Screening approaches for

product quality to enable

attribute-driven cell line

development with an eye

towards commercialization

23rd January 2017CMC Strategy Forum

Chris Sellick

Senior Scientist

Cell Culture and Fermentation Sciences

TiterProduct

quality

• Over the past six years the biologics pipeline has tripled in size and now

comprises about 50% of AstraZeneca’s pipeline

The changing pipeline

Non-mAb formats have

increased from 20% to 50%

of the Research Portfolio

utilising up to 13 different

technologies/formats

2

No

n-m

Ab

pro

jec

ts a

s a

%

of

Re

se

arc

h p

ort

foli

o

0

10

20

30

40

50

60

2010 2011 2012 2013 2014 2015

3

Combined Functional and Developability Focus

• Molecular design with functional focus & developability as a

fundamental input

Developability

Target

Profile

Functional

Target

Profile

Target

Molecule

Physiochemical properties

Manufacturability

Stability and integrity

PK/PD

Bioactivity

Specificity

Species cross-reactivity

Pre-Clinical/

Clinical

Launch &

PLC/LCMDiscovery

Pre-Clinical

DevelopmentEarly

Development

• Product knowledge sharing between Research and BPD- Structure/Function relationships- Mechanism of Action- Developability

• “What are the quality attributes we need to get right?”

• “The product defines the process and required analytics”

4

Project R&D Interface: A Critical Change

Research CMCResearch CMC

5

Attribute Focused Analytical Development

Define initial pCQAs

Evaluate methods for each

pCQA

Implement method to support

CLD and process development

Define method purpose &

requirements for each pCQA

Gain Early Product Knowledge

from R&D Interface &

Developability

Identify likely cell line /process-

dependent attributes

DevelopmentResearch

Overview of Cell Line Development (CLD)

Launch &

PLC/LCMDiscovery

Pre-Clinical

DevelopmentEarly

DevelopmentPre-Clinical/

Clinical

CLD Project Start

Vector

Construction

Creation of Pools and

Parental Cell Lines

Clonal Cell Line

Isolation

Cell Line

Characterisation

CLD generates clonal cell lines to produce product suitable for GMP manufacture

~20 weeks

6

Current CLD platform

Shaking 24 well-plate

overgrows

BULK

Expression

vector

CHO Host

Cell Line

AMBR

Research Cell Banks (RCB) for

top cell lines

Titre screening to select the best producing cell lines

ClonePix

7

8

Requirements for process analytics

Process Stage

Early multi well screen

AMBR

Number of

vessels

200 to

400

1 to 8

24 to 48

Method

Requirements

• Analysis in cell

culture media

• Sample volume

<0.5ml

• Concentration

potentially

<1mg/ml

• Rapid

turnaround

3L to 500L Bioreactor

9

Case study 1 – Changing a cell line to deliver improved yield

• Case study

– In-licensed human IgG2 monoclonal antibody

– Original cell line produced 0.9 ± 0.1 g/L average titer at 2000 L scale for

Phase I and II studies

– For commercial considerations a new cell line was required

Attribute Assay Criteria

Titre Protein A HPLC ≥ 4 g/L in a 14-day fed-batch process

Phenotypic Stability Protein A HPLC ≤ 30 % drop in titer between 40-60 PDL from RCB

Charge variants IECWithin target range (64% - 84%)

Main peak within historical range

GlycosylationOligosaccharide

profile

Man5/6 levels within historical range

Similar profile to Ref Std, no new peaks

Disulfides Peptide map Similar profile to Ref Std

Purity HPSEC ≥ 95.0%

10

Case study 1 – Cell line development

Static

Top 62 cell lines

Shaking

Top 9 cell lines

Top 9 Clones 3-7 fold titre improvement

Historiccell line

AMBR

Analysis of product from

the cell lines to assess:

• Purity

• Charge variants

• Glycosylation

• Disulphide profiles

• Phenotypic stability

11

Case study 1 – Cell line and product attributes

HPSEC

Gal:Agal ratio within 10% of Ref Std

Mannose ≤1SD from Ref Std

Glycans

Clone ID % Pre-peak% Main

peak

% Post-

peak

In

Spec?Main peak

SD range

Ref Std 20.4 72.0 8.3 1

F 16.3 71.7 12.0 1-2

G 18.2 71.4 10.5 1-2

E 20.4 69.1 10.5 2-3

H 19.7 67.5 12.8 2-3

I 20.5 66.7 12.8 2-3

C 22.4 64.7 13.0 >3

D 17.9 65.7 16.5 >3

A 21.3 63.4 15.3 X >3

B 18.4 63.2 18.3 X >3

IEC

64.0% to 84.0% main peak

≥ 95% monomer

12

Case study 1 – Summary of cell line performance

• Cell line G chosen based on:

– Product quality attributes most comparable to reference standard

– Titre and phenotypic stability meet acceptance criteria

CloneIEC

(% main peak)

Oligo(% high

mannose)

Disulfide profiles

Purity (%

monomer)

40 PDL Titer (g/L)

Phenotypic Stability Loss

(%)

Target range 64 - 84 2.3 - 4.0 Similar ≥ 95 ≥ 4 ≤ 30

Reference 72.0 2.3 N/A 98.9 ≤ 0.8 N/A

G 71.4 2.8 Similar 98.8 6.0 11

E 69.1 7.6 Similar 98.5 5.9 15

H 67.5 2.8 Similar 98.8 6.5 8

I 66.7 3.9 Similar 98.7 6.6 46

C 64.7 4.3 Similar 98.8 5.4 6

A 63.4 3.4 Similar 97.8 4.5 29

D 65.7 7.0 Similar 98.7 NT NT

F 71.7 5.3 Similar 99.1 NT NT

B 63.2 5.4 Similar 98.2 NT NT

Case study 1 – Summary

• New clonal cell line has comparable product attributes

– Used in-house expression vectors and CHOK1SV host

– New platform process (feed and media formulation changes)

– Achieved 6 g/L in AMBR and subsequently at 5 L & 50 L scale

• Comparable product quality

– This was achieved through the use of micro-scale bioreactor and cell line

product evaluation at critical point in the platform process

– Integrated analytics and cross-functional working was key to the successful

switch to a new cell line

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Case study 2 – MEDI-FP1

• Highly glycosylated Fc-fusion protein

• Molecule characteristics

– Potential fragmentation (LMWS) and aggregation (HMWS)

– Contains sialylated glycans

• N- and O-glycans

• Sialylation crucial for half life of molecule

• Aim

– Minimise LMWS and HMWS

– Achieve consistent glycosylation with high sialylation

– Understand the effects of cell line and process on molecule

characteristics

14

Case study 2 – Understanding the impact of cell line

and process on the product

15

0

200

400

600

800

1000

1200

1400

Cell Line 1 Cell Line 2 Cell Line 3 Cell Line 4 Cell Line 5

Tit

re (

mg

/L)

Media A Media B Media B +Temp Shift

• Productivity dependent on

cell line

• Cell lines vary significantly

in their response to media

and feed

• Productivity only part of the

story, product quality needs

to be controlled

Assay

requirements Titre Sialylation Fragmentation Aggregation

Assay Octet ??? SDS-CE (GXII) UPLC-SEC

HT

Process

samples

Low volume

Case Study 2 – Analytical requirements and method

development

• Can methods be developed for high throughput measurement of molecule

sialylation?

• Assays need to be in place before the start of the CLD campaign

16

Case study 2 – Octet-based lectin assay to screen

sialylated glycans

17

Biosensor

Biosensor Biosensor

Biosensor Biosensor

Biosensor

Biosensor Biosensor

Biosensor

Case study 2 – Octet-based lectin assay to screen

sialylated glycans

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Lectin Solution

Assay

requirements Titre Sialylation Fragmentation Aggregation

Assay Octet ??? SDS-CE (GXII) UPLC-SEC

HT

Process

samples

Low volume

Case Study 2 – Analytical requirements and method

development

19

Assay

requirements Titre Sialylation Fragmentation Aggregation

Assay OctetOctet-based

lectin assaySDS-CE (GXII) UPLC-SEC

HT

Process

samples

Low volume

Case Study 2 – Analytical requirements and method

development

• Process analytics in place before the start of the CLD campaign

– Allows informed attribute-led decisions to be made

• How and when should they be integrated into the platform process?

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Tailored CLD campaign

Shaking 24 well-plate

overgrows

BULK

ambr

Research Cell Banks (RCB) for

top cell lines

ClonePix

Titre screening to select high producing cell lines

MAL I/II binding assay to select optimal N- and O-glycan sialylation profiles

Non-reduced GXII to minimise fragmentation

UPLC-SEC to select the highest monomer purity21

Expression

vector

CHO Host

Cell Line

Case study 2 – PQ screening during CLD

22

Terminal sialic acid

Aggregation

Fragmentation

• Early interactions between Research and Development ensured potential

critical quality attributes (pCQAs) were identified

• Early development of analytics to monitor the pCQAs enabled them to be

used as an input for cell line development and process optimisation

• Process analytics were critical for controlling the product quality from

early material supply to toxicology batches Enabled timely decision making

• Defining the pCQAs early in the project lifecycle was essential for the

rapid progression of this project Process development was completed 3 months ahead of schedule

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Case study 2 – MEDI-FP1 summary

Case study 3 – MEDI-FP2

• Large multimeric Fc-fusion protein

• Molecule characteristics

– Requires correct assembly to form multimer

– Potential fragmentation (LMWS) and aggregation (HMWS)

– Fc glycans and an additional glycosylation site

• N-glycans

• Aim

– Confirm correct assembly

– Minimise LMWS and HMWS

– Achieve consistent glycosylation with low Man5

– Determine if additional glycosylation site is required

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Assay

requirements Titre

Oligomeric

state Aggregation Mannose 5

Assay Octet UPLC-SEC UPLC-SEC

Glycan

analysis

(GXII)

HT

Process

samples

Purified

samples

Low volume

Analytical requirements and method development

25

Case study 3 – Oligomeric assembly and aggregation

• UPLC-SEC on crude supernatant from different clones in AMBR

– Different amounts of aggregation

– Varying amounts of HCP’s

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Correct

assembly

Minus 2

subunitsHMWS

48 cell lines screened

Case study 3 – High throughput purification

• Atoll columns on a Tecan robot

• Protein A purification of up to 96 samples at a time

• Samples used directly for glycan analysis

27

Case study 3 – Fc glycan screening

• Cell line screening in AMBR system

• Three clones from one parent show high (>95%) Man5

– None of the clones from this parent were progressed

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• Tailored analytics that monitor glycosylation, oligomeric state and

aggregation were used to support attribute-led cell line selection

• High Man5 cell lines were removed early in the process

• Selected cell lines produced high % of hexameric product

• Early implementation of attribute focused screening methods allowed for

accelerated project delivery

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Case study 3 – MEDI-FP2 summary

Conclusion

• Understanding the potential critical quality attributes early allows optimal

cell lines and processes to be selected

– CLD and PD with an eye towards commercialization

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Late Stage

DevelopmentProcess

Development

Cell Line

DevelopmentResearch

Likelihood of optimal

Developability characteristics

ResourceTime

CostMonitor pCQAs

Developability

assessment

Control of

pCQAs

Streamlined late

stage development

Develop HT

analytics

Define pCQAs

Attribute-based

cell line selection

Commercialization

Acknowledgements

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• BioProcess Analytics

• Cross-functional Analytical Development (CAD) team

• Research

• Cell Line Development

• Cell Culture and Fermentation Sciences

• Purification Process Sciences

• Analytical Biotechnology

Questions?

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