Justyna Jozefczuk, Novartis

1. Justyna Jozefczuk, Thomas Jostock, Holger Laux, Annet Ritter, Juergen
Recktenwald and Burkhard Wilms
Integrated Biologics Profiling
Novartis Pharma AG
9th Annual BioInnovation Leaders Summit
Berlin, February 2016
Optimizing cell line development platform
© Novartis Pharma AG 2016

2. Challenge: Finding the few top perfomers fast and efficient
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Selection/screening
Diverse starting
population after
transfection
Identification
of top
performer

3. FACS vectors for selective cloning of high producing cells
Applying flow cytometry to selectively isolate high producing cells
Source: Department of Biology, Davidson College,
Davidson, NC 28036
http://www.bio.davidson.edu/COURSES/GENOMICS/method/FACS.html
Productivity of clones with 1 round of pre-enrichment: 24-well batch culture
0
100
200
300
400
500
600
700
800
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101106111
clones
mAb[mg/L]
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

4. Development and implementation of new technologies
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk4
Folic acid
receptor
selection system
Enchanced
CHO host cell
line
Evaluation of
additional new
technologies
Improved cell line development
platform
Monoclonality
assessment

5. Development and implementation of new technologies
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk5
Folic acid
receptor
selection system
Enchanced
CHO host cell
line
Evaluation of
additional new
technologies
Improved cell line development
platform
Monoclonality
assessment

6. Folic Acid Receptor: A new metabolic selection marker
Collaboration with Technion (Israel), Prof. Assaraf
Selection of transfected cells
by folic acid starvation
(no addition of toxins needed)
LC
HC
neo
amp
hu FOLRa ORF
prom
Intron
polyA
prom
Intron
polyA
phage f1prom
polyA
pA
promoter
FolR
vector
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

7. Combining Folic Acid Receptor and DHFR/MTX
Selection: Co-transfection
FolR/Neo Co-Transfection Dhfr/Neo
DHFR
vector
LC
HC
neo
amp
DHFR
prom
intron
polyA
prom
intron
polyAprom
polyA
prom
polyA
LC
HC
neo
amp
hu FOLRa ORF
prom
Intron
polyA
prom
Intron
polyA
prom
polyA
pA
promoter
FolR
vector
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

8. Clonal distribution: FR/dhfr co-selection vs G418->dhfr
Limiting dilution cloning and multiwell plate screening
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
0
50
100
150
200
250
300
350
400
450
500
1 3 5 7 9 11 13151719212325272931333537394143454749515355
mAB[mg/L]
clone
FRα/dhfr co-transfection/co-selection
0
10
20
30
40
50
60
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
mAb[mg/L]
clone
G418/dhfr sequential selection
! Average productivity of co-transfected/selected clones
about 40x higher than reference

9. 0
50
100
150
200
250
300
350
400
%titermodelmAb
Average of Top 5 Clones
Shake flask batch culture (13- 14 days)
NVS dhfr
NVS dhfr FACS
NVS dhfr-FACSvector
NVS FolR
NVS dhfr/folR
NVS dhfr/folR/FACSvector
Vector optimisation is a continuous process
Impact of vector technologies on platform performance
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Combining new vector technologies can significantly increase performance
Evaluation of further combinations of best performing tools is ongoing
Clone picking
Selection
Combination

10. Development and implementation of new technologies
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk10
Folic acid
receptor
selection system
Enchanced
CHO host cell
line
Evaluation of
additional new
technologies
Improved cell line development
platform
Monoclonality
assessment

11. Screening of the CHO transcriptome using microarray
technique
Analysis of 60 thousand transcripts in one experiment
Comparison of expression quantity of different genes
Through screening of 60 thousand transcripts pathway analysis is possible
1.
RNA
QC:
Agilent
TapeSta4on
3.
Affymetrix
GeneChip
hybridiza4on
4.
Washing
and
staining
5.
Laser
scanning
6.
Bioinforma4cs
2.
Automated
sample
prepara4on
Source: Affymetrix, Hamilton, Bimatix and Seedgenenetwork
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna
Jozefczuk
11

12. ! AIM: Identification of a set of genes between high-low
producing CHO clones
! Experimental design: Gene expression analysis using
Cricetulus griseus specific Affymetrix microarrays
" Signal intensity of 5 genes is significantly lower in high
producing CHO clones
Identification of new targets to improve productivity
Project
Low
producer
High
producer
Project
A
6
clones
6
clones
Project
B
6
clones
6
clones
Project
C
5
clones
5
clones
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

13. ! Based on orthologous data it could be predicted that these 5
genes are all located next to each other on a telomeric region
! This could be confirmed by hybridisation of a variety of BAC
clones encoding this region
(KF Wlaschin and WS Hu, Biotech and Bioeng, 2007)
All 5 down regulated genes are located at the same
chromosomal region
Telomeric
region
Karyogram analysis
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

14. Generation of parental CHO cells clones lacking
this telomeric region
single
cell
cloning
PCR
screen
of
gDNA
CHO
cell
pools
561
clones
grew
3
clones
iden4fied
Manipulated
CHO
cell
pools
manipula;on
steps
| 9th Annual BioInnovation Leaders Summit, Berlin,
February 2016 | Justyna Jozefczuk

15. Novartis next generation cell line development platform
Combining the enhanced host cell line with the folate receptor vector
CHO-­‐3
host
cell
line
Folate
Receptor
Vector
+
higher pool titer higher average clone titer higher clonal stability
Faster timelines with less clone screening
pNVS
vector
LC
HC
amp
DHFR
prom
intron
polyA
prom
intron
polyAprom
polyA
prom
polyA
hu FOLRa ORF
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

16. Easier material generation with higher producing pools
Faster and more!
+ and
20
days
34
days
From transfection
48
days
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
pNVS
vector
LC
HC
amp
DHFR
prom
intron
polyA
prom
intron
polyAprom
polyA
prom
polyA
hu FOLRa ORF

17. Evaluation combination of novel cell clone CHO-3 and
folate receptor vector approach for 4 projects
Evaluation of cell clone CHO-3 plus folate receptor selection verus
CHO plus G418/MTX selection
! 3.5 fold pool titer increase (average of fedbatch cultures (wave))
! 30% clone titer increase (average top 12 clones (fedbatch))
! 30% clone titer increase (top 3 clones in bioreactor)
Enabling for a 5 months reduction of the timeline from start of
antibody generation to IND
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk17

18. Clonal stability statistics
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Reference vs enhanced cell line platform
Post
implementation
projects
Total
%
Reference
benchmark
reduced folate
no MTX
reduced folate
no MTX
new platform new platform old platform
18

19. Development and implementation of new technologies
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk19
Folic acid
receptor
selection system
Enchanced
CHO host cell
line
Evaluation of
additional new
technologies
Improved cell line development
platform
Monoclonality
assessment

20. Evaluation of optimized UTRs/signal peptides
(Collaboration with UniTargetingResearch)
" Internal standard vector modified with UTR®Tech modules
" Standard and enhanced internal vectors as controls (Ctrl. S/E)
5’UTR
5’UTR
3’UTR
SP coding region
LC
3’UTR
HC
poly(A) site
poly(A) site
5’ module 3’ module
SP coding region
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

21. Screening of optimized UTRs and signal peptides
Combinatorial approach utilizing UTR®Betatech and Novartis internal elements
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Combinatorial mini-library approach to identify best combinations of
elements for mAb heavy and light chains
Elements used: UTR®Betatech and Novartis internal
5 LC libraries
5 HC libraries
360 combinations in total

22. Combining the best elements for LC and HC in tandem vectors
Combinatorial approach utilizing UTR®Betatech and Novartis internal elements
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
35 combinations
of LC and HC
cassettes

25. Development and implementation of new technologies
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk25
Folic acid
receptor
selection system
Enchanced
CHO host cell
line
Evaluation of
additional new
technologies
Improved cell line development
platform
Monoclonality
assessment

26. Visualizing “monoclonality”before sorting...
Cytena: Cy-Clone Single Cell Printer
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk26
! An “inkjet printer” for printing cells...
! Single-use cartridge to prevent cross-
contamination
! Cartridge filled manually with cell suspension
http://www.cytena.com/home.html
http://www.cytena.com/home.html

27. picture
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk27
one

28. picture
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk28
two

29. picture
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk29
three

30. picture
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk30
four

31. picture
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk31
goal...
Droplet volume:
150pl
and no room for
interpretation...

32. Monoclonality check
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk32
Example for seeded plate (CHO cell line)
in
silico
Predic4on
1
2
3
4
5
6
7
8
9
10
11
12
A
1
1
1
1
1
1
1
1
1
1
1
1
B
1
1
1
1
1
1
1
1
1
1
0
1
C
1
1
1
0
1
0
0
1
1
1
1
1
D
1
1
1
1
1
1
1
1
1
1
1
1
E
1
1
1
1
1
1
1
1
1
1
1
1
F
1
1
1
1
1
1
1
1
1
1
1
1
G
1
1
1
1
1
1
1
1
1
0
1
1
H
1
1
1
1
1
1
1
1
1
1
1
1
Readout
Cellavista
1
2
3
4
5
6
7
8
9
10
11
12
A
1
1
1
1
1
1
1
1
1
1
1
1
B
1
1
1
1
1
1
1
1
1
1
0
1
C
1
1
1
0
1
0
0
1
1
1
1
1
D
1
1
1
1
1
1
1
1
1
1
1
1
E
1
1
1
1
1
1
1
1
1
1
1
1
F
1
1
1
1
1
1
1
1
1
1
1
1
G
1
1
1
1
1
1
1
1
1
0
1
1
H
1
1
1
1
1
1
1
1
1
1
1
1
Limitation of imaging device:
6 wells pictures are not fully conclusive
No
cell
5
5%
Not
certain
0
0%
More
than
one
cell
0
0%
Single
Cell
91
95%

33. Cloning Efficiency
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk33
NVS production cell line
Cell line Plate 1 Plate 2 Plate 3 Average
Cytena [%]
Benchmark FACS [%]
(expectation)
CHO 70.3 67.6 77.8 71.9 25 to 50
• Cloning efficiency improved compared to FACS

34. Summary
! A novel selection marker has been established, which outperforms classical
selection systems
! Transcriptomics helped to identify target genes for higher productivity
! Combination of novel vector technologies and novel cell clone shows higher
pool and clonal productivity as well as shorter selection time and higher
clonal production stability
! Upgrade of cell line platform led to significant savings and performance
increase
! Further fine-tuning by evaluating additional technologies e.g UTR ® Beta/ ®
Tailortech is ongoing
! Cytena® in combination with an imager gives full traceability to proof
monoclonality to the health authorities (Picture of cell available before
seeding and after seeding in 96well plate) and higher cloning efficiency in
comparison to FACS
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk34

35. | 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Acknowledgements
! Thomas Jostock, Holger Laux, Annet Ritter
! Juergen Recktenwald, Rolf Koehler, Cathy Boscato, Helene
Lindecker, Rene Faller
! Delia Drewello, Sabine Lang, Fabienne Brogli, Sandra Haas,
Sven Dennler, Marco Brüstle, Johannes Wichter, Andrea Blötz,
Manuela Ortlepp, Mona Woerdehoff
! Sheri Nidositko, Zorica Dragic, Audrey Nommay, Sebastian
Schmidt, Corinne Ueberschlag
! Burkhard Wilms, Hans-Peter Knopf, Beat Gysin, Bernhard Helk
! External collaboration partners
• Yehuda Assaraf, Stavit Drori (Technion, Haifa, Israel)
• Beate Stern, Asta Optun, Melanie Liesenfeld (UniTargetingResearch)

36. Questions?
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

37. Backup
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

38. 0
50
100
150
200
250
300
350
400
%titermodelmAb
Average of Top 5 Clones
Shake flask batch culture (13-14 days)
NVS dhfr
NVS dhfr FACS
NVS dhfr-FACSvector
NVS enh SP
UTR®Tech
NVS FolR
NVS dhfr/folR
NVS dhfr/folR/FACSvector
Vector optimisation is a continuous process
Impact of vector technologies on platform performance
Combining new vector technologies can significantly increase performance
Evaluation of further combinations of best performing tools is ongoing
Clone picking
UTR/secretion
optimisation
Selection
Combination
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

39. Selecting the best clone
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk39
The cloning bottleneck
Cell
Pool:
highly
heterogeneous
popula4on
Vector
Parental
Cells
limita4on
one
FTE
=
x
cloning
projects
/year
task
-­‐
underline
monoclonality
-­‐
single
cell
to
colony
-­‐
maintenance
of
colonies
-­‐
select
best
30
clones
FACS:
selec4ve
cloning
maintain
diversity
3
pools
selected
for
cloning
Transfec4on
6
-­‐
8
AUTOMATION

40. Simple, boring and repetitive?
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk40
Get the right help
STARplus
(Hamilton)
C24
(Thermo)
Cellavista
(Innova4s)
SWAP
(Hamilton)
STX44
(Liconic)
Source:
HAMILTON
Robo4cs
GmbH

41. Cell maintenance
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk41
Keep them in good conditions
Day
15
Split
Day
12
No
Ac4on
Day
7
No
Ac4on
Day
0
No
Ac4on
Images taken with Cellavista:
- used to make well based decisions

42. Monoclonality
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk42
Monoclonality, a “construction area”
Evidence of monoclonality: FACS cloning
Automated Inspection
Colony count on images taken on day 7
For TOP 30 clones
Visual Inspection of images taken on day 0
Dedicated system to support
monoclonality
- Linear track incubator
- High resolution Imager
- Exclude non-monoclonal colonies
- Proceed with monoclonal colonies
Source:
HAMILTON
Robo4cs
GmbH

43. Selection stringency can be adjusted according to
needs of different applications (time vs titer)
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Increasing
selec;on
stringeny
prolongs
recovery
;me
Increasing
selec;on
stringeny
increases
pool
;ter
Increasing
selec;on
stringeny
increases
abundance
of
high
producers
Low
folate
Low
folate
+
1
nM
MTX
Low
folate
+
5
nM
MTX
Low
folate
+
10
nM
MTX
Low
folate
+
50nM
MTX
Lowfolate
Lowfolate

44. mAb
producing cell
Y
Y
Y
Y
FACS vectors for selective cloning of high producing cells
Translational Readthrough Approach: Principle
VH CH1 CH2 CH3 M1,2 pAProm
Vector/DNA
Transcription
Splicing + Translation
Protein
(2 heavy chain varaiants)
VH CH1 CH2 CH3
>=95% ?
VH CH1 CH2 CH3 M1M2
<=5% ?
Cell surface bound
mAb variant
Soluble mAb
variant
Processing + mAb assembly
mAb Heavy Chain
“leaky Stop codon”
mRNA
VH CH1 CH2 CH3 M1,2
AAAAAAAAA
“leaky Stop codon”
Y
FITC
Y
FITC
Y
Y
Y
Y
Y
Y
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

45. | 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Algorithm-generated signal peptide libraries based on best
combinations (UTR®Tailortech)

46. | 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
Evaluation of the rationally randomized signal peptide libraries
Pool productivity
Pool productivities of 2 different antibodies
(2 independent transfection experiments)
46

47. Combining Folic Acid Receptor and DHFR/MTX
Limiting Dilution Clones:Dhfr/FolR vs Dhfr Reference
Shake flask fed batch mAb1: Comparison of Top5-Clones
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
1 2 3 4 5
Clones
mAb(g/L)
dhfr/folR d16
dhfr Ref d17
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

48. Folic Acid Receptor: A new metabolic selection marker
Comparison of clonal performance with reference system
Fed batch shake flask model
0.00
0.50
1.00
1.50
2.00
2.50
1 2 3 4 5
Top 5 clones
mAb(g/L)
Traditional
New marker
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk

49. Benchmarking of combination vectors
Evolution of clonal performance
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk49

50. Mab 1 variants case study: Pool productivity
| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk
mAb1
mAb1 variant 1 mAb1 variant 2 mAb1 variant 3
50