Case study:Challenges and learning in implementing ATF

perfusion process

Dr. Jarno RobinPrincipal Scientist

CMC_Biopharm upstream developmentNovo Nordisk A/S, Hillerød, Denmark

Outline

• introduction to Novo Nordisk• case study: implementation of ATF perfusion process for

improving yield prior to fase3 production• background & timeline• perfusion technology• contraints• new perfusion process (lab & production scales)• process challenges & stabilisation

• conclusion:• yield improvement and quality• learnings

Novo Nordisk Way

Novo Nordisk Corporate strategy

Novo Nordisk around the world

R&D, production

Headquater,

Research Units,

R&D, production

production

Diabetes: A global pandemic

Part II

• case study: implementation of perfusion process for improving yield prior to fase3 production

• background & timeline• perfusion technology• contraints• new perfusion process (lab & production scales)• process challenges & stabilisation

Background & timeline

• Feb : 3-days-Draw & fill (D&F) process approved for phase3 production.

min0 10 20 30 40 50

LU

1

2

3

4

5

6

7

FLD1 A, Ex=280, Em=340 (DEF_1364 2008-01-09 10-23-29\001-0301.D)

33.62

3

36.75

6

40.17

2

min0 10 20 30 40 50 60

LU

0

10

20

30

40

50

60

FLD1 A, Ex=280, Em=340 (FIX\DEF_1364 2007-12-06 08-15-15\041-1001.D)

33.

909

37.

625

40.

854

44.

027

min0 10 20 30 40 50 60

LU

0

10

20

30

40

FLD1 A, Ex=280, Em=340 (FIX\DEF_1364 2007-11-27 12-06-56\016-1501.D)

1.7

57

31.

473

35.

473

38.

681

41.

823

min0 10 20 30 40 50 60

LU

0

5

10

15

20

25

30

35

40

45

FLD1 A, Ex=280, Em=340 (FIX\DEF_1364 2007-11-22 12-49-40\073-0301.D)

34.

174

37.

910

41.

134 4

4.32

3

min0 10 20 30 40 50

LU

0

5

10

15

20

25

30

35

FLD1 A, Ex=280, Em=340 (FIX\DEF_1364 2008-01-11 15-45-25\004-9101.D)

28.23

0

31.83

7

34.96

9

38.23

4

0 5 10 15 20 25 30 35 40 450

1

2

3

4

VCD (mio/ml) Act. (µg/l) Total ELISA (µg/l)

0

2500

5000

7500

Time (days)

VC

D (

mio

/ml)

FIX

(µg

/l)

min0 10 20 30 40 50 60

LU

0

2.5

5

7.5

10

12.5

15

17.5

20

FLD1 A, Ex=280, Em=340 (FIX\DEF_1364 2007-11-22 12-49-40\071-0101.D)

Area:

67.34

76

34.32

8

Area:

579.1

47

38.29

1

Area:

1883.

02

41.54

7 Area:

3574.

04 44.77

1• very stable & robust process (cell growth & product quality)

BUT• very poor productivity• Issues regarding production capacity

2008

Prod. Conc.

Background & timeline• Feb.: 3-days-Draw & fill (D&F) process approved for phase3 production.

• Oct.: process challenge meeting: approval for attempting to change the production process from D&F to a perfusion process based on expectations to increase yield significantly (shaked flasks simulation).

0

2000

4000

6000

8000

10000

24 48 72

Hours of culture

Cu

mm

ula

tive

FIX

10-

12

Gla

Pro

du

ced

No

rmal

ised

yie

ld

imp

rovm

ent

(%)

2008

Background & timeline• Feb.: 3-days-Draw & fill (D&F) process approved for phase3 production.

• Oct.: process challenge meeting: approval for attempting to change the production process from D&F to a perfusion process based on expectations to increase significantly yield.

• Nov-Dec.: Finalising the ATF purchase for lab- & production- scales.

• Jan-Jun.: Implementation of ATF-2 in lab scale and ATF-10 in production scale.

• Apr-Jul: TDR meetings:

• production medium chosen for phase3 production (3 upstream depts. involved).

• perfusion process using the ATF chosen for phase3 production (3 upstream + 1 downstream depts. involved, coordination meeting).

• Jul-Aug.: Master & production formulas in place for fase3 production planed from Oct. 2009 (2 upstream depts. involved).

• Q2/Q3: further process optimisation, MF/PF updated (2 upstream depts. involved).

• Q4-2011/Q2-2012: issue regarding impact of complex raw-material quality on process performance (2 upstream depts. involved).

• Q2/Q3: Process Justification & transfer to production support department.

2008

2009

2011

2012

Why ATF ? : Perfusion devices available

Applisens BioSepApplisens BioSep Spin filterSpin filter

good in lab up-scaling, pump 5-10% cells in harvest

good in lab (intern filter) up-scaling: extern at large scale (temp., pH, O2 stress, pump & shear stress) block at high cell conc. cells in harvest

LevelControl

FeedPump

WaterJacket

InletOutlet

D.O.

pHAgit.

Temp.

Hollow Fiber Unit

CirculationPump

PermeatePump

Gas inlet into headspace or liquid

LevelControl

FeedPump

WaterJacket

InletOutlet

D.O.

pHAgit.

Temp.

Hollow Fiber Unit

CirculationPump

PermeatePump

Gas inlet into headspace or liquid

Tangential Flow Filtration (TFF)Tangential Flow Filtration (TFF)

good in lab cell free harvest possible product binding pump, up-scaling

ATF (Alternating Tangencial Filtration)ATF (Alternating Tangencial Filtration)L EV EL

CONTROL

OF F

ON

AI RI NL ET

EXHAUST

ADDI T I ONPUMP

F L UI DI NL ET

V AL V E

QUI CK CONNECT

F I L TRATE PUMP

F I L TRATE

HF MODUL E OR SCREEN MODUL E

HOUSI NG

CONTROL L ER

PROCESS V ESSELDI APHRAGM

ATFPUMP

STANDF I L TER

L I QUI D L EV EL

simple & upscalable (to 5m3) no temp., pH, O2 stress no pump & low shear stress cell free harvest reasonable cost possible product binding

Bleed

Harvest

Harvest

Medium

ATF perfusion process at Novo Nordisk(Alternative Tangiencial Filtration)

Patent no.: WO2012045769

Pictures of ATF (“Darth Vader” sword)

New ATF-process: constraints

• Harvest stability of at least 2 days due to downstream process at large scale.

• Short timeline for a complete process change (Q1/Q2-2009,~6 months)

• Get a scalable perfusion process to the production facility using the ATF technology.

• Get similar product quality as the material produced for tox and fase1 clinical trial using the D&F process .

• Improve significantly the expression level (production capacity)

New process scalability from lab to production scale (2009)

Cell growth

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Time [days]

Via

ble

cell

den

sit

y [

x1

0^

6]

0

10

20

30

40

50

60

70

80

90

100

Via

bil

ity [

%]

VCD F9-C-003 F9-C-002 VCD 5L 1201

Viability [%] F9-C-003 Viability [%] F9-C-002 Viability 5L 1201

Challenges: Complex raw-material quality impact on process performance at large scale

Sammenligning af FI X batcheCelletal

0123456789

10111213141516171819202122232425

0 5 10 15 20 25 30 35 40

dage

Bio

masse (

x 1

0^

6 c

eller/

ml)

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (day)

Liv

ing

cell

(1E

6 ce

ll/m

l)

6276 (Y5UY963) 6121 (Y5UE480) 6119 (Y5UE480)

6299 (Y5UV515) 6310 (A5U6881) 6328 (YSC0784)

6329 (Y5UAG42) 6330 (A5UC040)

• Soy hydrolysate has been analysed:• tracer element,• free amino acids• NIR

Challenges: Complex raw-material quality impact on process performance. Confirmation at lab scale

Kultur nr. HySoy Zn Mo6299 Y5UV515 17,6 2,2611961216310 A5U6881 17,2 6,56328 YSC0784 3,6 2,66329 Y5UAG42 18,2 1,96330 A5UC040 6 6,56276 Y5UY963 15,8 2,1

4,9Y5UE480 16,1

NIR analyses on HySoy batches

PCA model of average NIR spectra (derivative spectra)Colored according to Cell Max results (5L)

YSC0784

Y5UY963

Y5UV515

Y5UAG42

Y5UE480

A5UC040

A5U6881A5U1709

A5U1707

t[2]

-0,002

-0,0015

-0,001

-0,0005

0

0,0005

0,001

0,0015

-0,005 -0,004 -0,003 -0,002 -0,001 0 0,001 0,002 0,003 0,004

t[1]

R2X[1] = 0,691 R2X[2] = 0,152 Ellipse: Hotelling's T2 (95%)

DS1.Cell Max (5L)

12

14

16

18

20

22

Missing

Work done by Erik Skibsted (ERSK)

Process stabilisation: focus on Zn concentrationSammenligning af FI X batche

Celletal

0123456789

10111213141516171819202122232425

0 5 10 15 20 25 30

dage

Bio

masse (

x 1

0^

6 c

eller/

ml)

018

019

020

021

022

023

025

026X

w/ Zn spiking

w/o. Zn spiking

Conclusion: Yield improvment and quality

Process

Normalised Productivity

(%)

Quality parameter 1

(%)

Quality parameter 2

(%)

D&F 100 ~95 0,03

ATF 250 90-95 0,06

ATF 600 ~90 0,07

Large scale yield for Factor 9, previous process vs. ATF process

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30 35 40 45

day

ATF process FIX

Previous process 1

Previous process 2

Conclusion: Learnings

• targets achieved:• new process development & implementation within short timeline (6

months)• scalable perfusion process• significant increase in yield (up to 600%)• identical product quality between D&F and perfusion process• process stabilisation after unexpected significant impact of complexe

raw-material on process performance• increased knowledge:

• solving challenges• increased collaboration across lab & production scales• complex raw-material characterisation• know-how for next generation process and/or products for early

implementation in the project plan:• Focus on correlation between cell line/medium/process

• Aknowledgements:• Dept.’s bioreactor team for dedicated technical work• Production & Research Unit, up- & down-stream for good collaboration• Vendors for support

Thank you !• for your attention