Bjorn Voldborg Technical University of Denmark

1. DTU Bioengineering
15 September 2022
WHICH SUGAR DO YOU WANT?
ENGINEERING CHO CELLS FOR THE
PRODUCTION OF RECOMBINANT
PROTEINS
Bjørn Voldborg
Head of The National Biologics Facility
Technical University of Denmark

2. DTU Bioengineering
15 September 2022
The DTU CHO Program:
Control the protein production
Quantity Quality Time
Controlled PTMs
(Safety and Efficacy)
- Proteolysis
- Glycosylation
- Charge variants
- Disulfides
- Etc
Purity
(Safety and Cost)
- Immunogenicity
- Degradation
- Purification loss
- Contamination
High Titers
(Cost)
- Transgene expression
- Translation
- Transport
- Effective folding
- Cell viability, density, longevity
Stable expression
- Stable yield
- Genome
stability
- Selective
sweeps
Development time
- Targeted
integration
- Predictable yield

3. DTU Bioengineering
15 September 2022
CHO Cells as engineerable entities

4. DTU Bioengineering
15 September 2022
Platform Cell Lines for controlled
production of biopharmaceuticals
• Fully Documentated (LIMS)
• CHO-S based (GMP “ready”).
• Engineered:
– GS selection (CLD)
– Quality (Glycosylation & Bioprocess)
– Zero Lactate (CHOZela) (Bioprocess)
– Glyco-engineered (geCHO) (Glycosylation)
– HCP reduced (cleanCHO) (Downstream)
– Targeted Integration (CLD & Bioprocess)
https://nbf.dtu.dk/

5. DTU Bioengineering
15 September 2022
Glyco-engineered geCHO Cell Lines for accurate control of
glycosylation of drug candidates
”geCHO panel”

6. DTU Bioengineering
15 September 2022
Glyco-optimize your glycoprotein for optimal efficacy:
Vaccines, mAbs, Enzymes, etc…
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Go to clinic with a sub-optimal product Go to clinic with optimal product

7. DTU Bioengineering
15 September 2022
Glycans specific to the producing cells:
Liver-secreted Plasma Proteins
• Glycoprofile of naturally occurring proteins
depends on the expressing cells/organ.
• Proteins secreted by the liver tends to be
bi-antennary non-fucosylated glycosylated.
• Can be critical to:
–Function
–Stability
–Specificity
–Immunogenicity
–..
Clerc F. et al: https://pubmed.ncbi.nlm.nih.gov/26555091/ PMID: 26555091

8. DTU Bioengineering
15 September 2022
Alpha1 Antitrypsin (AAT)
• Alpha-1 Antitrypsin Deficiency (Alpha-1) is a
genetic (inherited) condition
• Symptoms:
– Shortness of breath, Wheezing, Chronic
bronchitis, Recurring chest colds, Less
exercise tolerance, Year-round allergies,
Bronchiectasis, Liver failure
• Treatment:
– Augmentation therapy, lifelong weekly
infusion, doses from 4g, often increasing
over time.
• Approved products are all derived from
fractionated human plasma.
• There is currently no recombinant product on
the market.

9. DTU Bioengineering
15 September 2022
AAT from CHO vs Plasma derived
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AAT expressed in CHO-S AAT purified from human plasma

10. DTU Bioengineering
15 September 2022
plAAT (CfB)
rAAT – wt CHO (CfB)
AAT Glyco-Analysis
• Glyco-engineered CHO:
– KO of 9 Glycosyltransferases
– KI of 1 Sialyltransferase
• Glycoprofiles of AAT:
– AAT from CFB CHO is very
close to identical to AAT
purified from human plasma
and the AAT commercial drug.
(fractionated from human
plasma)
GlycoKO rAAT (CfB)
Commercial Drug

11. DTU Bioengineering
15 September 2022
Advantages of recombinant AAT over plasma
fractionated AAT
• Optimised variants:
– Glycovariants:
• Alternative glycosylation
• Extra glycosylation sites for e.g. improved ½-life
– Mutations:
• Stabilizing
• Changing Affinity
– Low batch-to-batch variation
– Secured Supply
– Formulation
– Concentration
– Protein-Fusions
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12. DTU Bioengineering
15 September 2022
Glycans on virus proteins
• Most virus proteins are glycosylated, dependent on how
the specific virus protein is processed by the host cell
during infection.
• Glycosylation has significant impact on the function of viral
proteins (and as such the virus)
• Glycoprofiles of virus from patients is very hard to get.
Li et al. https://doi.org/10.3389/fimmu.2021.638573

13. DTU Bioengineering
15 September 2022
Glycans on anti viral vaccines
H.-Y. Huang et al., Sci. Transl. Med.
10.1126/scitranslmed.abm0899 (2022).
Multiple SARS-CoV-2
spike protein glyco
variants could be
produced using geCHO
cells
Vaccination with SARS-
CoV-2 spike protein
lacking glycan shields
elicits enhanced
protective responses
in animal models

14. DTU Bioengineering
15 September 2022
Precision engineering of Vaccine candidate
Solving glycan shielding for antibody binding and neutralization
Neutralization
Antigenicity Comparison
Optimal glycans enhance immune response
hAb-1 hAb-2

15. DTU Bioengineering
15 September 2022
geCHO can go all the way.
• Transient Gene Expression (TGE) of vaccine candidate(s) in
relevant geCHO cells
• Purification
• Screening against patient derived Ab pools
• Larger scale production of promising candidates
• Neutralization test in animals.
• Cell Line development of monoclonal geCHO cell line producing
top candidate(s)
• Transfer to GMP
• Clinical trials
• Manufacturing for market
• Launch.
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16. DTU Bioengineering
15 September 2022
Targeted gene integration
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1.3% differentially expressed genes (160)
Transcriptome
Cell growth
Adj. p value <0.05, no LFC cut-off
Daria Sergeeva Nuša Pristovšek

17. DTU Bioengineering
15 September 2022
Dual site targeted insertion cell line
Daria Sergeeva Nuša Pristovšek

18. DTU Bioengineering
15 September 2022 18
Summary
Research and data driven toolbox for fast and
controlled development of optimized therapeutic
products and improved CHO cell lines, resulting in:
• Better protein based drugs
• Proteins that can not be made in CHO today
• Proteins with designed glycoprofiles.
• Faster from bench to pharmacy
• Cheaper development
• Cheaper production

19. DTU Bioengineering
15 September 2022
New Service: The Cell Line and Protein
Production Facility (CLPPF)
• Servicing academics and industrial partners in a strategic alliance with Bioneer
– Provide access to recombinant protein production
– Provide access to a capacity to develop pre-clinical grade production lines in 2-3
months, achieving g/L titres and well-defined protein properties.
– Access to a validated Master Cell Bank
– Consulting on Protein Development
• Access to Engineered cell lines:
– Developing an OpenCHO platform with MAD7, GS selection, etc
– Access to all the engineered cell lines from the CHO Program (under license with DTU)
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20. DTU Bioengineering
15 September 2022
DRUG DISCOVERY PRE-CLINICAL
DEVELOPMENT
CLINICAL TRIALS APPROVAL
MOLECULAR
CONSTRUCTION
CELL LINE
GENERATION
PROTEIN
EXPRESSION
PROTEIN
PURIFICATION
PROTEIN
CHARACTERIZATION
THE CELL LINE AND PROTEIN PRODUCTION FACILITY
Biopharmaceutical Drug Development pipeline
We are your partner from early drug discovery to pre-clinical development
https://nbf.dtu.dk/

21. DTU Bioengineering
15 September 2022
• Experts in in Molecular Construction, Cell Line generation, Protein Production and
Characterization for Drug Discovery and Preclinical phases
• We deliver everything from Expression Constructs over Purified Proteins to Production
Cell Lines that are ready to move to a CMO for GMP validation and GMP production
MOLECULAR
CONSTRUCTION
CELL LINE
GENERATION
PROTEIN
PURIFICATION
PROTEIN
CHARACTERIZATION
THE CELL LINE AND PROTEIN PRODUCTION FACILITY
PROTEIN
EXPRESSION
Cloning
High throughput
Protein Variants
Expression Vectors
Targeted Integration
Engineering
Stable Integration
Clonality
High producer
Characterization
Banking
Transient
Stable Pools
Producing Clones
Shake Flasks
Batch/Fedbatch
3 ml – 30 L scale
Tagged Protein
Untagged Protein
DSP Development
µg to g scale
Purity
Identity
Affinity
Interaction
Activity
Glycoprofile
https://nbf.dtu.dk/

22. DTU Bioengineering
15 September 2022
Flexible entry and exit points
MOLECULAR
CONSTRUCTION
CELL LINE
GENERATION
PROTEIN
PURIFICATION
PROTEIN
CHARACTERIZATION
THE CELL LINE AND PROTEIN PRODUCTION FACILITY
PROTEIN
EXPRESSION
PROTEIN OR DNA
SEQUENCE
EXPRESSION
CONSTRUCT
EXPRESSION
CONSTRUCT
EXPRESSION CELL
LINE
EXPRESSION
CONSTRUCT OR
CELL LINE
PROTEIN
CONTAINING
SUPERNATANT
PROTEIN
CONTAINING
SUPERNATANT
PURIFIED
PROTEIN
PURIFIED
PROTEIN
CHARACTERIZED
PROTEIN
https://nbf.dtu.dk/

23. DTU Bioengineering
15 September 2022
Competences CLPPF
• Cell Line development (From in silico DNA sequence to
production cell line):
– Random/targeted integration
– Reduced HCP contaminations
– Longer FedBatch runs
– Improved Quality
• Protein Production (From aa sequence to purified
protein):
– Transient (quick and flexible): µg to mg
– Stable Pool/Targeted integration (cheap): mg.
– Producer cell line (high yield): mg to g
• Protein Characterization: (From SDS-PAGE to activity)
– Purity
– Identity
– Glycan profile
– Activity
– Assay development
• Cell Line Engineering (Engineer the production
host):
– CRISPR/Mad7 based
– High throughput
– Targeted gene insertion
– Removal of contaminating proteins
– Tailored Glycans
• Advisory Function (use the expertise):
– Protein design
– Protein production
– Glycosylation

24. DTU Bioengineering
15 September 2022
Aknowledgements
• CLPPF Group:
– Sara Petersen Bjørn (Mol Bio)
– Johnny Arnsdorf (Cell Bio)
• Karen Kathrine Brøndum (Tech)
• Zulfiya Sukhova (Tech)
• Karoline Fremming (Tech)
– Sanne Schoffelen (Prot Chem)
• Daniel Duun (Tech)
– Anders Holmgaard Hansen (Glycans)
– Tune Wulf (Proteomics)
• CHO Program partners
– Johan Rockberg & Mathias Uhlen (KTH)
– Gyun Min Lee (KAIST)
– Bernhard Palsson (UCSD)
• NBF Partners
– Lars Nielsen (DTU)
– Bjarke Bak Christensen (DTU)
– Lise Grav (DTU)
– Tae Kwang Ha (DTU)
– Nathan Lewis (UCSD)
– Steffen Goletz (DTU)
– Andreas Laustsen (DTU)
• Bioneer
– Ryan Polito
– Peter Ravn
– Jette Asboe Lassen

25. DTU Bioengineering
15 September 2022
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