Historically cell line performance has been enhanced through media, feed and process optimization, primarily through trying to meet the basic nutritional requirements of the cells so that they can sustain high growth and productivity throughout the production runs.
However, the omics (genomics, transciptomics and metabolomics) era, sequencing of the CHO genome and enhancements in genome editing technologies over the past several years have enabled scientists to take a more direct route in cell line optimization through the modification of specific genes that have direct implications on cell culture performance, protein quality attributes and upstream and downstream manufacturing processes. These targets include but are not limited to genes that may be involved in cell cycle regulation, cellular metabolism, cellular transcription and translation, the secretory pathway and protein glycosylation or other post-translational modifications.
In this webinar we will discuss specific genetic modifications that have been made to CHO cell lines and how these modifications can lead to more efficient expression systems.
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Delivering More Efficient Therapeutic Protein Expression Systems Through Cell Line Engineering Applications
1. Trissa Borgschulte, PhD
Head of Cell Line Development and Engineering
Process Solutions Upstream R&D
Applications in Therapeutic Protein Expression Systems
ONLY FOR U.S. AND CANADA AUDIENCE
3. 3
Industry feedback
Webinar: Cell Line Engineering
Support the use of
concentrated feeds
Provide for reduced
analytical workload
Support clone stability
Maintain/enable favorable PQ
attributes
Reduce regulatory
compliance costs
Control/reduce metabolic
waste
Reduce product and process
related impurities
Reduce regulatory and risk
considerations
Increase process robustness
Promote high product yield
Support high growth rates
and cell densities
Scalable process
Provide batch to batch
consistency
Support single use
disposables
Downstream process friendly
Promote culture longevity
Maintain product stability
through harvest
Reduce product
heterogeneity
What makes a robust
therapeutic protein
manufacturing
process?
4. 4
Webinar: Cell Line Engineering
Cell Line Development and Engineering Strategy
Sources of Cell Line Engineering Targets
Discovery
Literature
Customer requests
Applications in Biomanufacturing
Selection mechanisms
Cell culture performance
Anti-apoptotic, metabolic engineering
Glycoengineering
Host cell proteins
Risk mitigation
Viral resistance
Targeted gene integration
Understanding
CHO Cell
Biology
Cell Line
Engineering
Media, Feed
and Process
Optimization
5. 5 Webinar: Cell Line Engineering
Trait Stacking to Improve Therapeutic Protein Manufacturing
Processes and Products
TI
Streamlined CLD Process and Enhanced
Performance
Homogeneity, stability
More Effective Biologics
Removal of immunogenic sugars, increased drug half-life
Robust CLD Process and High Performing
Recombinant Clones
Reduced time lines, high titers, increased stability
More Efficient Manufacturing Processes
Viral resistant cell lines, removal of contaminating host cell
proteins
Strong Base
Suspension adapted, CD media, bioreactor robust
Glyco-
engineering
Risk
Mitigation
CHO GS-/-
CHO K1
6. 6 Webinar: Cell Line Engineering
Genome Editing with ZFNs
Zinc Finger Nucleases
Introduce double strand breaks at sequence
specific genomic loci
Limited off-target effects
Cellular DNA Repair Mechanisms
NHEJ – imperfect repair resulting in INDELS
(gene KO)
HR – “perfect” repair by copying off of a template
(targeted integration)
Cellular
DNA
Repair
DNA
Binding
and
Cutting
7. 7 Webinar: Cell Line Engineering
Cell Line Engineering Workflow
ZFN Transfection
Activity Confirmation
(Cel 1 assay)
Single Cell Cloning
Expansion and
Further
Characterization
PCR and Seq Analysis
Phenotypic Assay
(if available)
8. 8 Webinar: Cell Line Engineering
Metabolic Selection: Benefits of a GS-/- Host Cell Line
GS-/- host cell lines lead to decreased CLD timelines and resources and
enhanced manufacturing performance
• Multiple rounds of
amplification are not
required
• Fewer clones evaluated
to identify high
performers
• Decreased CLD timelines
and resources
GS vs. DHFR
• MSX not required
• High performance in CD
processes
• Increased clone stability
• Fewer clones evaluated
to identify high
performers
• Decreased CLD timelines
and resources
GS-/- vs. GS+/+
• System saved 8 weeks
of development time
• Stable pool titers
exceeding 1g/L
• Clone titers averaging 2-
4.5 g/L
• Significant increase in
clone stability
• Fewer clones evaluated
to identify high
performers
GS-/- Industry
Feedback
9. 9 Webinar: Cell Line Engineering
CHO K1 GS-/- Cell Line Generation and Characterization
Genotype
Confirmation
•Genome sequencing
•qRT-PCR
•Western Blot
•Glutamine sensitivity
Phenotype
Characterization
•Robust growth/scalability in CD media
•Metabolic Profiling
•Transfectability
•Transient r-protein expression
•Cloning efficiencies
Performance
Validation
•Stable r-protein expression
•High pool and clone titers
•Long term stability
•Complex N-glycans
20 clones
3 clones
10. Transfection
-Gln Stable Pool Selection
(minipools)
Stable Pool Expansion and
Characterization
Single Cell Cloning of Lead Pools
Clone Expansion and
Characterization
10 Webinar: Cell Line Engineering
GS-/- Recombinant Cell Line Generation
7–9 weeks
7–9 weeks
11. 11 Webinar: Cell Line Engineering
GS-/- IgG Producing Stable Pools and Clones: Productivity Performance
Fed Batch TPP
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Stable Pools -
Fed Batch
Clones - Fed
Batch
g/L
Titer Range of Top Expressing
GS-/- Pools and Clones
12. 12 Webinar: Cell Line Engineering
GS-/- IgG Stable Clones: Stability Performance
8 out of 10 top clones maintain >70% titer over 60 generations
13. 13 Webinar: Cell Line Engineering
Glycoengineering for Enhanced Product Quality
14. 14 Webinar: Cell Line Engineering
N-Glycan Biosynthesis in CHO
Modified from: Hossler et al., (2009) Glycobiology
GGTA
15. 15 Webinar: Cell Line Engineering
Engineering a Mgat1- Cell Line for Recombinant Proteins with High
Man5 Glycoforms
Sources:
1. Betting, et al. (2009) Vaccine
2. Lam, et al. (2005) J of Immunology
Targeting applications for therapeutic
proteins
Cerezyme® targets the mannose receptor on
macrophages
Carbohydrate remodeling required
Increased efficacy of mannose receptor
targeted vaccines
• Insect cell derived antigens are more effective for
targeting mannose receptors on APCs1
• Antigens produced in Pichia Pastoris are more
potent at inducing CD4+ T cell proliferation2
X-ray crystallography
• Increased homogeneity in protein structure
16. 16 Webinar: Cell Line Engineering
Mgat1- Cell Line Engineering in a Recombinant IgG Producing CHO
Clone
Lectin Enrichment of ZFN Transfected Pools
Ricinus communis agglutinin-I (RCA-I)
Lectin isolated from castor bean seeds
Binds to terminal galactose residues
Toxic to CHO cells with WT glycan profiles
Ladder Mock
ZFN
DNA
ZFN
RNA
ZFN
RNA
ZFN
DNA
Percent Modified 0 3.8 5.5 35.7 33.2
-RCA +RCA
17. 17 Webinar: Cell Line Engineering
Man5 Glycoform is the Predominant Species in Lectin Enriched Pools
Mock-transfected
RCA-I enriched
18. 18 Webinar: Cell Line Engineering
Man5 Glycoform is the Predominant Species in Lectin Enriched Pools
0
10
20
30
40
50
60
70
80
90
100
PercentGlycanSpecies
Mock
Mgat1 ZFN DNA
Mgat1 ZFN RNA
Mgat1 ZFN DNA + RCA-I
Mgat1 ZFN RNA + RCA-I
19. 19 Webinar: Cell Line Engineering
Mgat1- Clones Have Similar Growth and Productivity to WT Clones
0
1
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12 14
VCD(10e6cells/ml)
Days in Culture
Original
WT1
WT2
KO1 -RCA
KO2 -RCA
KO3 +RCA
KO4 +RCA
KO5 +RCA
KO6 +RCA
0
200
400
600
800
1000
Original WT1 WT2 KO1 -
RCA
KO2 -
RCA
KO3
+RCA
KO4
+RCA
KO5
+RCA
KO6
+RCA
IgG(mg/L)
20. 20 Webinar: Cell Line Engineering
Mgat1- Clones Have Man5 as Predominant Glycan Species
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
PercentGlycanSpecies
Original
WT1
WT2
KO1 -RCA
KO2 -RCA
KO3 +RCA
KO4 +RCA
KO5 +RCA
KO6 +RCA
21. 21 Webinar: Cell Line Engineering
Mgat1- Cell Line Engineering in a CHO K1 GS-/- Host Cell Line:
Growth Performance
0
2
4
6
8
10
12
0 2 4 6 8 10 12
VCD(10e6cells/ml)
Days in Culture
WT
AB4
BC9
BD7
BH11
CG10
23. 23 Webinar: Cell Line Engineering
Man5 Glycoform is the Predominant Species in GS-/-/Mgat1- IgG Stable
Pools
GS-/-
IgG Stable Pool 1
GS-/-/Mgat1-
IgG Stable Pool 1
GS-/-/Mgat1-
IgG Stable Pool 2
24. 24 Webinar: Cell Line Engineering
Engineering CMAH-/- and GGTA-/- Cell Lines for Decreased
Biotherapeutic Immunogenicity
CMAH
CMP-Neu5Ac hydroxylase
Conversion of Neu5Ac to Neu5Gc sialic acid
N-acetylneuraminic acid (NANA or Neu5Ac)
N-glycolylneuraminic acid (NGNA or Neu5Gc)
Neu5Gc is not expressed in humans and can be
recognized as a foreign epitope
GGTA
N-acetyllactosaminide 3-α-galactosyltransferase-1
Immunogenic α-gal moiety
Expressed in murine cells but not in human
Humans have circulating antibodies to alpha-gal
Some CHO cell lines express functional Ggta1
25. 25 Webinar: Cell Line Engineering
CMAH-/-/GGTA-/- Cell Line Engineering in a CHO K1 GS-/- Host Cell
Line
GS-/- Host
CMAH ZFN Transfection
SCC and Genotyping
5 KO Clones
Clone Expansion and Characterization
Clone Banking
GS-/-/CMAH-/- Host
GGTA ZFN Transfection
SCC and Genotyping
1 KO Clone
Clone Expansion and Characterization
Clone Banking
26. 26 Webinar: Cell Line Engineering
GS-/-/CMAH-/-/GGTA-/- Host Cell Line Growth Characterization
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10
VCD(10e6cells/ml)
Days in Culture
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 2 4 6 8
PercentViability
Days in Culture
28. 28 Webinar: Cell Line Engineering
Targeted Gene Integration for Biomanufacturing
Reduced variability/enhanced cell line
performance
• Decreased integration site side effects
• Clone to clone consistency
• Molecule to molecule consistency
Decreased cell line development timelines
• More homogeneous stable pools
• Decreased clone screening
Increased pool and clonal stability
• Use of well characterized safe harbor sites
• Remove stability from the critical path
29. 29 Webinar: Cell Line Engineering
Targeted Gene Integration Project Focus Areas
321
• Short RFLP donors
• GFP reporters
• IgG and Fc r-proteins
• Decreasing NHEJ
• Increasing HR
• Tagged ZFNs
• Optimized donor designs
• TI landing pads
• Reverse engineering of
high expressing clones
• Transciptomis analysis of
constitutively expressed
genes
Increased EfficiencyFeasibility Hot Spot Identification
30. 30 Webinar: Cell Line Engineering
Targeted Integration of GFP at the Actin Locus
DonorOnlyZFN+Donor
1.2% GFP+
2.4% GFP+
decreased scaling
actual
GFP Reporter HAHA
Actin GeneGFP Reporter Actin Gene
31. 31 Webinar: Cell Line Engineering
Targeted Integration of GFP at the Actin Locus
Mock
DonorOnly
Donor+ZFN-Lsort
Donor+ZFN-HsortGFP Reporter Actin Gene
32. 32 Webinar: Cell Line Engineering
Targeted Integration on IgG at a Safe Harbor Site
Transfection
• CHO SH1 ZFN RNA
• IgG/GS Plasmid Donor
Stable Pool
Generation
• GS Selection
• FACS Enrichment
SCC
Generation
• jPCR/Seq, qPCR
• G&P
• Stability
Targeted Integration SCC
Random Integration SCC
33. 33 Webinar: Cell Line Engineering
Summary
4
3
2
1
Genome editing is a valuable tool for enhancing biomanufacturing
expression systems
GS-/- host cell lines can lead to more efficient cell line development
processes and higher performing recombinant pools and clones
Glycoengineering can lead to more efficient manufacturing processes
and the production of more effective therapeutic proteins
Targeted integration can result in increased homogeneity and stability
of recombinant stable pools and clones
5
Full extent of cell line engineering applications in biomanufacturing
remains to be discovered
35. Erika Holroyd
Product Manager, Cell Line
Development and Engineering
CHOZN® Program
erika.holroyd@sial.com
CONTACT
Trissa Borgschulte, PhD
Head of Cell Line Development and Engineering
CHOZN® Program | Process Solutions Upstream R&D
trissa.borgschulte@sial.com
Contact