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Tertiary Screening (FedBatch)

Stability Study

Thawing of ExpiCHO-S Host

Cell Line

Stable Transfection

Selection I

Selection II

Limited Dilution Cloning (LDC)

Clone Scale up & Primary and

secondary Screening

Shreya M. Lowmaster, Joaquín G. Canay, Mary E. Reynolds, and Andrew M. Campbell, Thermo Fisher Scientific, 3175 Staley Road,

Grand Island, NY, United States, 14072

Accelerating Bio-Production Using the New ExpiCHO™ Stable Production Medium

RESULTS

Figure 4. Selection Phases:Once cells are transfected and statically incubated for two days to

recover, they underwent two different selection phases based on

pressures previously determined by a kill curve. In each phase, cells are

passaged every 3 or 4 days at 2 and 3 x105 cell/mL respectively. The

passing criteria were as follows:

Selection I: 85% Viability and 1 x 106 cells/mL

Selection II: 90% Viability and 2 x 106 cells/mL

Figure 5. Stability Work Flow:After thawing the top 15 clones, selected during the screenings, the clones

were sub-cultured for 60 generations and passaged at a seeding density of

2 x105 cells/ml twice a week.

Each week, a seven day productivity assessment was set up for each clone

in a different shake flask. These assessments were seeded at 2 x105

cells/ml, fed 5g/L glucose on day 3 or 4, and viability, VCD and productivity

were measured on day 7. This was done throughout the entire study.

Figure 6. 14-day ambr15 Study:The same stably transfected clone was run in all conditions. AGT,

liquid conditions (n=12), and competitor media (n=3).

ECSPM showed to be consistent between formats and surpassing

the competitors’ media in both titer (A) and growth (B).

Figure 7. Direct Adaptation to ECSPM:Clones developed as previously described were passaged every 3

to 4 days into ECEM (red), ECSPM (blue), and two competitors

(grey). Cells were seeded at 2 x105 cell/mL in shake flasks.

ECSPM showed that no adaptation was required and obtained a

comparable growth with the control (ECEM). Cell growth was also

greater than the competitor media.

Figure 8. 14-day 50L SUB Run:A 50L SUB run was performed with the previously described

ExpiCHO-S clone. ECSPM supported scalability and high titers (A)

without a need for adaptation as shown in the growth profile (B). The

clone was frozen in ECEM with only 3 passages required prior to

inoculation.

ABSTRACT

Many research laboratories use the ExpiCHO™ Expression

System Kit to produce candidate molecules for further study.

However, after the therapeutic protein of choice is found, a more

stable, scalable, and consistent method of expressing the

protein of interest is needed. Thermo Fisher Scientific has

recently developed tools to help researchers that are interested

in generating ExpiCHO-derived stable clones capable of

producing high titers and scaling up into a large bio-production

process. The high costs and time involved to create and adapt

stable cell lines into a suitable production medium can be

substantial. ExpiCHO Stable Production Medium (ECSPM)

provides a seamless transition from the transient process for

ExpiCHO stable clones, requiring minimal to no cell adaptation.

We have produced stable cell lines that have shown up to 3.5

fold increase in titer from our previously generated CHO-S™

clones. With our new ECSPM, Thermo Fisher Scientific has

provided a clear path towards creating higher producing cell

lines while also supporting the scalability, time savings, and

product quality attributes in future cell line development.

INTRODUCTION

The transient ExpiCHO Expression System Kit offers a research

system to enable transient expression and produce high titers

of a protein of choice using the high density ExpiCHO-S™ host

cell line. When a specific protein of choice is selected, the need

to generate a stable clone arises. Once this clone is identified,

the next step is to move expediently into large bioreactors. To fill

this gap, ECSPM was created to simplify the process of scaling

up. ECSPM is offered in liquid and also in Advanced Granulation

Technology (AGT™) format to facilitate large scale

bioproduction while at the same time permitting high titers and

ease of use. The medium is formulated without hypoxanthine

and thymidine for use in dihydrofolate reductase (DHFR)-

amplified systems, without L-glutamine or GlutaMax™ for use in

glutamine synthetase systems, and without phenol red in order

to minimize the estrogen-like effects of phenol red. It is not

compatible for use as a medium during the transfection and

selection stages.

Using ExpiCHO Expression System Kit and host ExpiCHO-S

cells, high producing stable clones were generated. Cells were

passaged into ECSPM to run in bioreactors allowing production

over 3g/L of immunoglobulin G (IgG) titers.

MATERIALS AND METHODS

Cell Line Development:

Stable clones were generated following the work flow illustrated

in Figure 2. Using a proprietary ExpiCHO-S host cell line and the

ExpiCHO Kit. Cells were transfected with a vector containing a

human IgG. Two days after transfection the pool went through

two selection phases as shown in Figure 4. Selection I

pressures were low, while selection II pressures were 2-5 times

higher than selection I.

Limited dilution cloning (LDC) was performed as outlined in

Figure 3. Note: The primary five day assessment was set up in

shaking 6-well plates, seeded at 3 ×105 viable cells/mL in 3 mL

of ExpiCHO Expression Medium (ECEM) plus 3g/L glucose.

Incubated for 5 days before sampling for productivity. A total of

35 clones, all measuring over 100mg/L of IgG were Cryo-

banked.

A secondary (glucose only) and a tertiary (fed-batch) shake flask

assessments were performed after thawing the top 8 clones

based on the 1st assessment (not shown). Followed by a

stability study of the 15 top clones as shown in Figure 5.

Micro bioreactor 15 (ambr®15) Study:

An ExpiCHO-S clone was used. ECSPM, in liquid and AGT

formats, and two competitor media were used. The conditions of

the run were as follows: glucose fed when < 3g/L up to 6g/L,

37ºC, 50% DO, 7pH, 1000 rpm. Feed conditions were bolus fed

and followed manufacturer specifications. ECSPM were fed a

2% daily volume of 2x EfficientFeed™ C+ (EFC+) from day 3 to

12.

Thermo Scientific HyPerforma Single Use Bioreactor (SUB):

The ExpiCHO-S clone was seeded at 3 x105 cells/mL in ECSPM

liquid format. The conditions of the run were as follows: glucose

was continuously fed to maintain 4g/L, 37ºC, 26% DO, pH 7,

172 rpm and bolus fed 28% of final working volume of EFC+

(2x) from days 3 to 16.

GlycanAssure™ APTS Kit:

ExpiCHO-S transient (standard protocol) and ExpiCHO-S stable

samples were both harvested on day 14.

CONCLUSIONS

ExpiCHO Stable Production Medium allows a seamless

transition from ExpiCHO Expression Medium and supports

large scale bioproduction runs with minimal to no cell

adaptation with high producing stable clones. ECSPM shows

consistent growth and titers between formats and surpasses

competitors’ media. Product quality attributes also show

consistency between transient and stable clones. In

conclusion, ECSPM provides a scalable solution for stable

protein production.

The new cGMP ExpiCHO-S cells will be available September

2018 for purchase. ECSPM is available in both liquid and AGT

formats to suit all our customers’ needs.

ACKNOWLEDGEMENTS

We would like to thank the BID ExpiCHO Expression System

Kit team, Bio Production Cell Culture (BPD) analytical team,

Gino Stolfa, BPD ExpiCHO SPM team for their continued

support.

TRADEMARKS/LICENSING

© 2018 Thermo Fisher Scientific Inc. All rights reserved. All

trademarks are the property of Thermo Fisher Scientific and its

subsidiaries unless otherwise specified. ambr15 is a registered

trademark of Sartorius Stedim Biotech.

For Research Use Only. Not for use in diagnostic procedures.

Passage clones twice a week

for 60 generations or 12 weeks

Thaw of top

15 clones

7-Day Productivity

Assessments

of each clone

(once per week)

Freeze clones at

middle point at

~30 generations

Identify

stable clones

Select Clones to Expand into

125mL Shake Flasks

Small Working Cell Bank

ExpiCHO-S Pool

½ Cell/Well in 96 Well Plates

Static Incubation for 12 days (on day 7 check for monoclonality)

Expand to 24 Well Plates with

2mL of Medium

Expand to 6 Well Plates with

2mL of Medium

5 Day Assesment in 6 Well

Shaking Plates

Figure 2. Clone Work Flow:Steps taken to generate a new

stable cell line. After transfection,

two selection phases were

performed to select pools via the

vector’s specific selection

markers. LDC was used to select

individual clones which were

scaled up, and screened for viable

cell density, viability, and

productivity in both a glucose only

and fed batch process. A stability

study of the top clones were

performed to determine the four

most stable clones for future use.

Figure 3. LDC Work Flow:After selection, the pool was

diluted down to ½ a cell/well and

plated into 96 well plates.

Followed by static incubation for

two weeks and the expansion of

the clones into 24 well plates, 6

well plates, and finally shake

flasks. The clones were

evaluated via a small five day

primary screening, and small cell

banks of the thirty five top

producing clones were cryo-

frozen.

Figure 1. Transient vs Stable Work Flow:An overall graphical representation for both types of transfection

workflows.

B

A

B

A

Figure 9. Glycosylation Profiles:Transient titers in shake flasks and stable clone titers in a 50L Thermo

Scientific™ HyPerforma™ single-use bioreactor were greater than 3.0

g/L in a fed-batch process with comparable product quality.