Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com

Ofira Carmi, Gad Beck; Valin Technologies LTD, Yavne, Israel

Kimberly Prohaska, Andrew Campbell, Irina Bauer; Thermo Fisher Scientific, Grand Island, NY

Gibco Freedom CHO-S for stable expression of rProteins in GMP production:

A case study

FREEDOM™ CHO-S CELL LINE DEVELOPMENT WORKFLOW SUMMARY

The Gibco™ Freedom™ CHO-S™ Kit is an easy-to-use, beginning-to-end product, for cloning

and expression of recombinant proteins in Chinese Hamster Ovary (CHO)-derived suspension

culture. Here, we report the implementation of the system to develop recombinant Fc fusion

protein-producing clones and transition of the lead clone to early stage process development in

bench-scale bioreactors. A Freedom pCHO 1.0-based vector expressing a protein of interest (a

recombinant Fc fusion protein) was used to transfect cGMP CHO-S cells to generate stable

pools. The cells were selected using multiple concentrations of methotrexate (MTX) and

puromycin. Following Phase 2 selection, the lead conditions resulted in titer ranges from 256-

339 mg/L in shake flask culture. Single cell limited dilution cloning (LDC) was conducted and

the resulting clones were screened and ranked based on specific productivity (pg/cell/day) in a

shake flask simple fed-batch (SFB) assay (glucose feed only). The top producing clone

demonstrated a specific productivity of 25.04 pg/cell/day and was selected for further fed-batch

assessment. This lead clone was studied in duplicate in a 2L working volume bench-scale

bioreactor system using Dynamis™ AGT™ production medium with the EfficientFeed™ C+

(Plus) AGT™ Supplement (reconstituted at a 2X concentration) added at a total feed volume of

35% (v/v). Results from the bioreactor runs show an average harvest titer of 2.4 g/L and an

average specific productivity of 22.3 pg/cell/day. Further process development and scale-up

studies are in progress.

GOALS

The main goals of this development study are:

High titers

Satisfactory product quality

A short timeline to clinical studies, including:

Regulatory compliance

Manufacturing friendliness

Flexibility

Cost of goods efficiency

Quality assurance

MATERIALS AND METHODS

Freedom CHO-S Kit Components

Host cells: CHO-S cGMP banked by Life Technologies in 2010

Cloning vector (Freedom pCHO 1.0; ProBioGen): single vector for expression of one or two

sub-unit proteins

Double selection: use of MTX and puromycin enhances integration of the gene(s) of

interest

Promoters are CMV-based but different enough to avoid recombination and down-

regulation of the downstream gene. We have found that this design drives higher

expression of the gene of interest.

Transfection reagents (FreeStyle™ MAX, OptiPRO™ SFM)

Cell culture medium (CD FortiCHO™ Medium)

Complete protocol

Methods

Transfection of 4 plasmids of the chimeric protein

Dual phase selection and Phase 2 pool productivity assessment

Limited dilution cloning (LDC)

Clone growth productivity assessments/feed response testing

Second round of LDC for top 4 producing clones

Direct subculture of top 4 clones into Dynamis production medium

Lead clone bioreactor assessment

CONCLUSIONS

Goals achieved

A short timeline of 27-30 weeks to lead clone identification including quality and stability

assessment

High titers without media or process optimization (2.4g/L)

Easy adaptation of top clones to a production medium (Dynamis)

Freedom CHO-S Kit summary:

1. CHO-S cGMP banked by Life Technologies in 2010

2. Off-the-shelf cells, vector, and cell culture medium (CD FortiCHO Medium)

3. Availability of a production medium (Dynamis) and matched feed (EfficientFeed C+)

4. Simple process of clone generation within 4 months

5. Fast transition to GMP production - The Freedom kits facilitate the generation of

commercially ready cell lines

6. One-time license fee with no royalty payments

TRADEMARKS/LICENSING

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