Novartis BILS 2016

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

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

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 96 101 106 111 clones

mA

b [m

g/L]


Development and implementation of new technologies

| 9th Annual BioInnovation Leaders Summit, Berlin, February 2016 | Justyna Jozefczuk 4

Folic acid receptor

selection system

Enchanced CHO host cell

line

Evaluation of additional new technologies

Improved cell line development platform

Monoclonality assessment



Folic acid receptor

selection system


line




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 f1 prom

polyA

pA

promoter FolR

vector


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

polyA prom

polyA

prom

polyA

LC

HC neo

amp

hu FOLRa ORF prom

Intron

polyA

prom Intron

polyA prom

polyA

pA

promoter

FolR vector


Clonal distribution: FR/dhfr co-selection vs G418->dhfr Limiting dilution cloning and multiwell plate screening


0 50

100 150 200 250 300 350 400 450 500

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55

mA

B [m

g/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

mA

b [m

g/L]

clone

G418/dhfr sequential selection

!  Average productivity of co-transfected/selected clones about 40x higher than reference

0 50

100 150 200 250 300 350 400

% ti

ter m

odel

mA

b

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


Combining new vector technologies can significantly increase performance

Evaluation of further combinations of best performing tools is ongoing

Clone picking

Selection

Combination



Folic acid receptor

selection system


line




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


11

! 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


! 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


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


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

polyA prom

polyA

prom

polyA

hu FOLRa ORF


Easier material generation with higher producing pools Faster and more!

+ and

20 days

34 days From transfection

48 days


pNVS vector

LC

HC amp

DHFR prom

intron polyA prom

intron

polyA prom polyA

prom polyA

hu FOLRa ORF

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


Clonal stability statistics


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



Folic acid receptor

selection system


line




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


Screening of optimized UTRs and signal peptides Combinatorial approach utilizing UTR®Betatech and Novartis internal elements


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

Combining the best elements for LC and HC in tandem vectors Combinatorial approach utilizing UTR®Betatech and Novartis internal elements


35 combinations of LC and HC cassettes



Folic acid receptor

selection system


line




Visualizing “monoclonality”before sorting... Cytena: Cy-Clone Single Cell Printer


! 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

picture


one

picture


two

picture


three

picture


four

picture


goal...

Droplet volume: 150pl

and no room for interpretation...

Monoclonality check


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%

Cloning Efficiency


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

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



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)

Questions?


Backup


0 50

100 150 200 250 300 350 400

% ti

ter m

odel

mA

b

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


Selecting the best clone


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

Tran

sfec4on

6 -‐ 8

AUTOMATION

Simple, boring and repetitive?


Get the right help

STARplus (Hamilton)

C24 (Thermo)

Cellavista (Innova4s)

SWAP (Hamilton) STX44 (Liconic)

Source: HAMILTON Robo4cs GmbH

Cell maintenance


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

Monoclonality


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

Selection stringency can be adjusted according to needs of different applications (time vs titer)


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

Low

fola

te

Low

fola

te

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 pA Prom

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



Algorithm-generated signal peptide libraries based on best combinations (UTR®Tailortech)


Evaluation of the rationally randomized signal peptide libraries

Pool productivity Pool productivities of 2 different antibodies (2 independent transfection experiments)

46

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

mA

b (g

/L)

dhfr/folR d16 dhfr Ref d17


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

mA

b (g

/L)

Traditional New marker


Benchmarking of combination vectors Evolution of clonal performance


Mab 1 variants case study: Pool productivity


mAb1

mAb1 variant 1 mAb1 variant 2 mAb1 variant 3

50

Presentations & Public Speaking

Novartis BILS 2016