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
© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of
Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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