control strategy for glycosylation€¦ · • amgen team: joseph phillips (lead), bob kuhn •...

7/13/2010

1

Control Strategy for GlycosylationUsing a QbD Approach:

Monoclonal Antibody with EffectorFunction from the A-Mab Case Study

CMC Forum Washington, DCWorkshop I - CQAs

July , 2010

Presented by Victor Vinci, Eli Lilly

CMC BWG – A-Mab Case StudyWorking Group Members

• Amgen Team: Joseph Phillips (Lead), Bob Kuhn

• Abbott Team: Ed Lundell (Lead), Hans-Juergen Krause, Christine Rinn, Michael Siedler, and Carsten Weber

• Eli Lilly Team: Victor Vinci (Lead), Michael DeFelippis, John R Dobbins, Matthew Hilton, Bruce Meiklejohn, and Guillermo Miroquesada

• Genentech Team: Lynne Krummen (Lead), Sherry Martin-Moe, and Ron Taticek

• GSK Team: Ilse Blumentals (Lead), John Erickson, Alan Gardner, Dave Paolella, Prem Patel, Joseph Rinella, Mary Stawicki, Greg Stockdale

• MedImmune Team: Mark Schenerman (Lead), Sanjeev Ahuja, Laurie Kelliher , Cindy Oliver , Kripa Ram, Orit Scharf, and Gail Wasserman

• Pfizer Team: Leslie Bloom (Lead) and Amit Banerjee, Carol Kirchhoff, Wendy Lambert, Satish Singh

• Facilitator Team: John Berridge, Ken Seamon, and Sam Venugopal

• Plus help from many others

2Vinci/Defelippis - CMC BWG

QbD Case StudyLilly - Company Confidential 2010

3

QbD Development ParadigmCreation of a Control Strategy

Product Quality

Attributes

Criticality

Assessment

1.Quality attributes to be

considered and/or controlled

by manufacturing process

2. Acceptable ranges for

quality attributes to ensure

drug safety and efficacy

Attributes that do not need to

be considered or controlled

by manufacturing process

Safety and

Efficacy Data

Process Targets

for Quality

Attributes

Process

Development and

Characterization

Co

ntin

uo

us P

roce

ss V

erifica

tio

nProcedural Controls

Characterization &

Comparability Testing

Process Parameter

Controls

Specifications

Input Material Controls

In-Process Testing

Process Monitoring

Co

ntr

ol S

tra

teg

y E

lem

en

ts

High Criticality

Attributes

Low Criticality

Attributes

Product Understanding Process Understanding

Clinical

Studies

Animal

Studies

In-Vitro

Studies

Prior

Knowledge

Design

Space

Process Controls

Testing

Vinci/Defelippis - CMC BWG QbD Case

StudyLilly - Company Confidential 2010

7/13/2010

2

Creating a Biotech Case Study:“A-Mab”

• Based on a monoclonal antibody drug substance and drug product– “A-Mab”– Humanized IgG1 (w/ effector function)

– IV Administered Drug (liquid)– Expressed in CHO Cells– Treatment of NHL– Molecule designed to maximize

clinical outcomes and minimize impact on quality attributes (TPP)

• Publically and freely available as a teaching tool for industry and agencies at CASSS or ISPE

Why Monoclonal Antibody?

Represents a significant number of products in development

Good product and process exp. in dev. & manufacture

Reasonable level of complexity

Vinci/Defelippis - CMC BWG

QbD Case Study4Lilly - Company Confidential 2010

CQA Risk Ranking & Filtering ToolA Continuum of Criticality (Tool #1 Ex.)

• Assess relative safety and efficacy risks using two factors:– Impact and Uncertainty used to rank risks

• Impact = impact on safety or efficacy, i.e. consequences– Determined by available knowledge for attribute in question (prior, clinical, etc)

– More severe impact = higher score

• Impact on biological activity, PK/PD, immunogenicity, adverse effects

• Uncertainty = uncertainty that attribute has expected impact– Determined by relevance of knowledge for each attribute

– High uncertainty = high score (no information with variant or published lit. only)

– Low uncertainty = low score (data from material used in clinical trials)

• Severity = risk that attribute impacts safety or efficacy

Severity = Impact x Uncertainty


QbD Case Study5Lilly - Company Confidential 2010


StudyLilly - Company Confidential 2010 6

Attribute

Prior

Knowledge

In-vitro

Studies

Non-clinical

Studies

Clinical

Experience

Claimed

Acceptable

Range

Galactose

Content

Clinical

experience of 10-

40% G0 for Y-

Mab, another

antibody with

CDC activity as

part of MOA; no

negative impact

on clinical

outcome

0-100% has

statistical

correlation with

CDC activity with

A-Mab

No animal studies 10-30% 10-40%

aFucosylation 1-11%; Clinical

experience with

X-Mab and Y-

Mab; both X-Mab

and Y-Mab have

ADCC as part of

MOA

A-Mab with 2-

13% afucosylation

tested in ADCC

assay; linear

correlation; 70-

130%

Animal model

available;

modeled material

(15%) shows no

significant

difference from

5%

5-10%;

Phase II and

Phase III

2-13%

Platform and Product Specific Experience

7/13/2010

3

Criticality Ratings for Glycosylation

Attribute Criticality

Aggregation 60

aFucosylation 60

Galactosylation 48

Deamidation 4

Oxidation 12

HCP 36

DNA 6

Protein A 16

C-terminal lysine

variants (charge

variants)

4

Glycoslyation - High Criticality

• Example is for afucosylation

and galactosylation; other

glycan structures require

individual consideration

• Primarily impacted by

production BioRx

• No clearance or modification in

DS

• Not impacted by DP process or

stability

Vinci/Defelippis - CMC BWG QbD

Case Study7Lilly - Company Confidential 2010

Note: Assessment at beginning of development

Experimental DesignProgression of Studies for Production Bioreactor

Prior knowledge and risk assessments inform designed experiments:

• Risk analysis tools guide informed assessments

• Risk assessment links product attributes with parameters

• DOE’s allow understanding of the impact of process parameters and attributes

• Risk assessments are iterative and continue through the lifecycle of product

8Vinci/Defelippis - CMC BWG QbD

Case StudyLilly - Company Confidential 2010

9

Risk Assessment ApproachMultiple Assessments Throughout the

A-Mab Development Lifecycle for Entire Process

Prior Knowledge

Process Understanding

Product Understanding

Process

Development

Risk

Assessment

Process

Characterization

Risk

Assessment

Risk

Assessment

Process

Performance

Verification

Risk

Assessment

Life Cycle

Management

Final Control

Strategy

Process

Parameters

Quality

Attributes

Design Space

Draft Control

Strategy

Process 2

Process 1 2



You Are Here

7/13/2010

4

10

Example of Risk Assessment ToolApproach to Process Characterization

Aggredates

Fucosylation

Galactosylation

CEX AV

HCP

DNA

N-1 Bioreactor

FeedGlucose Feed

Production

BioreactorHarvest

Medium

Procedures

Temperature

pH

Seed

In Vitro Cell

Age

Seed Density

Viability

Operations

Time of Feeding

Volume of

Feed

Preparation

Concentration

pH

Age

DO

pH

Temperature

CO2

Agitation

Shear/

Mixing

Gas

Transfer

Airflow

Antifoam

Scale

Effects

Amount Delivered

Number of

Feeds

TimingPreparation

[Glucose]

Osmolality

Concentration

Procedures

Age

Duration

Working

Volume

[NaHCO3]

Pre-filtration

hold time

Storage

Temperature

[Antifoam]

Procedures

Age

Storage

Temperature

Pre-filtration hold

time

Filtration

Filtration

# of

Impellers

Vessel

Design

Baffles

Control

Parameters

Operations

Impeller

Design

Sparger Design

Nominal

Volumne

Step 1. Use a Fish-bone (Ishikawa) diagram to identify parameters and attributes that

might affect product quality and process performance


StudyLilly - Company Confidential 2010

11

A-Mab: Mid-Development Risk Assessment Approach

Quality Attributes Process Attributes Risk Mitigation

Process Parameter in Production Bioreactor

Agg

rega

te

aFuc

osyl

atio

n

Gal

acto

syla

tion

Dea

mid

atio

n

HC

P

DN

A

Pro

duct

Yie

ld

Via

bilit

y at

Har

vest

Tur

bidi

ty a

t

harv

est

Inoculum Viable Cell Concentr DOE

Inoculum Viability Linkage Studies

Inoculum In Vitro Cell Age EOPC Study

N-1 Bioreactor pH Linkage Studies

N-1 Bioreactor Temperature Linkage Studies

Osmolality DOE

Antifoam Concentration Not Required

Nutrient Concentration in medium

DOE

Medium storage temperature Medium Hold Studies

Medium hold time before filtration

Medium Hold Studies

Medium Filtration Medium Hold Studies

Medium Age Medium Hold Studies

Timing of Feed addition Not Required

Volume of Feed addition DOE

Component Concentration in Feed

DOE

Timing of glucose feed addition

DOE-Indirect

Amount of Glucose fed DOE-Indirect

Dissolved Oxygen DOE

Dissolved Carbon Dioxide DOE

Temperature DOE

pH DOE

Culture Duration (days) DOE

Remnant Glucose Concentration

DOE-Indirect

Potential impact to

significantly affect a

process attribute

such as yield or

viability

Potential impact to QA

with effective control of

parameter or less

robust control

Rank parameters and attributes from Step 1 based on severity of impact and control capability.

Identify interactions to include in DOE studies


QbD Case StudyLilly - Company Confidential 2010

Note: pH is red or

critical at this stage due

to linkage to

glycosylation

MCC BioreactorControl Strategy Elements by System - pH

Raw Materials (Reg/QMS) – vendor qualification; media (or buffer) make-up based on instructions, weight based; pH check post make-up

Equipment (QMS) – bioreactor design (probe type/placement), probe vendor qualification, receipt verification, linked to IQ/OQ and PV for bioreactor

Automation (QMS) – control loop qualified (CSV) and controlled via DCS, alert/action alarms aligned with process, data monitored continuously and archived

DOE and Models (QMS/Reg) – small-scale models use parameter ranges intended for large-scale; confirm during pivotal and commercial tech transfer

In Process/Operations (QMS) – pH probe calibration (pre-run), batch record instructions on how to do daily check and adjustment, data trended

Specification Limits/Tests (Reg/QMS) – Control Strategy in place, validated methods reflecting QbD analytical development

Process Verification/Continuous Monitoring (QMS/Reg) – MVA (PLS) or SPC monitoring of performance over manufacturing lifecycle


QbD Case StudyLilly - Company Confidential 2010 12

7/13/2010

5

13

DOE Studies to Define Design SpaceBringing Together Process and Product Attributes

Example of DOE Results from Screening Study (Process 2). N=20.

3

4

5

Tite

r (g

/L)

3.7

43131

±0.076052

4

6

8

aF

ucosyla

tion

6.4

39933

±0.226948

24

28

32

Gala

cto

syla

tion

(%)

29.2

8939

±0.674582

4e+5

6e+5

8e+5

1e+6

HC

P (

ppm

)

695538

±16518.3

1500

2000

2500

DN

A (

ppm

)

1935.3

43

±89.55908

24

28

32

CE

X %

Acid

ic

Variants

27.6

6898

±0.480814

1.8

2.2

2.6

3.0

Aggre

gate

s

(%)

2.5

15119

±0.03524

34

34.5 35

35.5 36

35

Temperature

(C)

30

40

50

60

70

50

DO (%)

40

60

80

100

120

140

160

100

CO2 (%)

6.6

6.7

6.8

6.9 7

7.1

6.85

pH

.8 1

1.2

1.4

1.6

1.2

[Medium]

(X)

360

380

400

420

440

400

Osmo (mOsm)

9

10

11

12

13

14

15

12

Feed (X)

.7 .8 .9 1

1.1

1.2

1.3

1

IVCC (e6

cells/mL)

15

16

17

18

19

17

Duration

(d)

-0.1 .1 .3 .5 .7 .9

1.1

0.21

Curvature

Prediction Profiler



Influence of Glycosylation on ADCC and CDC Effector Functions


Case StudyLilly - Company Confidential 2010 14

CQA Linkage to Process Knowledge

afucosylation and galactosylation are assigned as CQAs due to linkage to ADCC and CDC activity and proposed NHL therapeutic need

Analytical characterization method for afucosylation and galactosylation is CE-LIF:

Bioassay development led to a robust assay with a linear correlation between aFuc (2-13%) and ADCC activity (range of 70 – 130%)

Bioassay for CDC showed no impact over the range of galactosylation (10 – 40%)

Ranges of afucosylation and galactosylation can be ensured by control of bioreactor process parameters found to have influence on these structures.

Release testing of Biopotency assay (acceptance criterion 70 – 130%) confirms appropriate product quality.



7/13/2010

6

Continuity of RangesAttributes and Parameters in Study



Levels of CQAs:

CQA Lower Limit Higher LimitAfucosylation (%) 2 13Galactosylation (%) 10 40

Parameter Ranges:Platform (2 liters and at-scale FHD) pH 6.6 – 7.1 (initial set pt*)

Screening Study (Central Composite) pH 6.6 – 7.1 (2 liter)

Design Space Proposal pH 6.6 -7.1 (commercial)

Batch Record (Pivotal and Comm.) pH 6.95 (initial ref pt)

Automation Alarms pH 6.85 lo/pH 6.95 hi alert-control space

pH 6.7 lo/pH 7.1 action-design space*Note that pH variable is set at initial as ref pt and moves through low (base) and high (acid or CO2) control

Moving Toward Design SpaceFollow-up Studies and Analysis

Augment the screening design to enable estimation of a full response surface:

all main effects

two-way interactions

quadratic effects

Additional runs form Central Composite Design (when comb. w/ previous runs):

8 additional runs form full factorial on important parameters.

8 axial points allow to estimate non-linear relationships

4 parameters and 6 QA’s (responses)

8 center points total

17Vinci/Defelippis - CMC BWG QbD


Response surface model captures all input – output

relationships and is suitable to define the design space

N=40 total bioreactor runs

(4 blocks of 10, ~12 weeks)

18

Develop Multivariate Models to define Design Space

A better way to look at the data:

One model for each CQA: describes

relationships with CPPs

Intersection of all CQA models define the

Design Space

For the production bioreactor the limits of

Design Space are defined by a subset of

CQAs:

Galactosylation

aFucosylation

All other CQAs did not exceed Quality Limits

when process operated within Knowledge

Space & Design Space

*Note that DO and Feed Conc from earlier study

are controlled in same range

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)

Culture Duration (days)

Factor

34.999293

50

100

6.85

1.2

440

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )

CEX % Acidic Variants

Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.2

5.7

30.3

490873.2

1498.3

26.7

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

CO2

Osmolality

< 2%

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

11

40

15000000

19000

40

3

Contour

4.8

8.3

33.4

513494.5

1471.7

28.2

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

>11%

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

11

40

15000000

19000

40

3

Contour

5.2

9.8

36.8

469303.1

1465.4

33.1

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

4.7

7.8

33.7

495754.0

1552.2

30.2

1.2

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

360mOsm 440mOsm HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

4.5

6.6

34.4

458789.8

1479.0

31.0

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

160 mmHg

40 mmHg

100 mmHg

400mOsm

>40% >40%

>40%

<20%

<20%

>11%

>11%

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

34.999293

50

100

6.85

1.2

440

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.2

5.7

30.3

490873.2

1498.3

26.7

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

CO2

Osmolality

< 2%

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

11

40

15000000

19000

40

3

Contour

4.8

8.3

33.4

513494.5

1471.7

28.2

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

>11%

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

11

40

15000000

19000

40

3

Contour

5.2

9.8

36.8

469303.1

1465.4

33.1

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

4.7

7.8

33.7

495754.0

1552.2

30.2

1.2

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

360mOsm 440mOsm HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

4.5

6.6

34.4

458789.8

1479.0

31.0

1.3

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

160 mmHg

40 mmHg

100 mmHg

400mOsm

>40% >40%

>40%

<20%

<20%

>11%

>11%

Design Space for Culture Duration 15 Days

CO2

Osmolality

360mOsm 440mOsm

160 mmHg

40 mmHg

100 mmHg

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

5.0

6.5

29.8

697946.1

2040.3

30.2

1.5

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

440

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.6

5.2

25.7

694855.9

1966.2

26.9

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylationGalactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

400mOsm

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.1

6.3

29.1

702394.3

1965.8

28.4

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

5.7

7.5

33.5

669715.6

1973.9

32.9

1.5

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.9

5.9

30.9

674274.3

1961.3

30.7

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

>40%

<20%

< 2%<20%


CO2

Osmolality

360mOsm 440mOsm

160 mmHg

40 mmHg

100 mmHg

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

5.0

6.5

29.8

697946.1

2040.3

30.2

1.5

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

440

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.6

5.2

25.7

694855.9

1966.2

26.9

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH


34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

400mOsm

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.1

6.3

29.1

702394.3

1965.8

28.4

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

5.7

7.5

33.5

669715.6

1973.9

32.9

1.5

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.9

5.9

30.9

674274.3

1961.3

30.7

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

>40%

<20%

< 2%<20%

CO2

Osmolality

360mOsm 440mOsm

160 mmHg

40 mmHg

100 mmHg

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

5.0

6.5

29.8

697946.1

2040.3

30.2

1.5

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

440

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.6

5.2

25.7

694855.9

1966.2

26.9

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH


34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

400mOsm

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.1

6.3

29.1

702394.3

1965.8

28.4

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

40

15000000

19000

40

3

Contour

5.7

7.5

33.5

669715.6

1973.9

32.9

1.5

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

17

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.9

5.9

30.9

674274.3

1961.3

30.7

1.6

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

>40%

<20%

< 2%<20%



CO2

Osmolality

360mOsm 440mOsm

160 mmHg

40 mmHg

100 mmHg

400mOsm HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.4

5.2

27.3

889758.9

2443.6

30.5

1.9

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

6.1

5.3

30.2

870128.1

2482.5

32.8

1.8

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.4

4.3

24.8

891294.0

2459.8

28.6

1.8

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

440

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.9

4.7

21.1

898827.7

2434.0

27.0

1.9

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.4

5.1

25.9

900138.3

2528.5

30.1

1.8

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

<20%

<20%

< 2%

<20%


CO2

Osmolality

360mOsm 440mOsm

160 mmHg

40 mmHg

100 mmHg

400mOsm HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

440

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.4

5.2

27.3

889758.9

2443.6

30.5

1.9

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

40

6.85

1.2

360

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

6.1

5.3

30.2

870128.1

2482.5

32.8

1.8

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

360

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.4

4.3

24.8

891294.0

2459.8

28.6

1.8

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

100

6.85

1.2

440

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

4.9

4.7

21.1

898827.7

2434.0

27.0

1.9

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

Galactosylation (%)

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

HorizVert

Tem perature (C)

DO (%)

CO2 (%)

pH

[Medium] (X)

Osm olality (m Osm)

Feed (X)

IVCC (e6 cells /mL)


Factor

35

50

70

6.85

1.2

400

12

1

19

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm )

DNA (ppm )


Aggregates (%)

Response

0

2

20

15000000

19000

40

3

Contour

5.4

5.1

25.9

900138.3

2528.5

30.1

1.8

Current Y

.

2

20

.

.

20

.

Lo Limit

.

11

40

15000000

.

40

3

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

34 34.5 35 35.5 36

Tem perature (C)

Contour Profiler

<20%

<20%

< 2%

<20%



7/13/2010

7

Horiz Vert

Temperature (C)

DO (%)

CO2 (mmHg)

pH

[Medium] (X)

Osmo (mOsm)

Feed (X)

IVCC (e6 cells/mL)

Duration (d)

Factor

35

50

40

6.85

1.2

360

12

1

15

Current X

Titer (g/L)

aFucosylation

Galactosylation (%)

HCP (ppm)

DNA (ppm)


Response

3

11

40

675000

2250

40

Contour

5.3408326

9.1879682

38.227972

466955.66

1382.1644

34.420095

Current Y

3

.

.

.

.

.

Lo Limit

.

11

40

.

.

.

Hi Limit

6.6

6.7

6.8

6.9

7

7.1

pH

aFucosylation

Galactosylation (%)

34 34.5 35 35.5 36

Temperature (C)

Contour Profiler

Design Space Based on Process Capability Understanding Variability



Galact >40%

aFucos >11%

34 34.2 34.4 34.6 34.8 35 35.2 35.4 35.6 35.8 366.6

6.65

6.7

6.75

6.8

6.85

6.9

6.95

7

7.05

0.250.250.25

0.2

5

0.50.5

0.5

0.5

0.70.7

0.7

0.7

0.80.8

0.8

0.8

0.9

0.9

0.9

0.9

0.950.95

0.95

0.95

0.99

0.99

0.99

0.9

9

Example: Day 15, Osmo=360 mOsm

and pCO2=40 mmHg >99%

confidence of

satisfying all

CQAs

50% contour

approximates “white”

region” in contour plot

pH pH

Temperature (C) Temperature (C)

20

Risk Assessment ApproachMultiple Assessments Throughout the

A-Mab Development Lifecycle for Entire Process

Prior Knowledge

Process Understanding

Product Understanding

Process

Development

Risk

Assessment

Process

Characterization

Risk

Assessment

Risk

Assessment

Process

Performance

Verification

Risk

Assessment

Life Cycle

Management

Final Control

Strategy

Process

Parameters

Quality

Attributes

Design Space

Draft Control

Strategy

Process 2

Process 1 2



You Are Here

Control Strategy for Upstream Production

Step 2

Seed Culture Expansion

in Fixed Stirred Tank

Bioreactors

Step 3

Production Culture

Step 4

Centrifugation and Depth

Filtration

Working Cell Bank

Clarified Bulk

Step 1

Seed Culture Expansion

in Disposable Shake

Flasks and/or bags

In-Process

Quality Attributes

Bioburden

MMV

Mycoplama

Adventitious Virus

Product Yield

Turbidity

Viable Cell Concentration

Viability

Product Yield

Viability at Harvest

Turbity at Harvest


Viability

Key Process

Attributes


Viability

Quality-linked

Process Parameters

(WC-CPPs)

Temperature

pH

Dissolved CO2

Culture Duration

Osmolality

Remnant Glucose

Temperature

pH

Dissolved Oxygen

Culture Duration

Initial VCC/Split Ratio

Antifoam Concentration

Time of Nutrient Feed

Volume of Nutrient Feed

Time of Glucose Feed

Volume of Glucose Feed

Dissolved Oxygen

Flow Rate

Pressure

Temperature

Culture Duration

Initial VCC/Split Ratio

Key Process

Parameters

(KPPs)

Temperature

Time

Controlled within the

Design Space to

ensure consistent

product quality and

process performance

Controlled within acceptable

limits to ensure consistent

process performance

Assay results part

of batch release

specifications

Slide 21Vinci/Defelippis - CMC BWG QbD


7/13/2010

8

Example of Control Strategy for Selected CQAs

CQA Criticality Process

CapabilityTesting Criteria

Other ControlElements

Aggregate High (48) High RiskDS and DP

releaseYes

Parametric Control ofDS/DP steps

aFucosylation High (60) Low RiskDS Process Monitoring

YesParametric Control of

Production BioRx

Galactosylation High (48)Low Risk DS Process

MonitoringYes

Parametric Control of Production BioRx

Host Cell Protein

High (24)Very Low

RiskCharact.

ComparabilityYes

Parametric Control of Prod BioRx, ProA, pH inact, CEX , AEX steps

DNA High (24)Very Low

RiskCharact.

ComparabilityYes

Parametric Control of Prod Biox and AEX

Steps

Deamidated Isoforms

Low (12) Low RiskCharact.

ComparabilityNo

Parametric Control of Production BioRx


Case Study22Lilly - Company Confidential 2010

Lifecycle Management of Design SpaceDynamic Modeling

Challenge:

• Data from a limited number of batches is required for process validation

ex: n=5 or more for 3 bioreactors ; costly and often critical path

• Limited replicates are not statistically significant – at best test the “system” including facility, equipment, process, operators, etc

Alternative Lifecycle Approach or Continuous Process Verification:

• Quality Mgt System assures site’s readiness and compliance

• Use 1 or 2 batches to confirm or demonstrate validity of design space

• Utilize a multivariate statistical partial least squares (PLS) model for continuous process verification as commercial experience grows in number of runs

• Scheduled reviews of product quality data trends and design space validity during the product lifecycle



Design Space and Elements of Control

Successful acceptance or utilization of our evolving view of design space relies on linking the multiple elements of documented knowledge and systems:

Facilitated formal attribute rankings and parameter risk assessments to guide DOEs

Linkage of all attribute and parameter ranges used for modeling and scale

Delineation of how lifecycle oversight (control strategy) of critical and non-critical parameters and specification/limit testing occurs

Movement to best practices for engineering first principles/mechanistic models and statistical modeling as they apply to QbD paradigm


StudyLilly - Company Confidential 2010 24

7/13/2010

9

Upstream Development Team

Ilse Blumentals GSK

Guillermo Miroquesada MedImmune

Kripa Ram MedImmune

Ron Taticek Genentech

Victor Vinci Lilly

*Help from many others – CMC BWG member company reps and internal resources at each company


QbD Case StudyLilly - Company Confidential 2010 25

control strategy for glycosylation€¦ · • amgen team: joseph phillips (lead), bob kuhn •...

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