leap-in transposase th & transposon for improved protein … · 2019. 9. 23. · leap-in...

Leap-In Transposase®

& Transposon for Improved Protein Production

Sowmya Balasubramanian, Ph.D.

ATUM Cell Line Development

14th PEACe conference

Newport, Rhode IslandSeptember 23, 2019

• Founded in 2003 as DNA2.0

• Organic growth, Employee owned

• ~100 employees

• >25 issued patents

• >50 peer-reviewed papers

• Services in >2,500 publications

ATUM

Classic CLD workflow is slow, tedious,uncertain and labor intensive

Stable pool

generation and rankingSingle cell cloning

and screening

RCB manufacturing

and release

6-9 weeks 9-10 weeks 4-5 weeks6-8 weeks

27-36 Weeks

Genetic and

expression stability

2-4 weeks

Vector design

and synthesis

Titer based ranking

Screen 1000s of clones

On multiple top clones

The life of a transposon-transposase pair

TTAA

Chromosomal target site

ITR ITR

Transposase gene TTAATTAA

Transposase

• 4 billion years of successful evolutionary history

• Cut-paste mechanism

• Single copy integration at each site

• Perfect integration of elements between ITR’s

Transposon

ITR ITR

Transposase gene TTAATTAA

Transposase applied to stable cell line development

TTAAGOI ORFs, regulatory elements

ITR ITR

TTAA TTAA

Chromosomal target site

TTAA

• Transient exposure to transposase = Stable insertion

• Single copy integrations at each site

• Multiple insertions (5 – 60) across the genome

• Structural integrity maintained

• No size limitation

Expression construct

Transient transposase(mRNA)

ITR ITR

GOI ORFs, regulatory elements TTAATTAA

Consistent, uniform presentation of Leap-In® transgenes

Intact constructs maintained at every integration site

VectorGPS® on Leap-In® transposon

• Multiple transcriptional units/construct

• Catalog of selectable markers

• Catalog of promoters

• Catalog of insulators

• Other regulatory elements

• Signal peptides,

• mRNA transport sequences,

• IRES etc.

Controlling ratios with construct design – 2 ORFs

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Re

lativ

e e

xp

ress

ion

Construct number

ORF 1 ORF 2

Bispecific Antibodies

chain ratio modulation

Controlling ratios with construct design – 3 ORFs

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1 2 3 4 5 6

Re

lativ

e e

xp

ress

ion

Construct number

Bispecific Antibodies

chain ratio modulation

ORF 2ORF 1 ORF 3

Leap-In® generates high expressing homogeneous pools

Random

IntegrationLeap In

Balasubramanian, S, et. al., 2018, Biotechnol. J

Intracellular Staining of stable pools

Clone-like distribution of cell pool

GFP expression in cell pools

0

2000

4000

6000

8000

10000

12000

Random Integration Leap-In

FAC

S

Me

an

Flu

ore

sce

nc

e

15x higher mean fluorescence

Leap-In® + VectorGPS® = Drug free selection

• Use vector elements to modulate stringency of selection

• Drug free selection = absence of Glutamine

• Recovery in <2 weeks

• Low impact of selection dip on pool titers

0

20

40

60

80

100

120

0 5 10 15

Via

bili

ty (

%)

Culture Duration (days)

Leap-In1

Leap-In2

Leap-In3

0

500

1000

1500

2000

2500

3000

3500

Leap-In1 Leap-In2 Leap-In3

Tite

r (m

g/L

)

Drug Free Selection Fed-batch titer

Productivity in Leap-In® generated stable pools

Pool Titers

Protein Volumetric productivity Specific productivity

IgG1 4.2 g/L 42 pcd

IgG1 4.0 g/L 44 pcd

IgG1 4.3 g/L 22 pcd

IgG1 5.9 g/L 39 pcd

IgG1 4.2 g/L 33 pcd

IgG4 5.0 g/L 43 pcd

IgG4 5.0 g/L 49 pcd

Population shift towards high producing clones

https://www.berkeleylights.com/

• 62% of clones in top quartile of expressers

• 82% of clones in top half of expressers

• 99% probability in under 200 clones

0

200

400

600

800

1000

1200

1400

1600

1800

Tite

r (m

g/L

)

Clones

62% 20% 14% 4%

Leap-In Transposase®

• High producers rare

Random Integration

(mg

/L)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

166 clones Static 96 well plate

22 clones24 deep-well scale

7 day fed batch

0

1000

2000

3000

4000

5000

6000

7000

5 10 15

Time (Day)8 clones 125 mL shake

14 day fed batch

Monoclonality

established

Lead Clone125 mL shake

14 day fed batchMedia evaluation

Leap-In® + VIPS™ = reduced clone ranking effort

Robust expression and copy number stability

Consistent genetic stability over >60 population doublings

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9

Co

py n

um

be

r/re

fere

nc

e

Clones

Copy number stability

T0 PD60-Gln PD60+GlnPD0

0

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7 8 9

Tite

r (m

g/L

)

Clones

Expression stability

T0 PD60-Gln PD60+GlnPD0

0

10

20

30

40

50

60

70

80

90

100

100% 90-100% 80-90% 70-80%

Fra

ctio

n o

f c

lon

es

(%)

Copy number at PD60 (in % of PD0 copies)

Copy number stability

0

10

20

30

40

50

60

70

80

90

100

100% 90-100% 80-90% 70-80%

Fra

ctio

n o

f c

lon

es

(%)

Productivity at PD60 (in % of PD0 productivity)


Genetic stability is not a clone ranking parameter

Genetic Stability Statistics

Structural stability of the integrated expression constructs

PD90PD0

Perfect nucleotide level stability over 90 generations

Case Study: Hard to express non-CHO

Pool titers are predictive of clone titers

0

500

1000

1500

2000

2500

3000

3500

4000

Tite

r (m

g/L

)

Project goal Pool Clones

Case study: Intensified fed-batch

65

70

75

80

85

90

95

100

0 2 4 6 8 10 12

Via

bili

ty (

%)

Culture duration (days)

Viability

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12

Tite

r (g

/L)


Productivity

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10 12

VC

D (

E+

06

/mL)


Viable Cell Density

Case study: Stable pools predict derivative clone titers

0

2

4

6

8

10

SF SF ambr250

Txn Pool Clones

Tite

r (g

/L)

Molecule 1

0

2

4

6

8

10

SF SF ambr250

Txn Pool Clones

Tite

r (g

/L)

Molecule 2

Classic CLD workflow

Stable pool

generation and ranking

Single cell cloning

and screening

RCB manufacturing

and release

Classic CLD workflow is slow, tedious,uncertain and labor intensive

6-9 weeks 9-10 weeks 4-5 weeks6-8 weeks

27-36 Weeks

Genetic and

expression stability

2-4 weeks

Vector design and

synthesis

Leap-In CLD workflow - Transfection to RCB in 12 weeks

1 – 3 weeks 4 – 6 weeks

CONSTRUCT DESIGN

CODON OPTIMIZATION

SIGNAL SEQUENCE SELECTIONGENE SYNTHESIS

MOLECULAR CLONING

6 – 9 weeks4-5 weeks

~9 weeks

TRANSFECTION/SELECTION

PRODUCTIVITY ASSESSMENT

PRODUCT QUALITY

ASSESSMENT

MONOCLONALITY

VIA IMAGING

VIABILITY AT THAW

STERILITY, MYCOPLASMA

60 PD GENETIC STABILITY

Gene synthesis & vector construction

Stable pool generation & characterization

Cell line cloning and ranking

RCB manufacturing and testing

RCB

Representative pool Clones available

Rapid Timelines• Efficient and robust integration = Predictable selection• From transfection to RCB in ~12 weeks• Predictive stable pools

High Titer• Leveraging > decade of ATUM proprietary vector elements and algorithms• Highly uniform cell pools up to 5+g/L and clones in excess of 14g/L

Robust Stability• Transposase mechanism provides very high genetic stability• No loss in productivity or transgene copy numbers after 60+ doublings

Enabling for Next Generation Biologics• Compatible with very large inserts (e.g. >100kb)

• Able to co-express multiple genes and tune ratios• Multiple transposases enable unique genetic engineering strategies

Summary of the Leap-In Transposase® Platform

Cell engineering using Leap-In®

Improving product quality through cell engineering

0

1

2

3

4

5

6

7

Parent clone Parent clone + media

additives

Leap-In engineered

clone

Leap-In engineered

clone + media additives

Fold

inc

rea

se in

att

ribu

te 5.5X

6.5X

Complex large molecules

help with folding,

glycosylation and secretion

Biosimilars

specific product quality attributes

Cell Pools for speeding timeline

Low titer


Pool product quality ≠ Clone product quality

Cell pools – Risks Cell pools – Advantages

Shorter timelines

Reduced cost

Cell Line Development off critical path

Clone like expression titer


Comparable product quality to derivative clones

Cell pool – Requirements

Leap-In® pools: High productivity

Pool Titers

Protein Volumetric productivity Specific productivity

IgG1 4.2 g/L 42 pcd

IgG1 4.0 g/L 44 pcd

IgG1 4.3 g/L 22 pcd

IgG1 5.9 g/L 39 pcd

IgG1 4.2 g/L 33 pcd

IgG4 5.0 g/L 43 pcd

IgG4 5.0 g/L 49 pcd

Leap-In® pools: Predict derivative clone titers

Pool titers predictive of clone titers

0

1000

2000

3000

4000

5000


Tite

r (m

g/L

)

Pool

Clone1

Clone2

Clone3

0

1000

2000

3000

4000

Tite

r (m

g/L

)

Pool Clones

0

2

4

6

8

10

SF SF ambr250

Txn Pool Clones

Tite

r (g

/L)

0

2

4

6

8

10

SF SF ambr250

Txn Pool ClonesTi

ter

(g/L

)

Leap-In® pools: Stable expression

0

500

1000

1500

2000

2500

3000

3500


Tite

r (m

g/L

)

PD0

PD30

24.5%

20.5%

17.8%

• ~80% productivity maintained

• Acceptable criteria for expression stability

Leap-In® pools: Predict derivative clones product quality

Comparable product quality of pools and clones

0

5

10

15

20

25

30

35

40

45

A2G0 A2G0F-N A2G0F A2G1F A1G1F-N A2G1 A2G2F Man3 Man5

Gly

co

form

(%)

Pool Clone1 Clone2 Clone3

0

10

20

30

40

50

60

70

80

Basic Main Acidic

Ch

arg

e v

aria

nt

(%)

Pool Clone1 Clone2 Clone3

Glycan abundance Charge

Leap-In® pools: : Product quality stability

Consistent product quality of stable pools

0

20

40

60

80

100

120

PD0 PD30 PD0 PD30 PD0 PD30

Low Medium-1 Medium-2

Gly

co

form

(%

)

A2G2F

Man5

A2G1

A1G1F-N

A1G1-N

Man3

Man3-F

A2G1F

A2G0F-N

A2G0F

A2G0

Glycan abundance


0

20

40

60

80

100

120

PD0 PD30 PD0 PD30 PD0 PD30

Low Medium-1 Medium-2%

Ch

arg

e V

aria

nt

Acidic

Main

Basic

Charge


Cell Pools speeding timeline to IND

Low titer


Pool product quality ≠ Clone product quality

Cell pools – Risks Cell pools – Advantages

Shorter timelines

Reduced cost

Cell Line Development off critical path

Cell pools – Requirements

Clone like expression titer


Comparable product quality to derivative clones

Leap-In® pool ranking more critical than clone ranking

Applications for cell pools

Screen vector constructs

Screen sequence variants

Process development

Media optimizations

Cell engineering

Purification method development

Analytical and formulation development

Generating material for IND enabling tox

Generating Ph. I lot

• The Leap-In Transposase family is expanding

• Engineered CHO host cell lines

• Optimization of alternative host cell lines

• Cell and gene therapy modalities

Ongoing leap-In platform innovations

Thank You

Sowmya Balasubramanian

[email protected]

CLD Partners

SolentimVIPS™ clonality verification

Horizon DiscoveryGS null CHO K1 cell line

Technology presented is protected by

issued US patents 10287590, 10253321,

10233454, 10041077, 9771402, 9580697,9574209, 9534234, 9493521, 9428767,

9290552, 9206433, 9102944, 8975042,8825411, 8635029, 8412461, 8401798,

8323930, 8158391, 8126653, 8005620,

7805252, 7561973, 7561972 andpending applications

leap-in transposase th & transposon for improved protein … · 2019. 9. 23. · leap-in...

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