the same

efficiency to remove Anti-A & Anti-B hemagglutinins

Internal monograph to control ISOA and ISOB

gels at receipt

Product controlStage ID Routine testing Characterisation Stability Monitoring Method Status

Affinity Chromatography VIII NoFlow Cytometry used during

manufacturing development to quantify process step efficiency

NA Qualified

Drug product According to Ph. Eur. (2.6.20) Yes No Ph. Eur. method

Compendial requirements restrict the benefits of a QbD Control strategy by imposingsome QC testing which could be avoid by implementing a Design Space.

Example of haemagglutinins A&B

Conclusion (1)

The QbD approach, by integrating DoE robustness studies to a risk analysisprocess and although time and resources consumming, has generated verypositive outputs:

█ A strong rationale for process validation design,

█ A justification for product specifications,

█ An enhanced knowledge of the manufacturing process with proven links between CPPs and CQAs,

█ An improved process control strategy with proven links between CPPsand CQAs and with demonstration of PARs for all Process Parameters(CPPs + non-CPPs),

18

Conclusion (2)

The QbD approach limitations:

█ Due to complex purification process for Biologicals products leading to non-orthogonal steps, design space does not seem to be achievable for all CQAs

█ Regulatory requirements restrict the benefits of a QbD-based Control Strategy by imposing some QC testing on the DP,

The QbD development approach remains an investmentfor future commercial manufacturing for:

• Treatment of deviations• Change controls (comparability exercises rationale)• Rationale for continuous process verification

19