CHO cell line engineering

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Ambrose Alexander
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1 CHO cell line engineering Protein quantification on the Octet RED96 P4EU Vienna, Stefan Kol

CHO Core Group Generate the engineered CHO cell lines

engineered cell lines Growth rates, morphology,

glycomics Expression and purification of model proteins

Analysis of model proteins Glycosylation, activity,

2 CHO Core Group Generate the engineered CHO cell lines Design Knock-In/Knock-out constructs Analysis of the engineered cell lines Growth rates, morphology, apoptosis, transcriptomics, metabolomics, proteomics, glycomics Expression and purification of model proteins Erythropoietin, Rituximab, Enbrel, C1-inh, α1-at Analysis of model proteins Glycosylation, activity, titer, other PTM s. 5 DTU The Novo Nordisk Foundation Center for Biosustainability,

3 Biolayer Interferometry Monitoring the interference pattern vs. time provides kinetic data on molecular interactions nm shift Time 6 DTU The Novo Nordisk Foundation Center for Biosustainability,

4 Quantification by Biolayer Interferometry 7 DTU The Novo Nordisk Foundation Center for Biosustainability,

The proteins EPO - controls red blood cell

change in optical thickness close to the

inhibits a wide variety of proteases 8 DTU

5 The proteins EPO - controls red blood cell production As the Octet measures the change in optical thickness close to the surface of a biosensor tip, the use of a V H H based affinity ligand for capture should lead to higher sensitivity AAT - inhibits a wide variety of proteases 8 DTU The Novo Nordisk Foundation Center for Biosustainability,

6 EPO Quantification Specificity and ph 9 DTU The Novo Nordisk Foundation Center for Biosustainability,

7 EPO Quantification Sensitivity 10 DTU The Novo Nordisk Foundation Center for Biosustainability,

8 EPO Quantification Reduce unspecific binding in CHO SN 11 DTU The Novo Nordisk Foundation Center for Biosustainability,

9 EPO Quantification Regeneration 12 DTU The Novo Nordisk Foundation Center for Biosustainability,

10 EPO Quantification BLI vs ELISA 13 DTU The Novo Nordisk Foundation Center for Biosustainability,

625 ug/ml) 6: CHO S supernatant 7: CHO S supernatant + AAT (40, 20, 10, 5, 2.5, 1.25, 0.

11 + 500 mm NaCl AAT Quantification Set up 1: PBS 2: PBS + 5 ug/ml AAT V H H 3: Quench 1 ug/ml biocytin 4: PBS 5: PBS + AAT (40, 20, 10, 5, 2.5, 1.25, ug/ml) 6: CHO S supernatant 7: CHO S supernatant + AAT (40, 20, 10, 5, 2.5, 1.25, ug/ml) 8: CHO S transient transfected with AAT (A8, B8: undiluted; C8, D8: 2x diluted; E8, F8: 4x diluted) 11: 2 M MgCl 2 12: PBS 14 DTU The Novo Nordisk Foundation Center for Biosustainability,

12 AAT Quantification Calibration Plate Sensor Sample Sample ID Binding Rate Know n Conc Well Conc. Dilution FactoCalc Conc. R2 1 A1 A8 CHO AAT sample N/A B1 B8 CHO AAT sample N/A C1 C8 CHO AAT sample N/A D1 D8 CHO AAT sample N/A E1 E8 CHO AAT sample N/A F1 F8 CHO AAT sample N/A DTU The Novo Nordisk Foundation Center for Biosustainability,

13 AAT Quantification BLI vs ELISA 16 DTU The Novo Nordisk Foundation Center for Biosustainability,

Conclusions and outlook EPO assay on Octet is robust and quick Compares well with ELISA AAT assay seems quite easy to set up But does not compare well with

14 Conclusions and outlook EPO assay on Octet is robust and quick Compares well with ELISA AAT assay seems quite easy to set up But does not compare well with ELISA Publish EPO quant paper Look into AAT problems (HPLC quant?) C1-inh V H H is coming up 17 DTU The Novo Nordisk Foundation Center for Biosustainability,

Acknowledgments CHO Core group Bjørn Voldborg

BAC BV (part of Thermo) Pim Hermans Simon Adema

15 Acknowledgments CHO Core group Bjørn Voldborg (Project leader) Sara Petersen Bjørn (Mol Bio) Johnny Arnsdorf (Cell Biologist) Helle Munck Petersen (Tech) CHO scientific Thomas Kallehauge BAC BV (part of Thermo) Pim Hermans Simon Adema 18 DTU The Novo Nordisk Foundation Center for Biosustainability,

SUPPLEMENTAL INFORMATION

SUPPLEMENTAL INFORMATION EXPERIMENTAL PROCEDURES Tryptic digestion protection experiments - PCSK9 with Ab-3D5 (1:1 molar ratio) in 50 mm Tris, ph 8.0, 150 mm NaCl was incubated overnight at 4 o C. The