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Joan Hilly Foster M.S.Senior Field Application ScientistBioProcess International ConferenceOctober 5, 2016
Accelerating Biotherapeutic Development Using
CHO Cell Transfection
Outline
• Brief Overview of MaxCyte Scalable Electroporation
• CHO Cell Transfection for Bioproduction
o Transient protein production
o Stable pool production
o Stable cell line production
• Transient and Stable Cell Product Quality
• Questions and Answers
Benefits of MaxCyte Scalable Electroporation
• Work with the appropriate cell type, maximizing the relevance of the data produced
• Capable of producing a wide range of molecules
• Routinely reach levels of reproducibility and scalability higher than those achieved using reagent-based transfection methods
• Higher efficiency, viability, and improved recoveries deliver improved productivity
• Produce sufficient material to drive development in weeks instead of months
• Shorten timelines to generate stable pools and clones
• Accelerate development and reduce costs
Why Use MaxCyte Electroporation?
Features
Capabilities Multiple Applications
Transfection efficiency Consistently > 90%
Cell viability Consistently > 90 %
Diverse loading molecules DNA(s), mRNA, siRNA, proteins, lysate
Cell type diversityMammalian cell lines, primary cells, stem cells& difficult-to-transfect cells
Scalability 0.5 x 106 ⎯ 2 x 1011 cells
Single-useSterile, single-use, processing assemblies for a range of volumes & cell numbers
QualityDesired product attributescGMP compliant – Process and Product
>95% Transfection Efficiency
CAP-T
SF-9
HEK 293F
CHO-S
Cells transfected with pGFP assayed 24-48 hours post-EP
GFP+
GFP+
GFP+
GFP+
EP, No DNA EP + pGFP
Preprogramed to Accommodate 81 Cell Lines
• 10T½• 1321N1• A549• Ba/F3• B16• B65• BHK-21• C2C12• C6• CaCo-2• CAP/CAP-T• CaGo-K-1 • CHO• COS-1• Cos-7• CV-1• DLD-1• DT40• EL4• H1299• HCT 116
• HEK 293• Hela• Hep G2• HOS• Huh-7• HT1080• HT29 • JCOCB• Jurkat• K562• L5278Y• L6 • LLC-PK1• LNCaP• MCF7• MDA-MB-231• MDCK• ME 180 • Mesenchymal
Stem Cells
• RLE• S2• SAOS-2• SF21• SF9• SH-SY5Y• SK-BR-3 • SK-MES-1• SK-N-SH• SK-OV-3 • SL3• Snu-1• SP2/0• SW403• THP-1• U2OS• U937• Vero• YB2/0
• MG63• Min-6• ND7/23 • Neuro2a• NIH 3T3• NIH3T3L1• NRK• NS0• Opossum Kidney• P3U1• Panc-1• PC12• PC-3• Per.C6• Primary
Fibroblasts• Ramos• RAW 264.7• RBL• Renca
MaxCyte Application – Cell Therapy
• Multiple (10+) human clinical trials underway (US, Canada, Singapore)
• Commercially marketed therapy in Japan
• FDA master file available
MaxCyte Application – Cell-based Assays
• Diversity of Targets:
– GPCRs
– Ion Channels
– Nuclear Receptors
– Kinases
• Diversity of Assay Types:
– High-throughput screening
– Profiling
– Reporter genes
– Receptor signaling
MaxCyte Application – Bioproduction
• Antibody Production
• Recombinant Protein Production
• VLPs and Viral Vectors
• Vaccines
• Cell Line Development
MaxCyte CHO Cell Transfection for Development through Bioproduction
MaxCyte Transfection Platform Delivers Scalability
STX Scalable Transfection System 5 x 105 to 2.0 x 1010 cells
in < 30 min
VLX Large Scale Transfection System Up to 2.0 x 1011 cells in < 30 min
Selecting the Best Cell System is Critical for Success
Choose a cell type suitable for both R&D & manufacturing
Picture from Cevec, Croset, A. etc. (2012) Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells. J. Biotech.Zeck, A. etc. (2011) Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa. J. Proteome Res.Bulter M and Spearman, (2014) The choice of mammalian cell host and possibilities for glycosylation engineering. Current Opinion in Biotech.
Highly Efficient DNA Loading in CHO Cells
GFP Transfection: >95% Viability & Transfection Efficiency
Untransfected Cells
CHO-S cells 24 hours post-EP with pGFP
FACS Data
96% GFP+, 96% Viable
High performance also seen with CHOZN®, CHO-K1, CHO-K1SV, CHO EBNA
EP Outperforms other Transfection Methods
Non-antibody Therapeutic Protein (CHO-K1 Cells)
MaxCyte 1
MaxCyte 2
MaxCyte 3
Lipid 1
Lipid 2
Polymer 1
Polymer 2
Other EP 1
Other EP 2
No process development Customer Data
]
IgG IgG IgG VralProtein Bi-Specific
Ab
Bi-Specific
Ab
Ig4 IgG Bi-Specific
Ab
Secreted
Protein
Rabbit
antibody
IgG Cytokine
fusedIgG
Antigen
ProductivityMaxCyte EP&OtherMethods
OtherMethods MAXCYTEEP
Customer Data
Comparative Study ⎯ Customer Experience
STX Electroporation Provides Seamless Scalability
h-IgG1 Expression Plasmid
CHO-S cells cultured in commercial media using commercial supplements
Make grams of antibody!28 mg to 3.42 g < 2 weeks
Without Process Development
Accelerate R&D to Market using Transient Material
What could you do with grams of protein earlier in development?
Analytical study & Assay development
New drug study Proof of concept
Formulation development and stability
Downstreamprocess & purification
development
Large animal PK & PD
Maximizing Expression through Process Development
Pre-transfection
Expression vector
DNA Quality
Host cell line
Cell generationCell density
Growth state
During Transfection
High transfection efficiency
High viability
Post-transfection
Inoculum cell density
Cell growth controls
Temperature
Nutrient balance
Toxic waste control
pH and DO control
Process Dev. Improves Titer & Productivity
Titers by ELISA were verified by Protein A Capture Assay
Up to 2.7 Grams per Liter Specific Cell Productivity
Perform Transient & Stable Transfection Simultaneously
Selection & stable pool
STX Transfection
CL-2
Transient protein expression
6-8 weeks2 weeks
2e10 cells
8e7 cells
Milligrams to grams of purified protein
Clonal selection, production culture & cell bank
Purification, analysis & functional assays
Shorten Timeline to Generate Stable Pools
Rapid, High Yield Stable CHO Cell Line Generation
Titers verified by ELISA and Protein A Capture AssaysA non-proprietary ACF process
Titer mg/L Cell-specific productivity
Product Attributes: Transient vs. Stable Cell Lines
• Equivalence of Transiently & Stably Produced Proteins
- Post-translational modifications- Protein integrity
• Consistency of Product Produced by Stable Cell Lines for 60 Gen
Consistent Glycosylation: Transient EP vs. Stable EP
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
Man5 G0 G0F G1 G1F G2F G2
Re
lati
ve A
bu
nd
ance
(%
)
Glyco-form
TGE S10
TGE S4
TGE S6
TGE S12
Stable 17
Glycosylation Analysis of Antibody Captured by Protein A
Consistent LC/HC: Transient & Stable Expression
SDS PAGE AnalysisProtein A - Purified Antibody (2 μL)
HC: Heavy ChainLC: Light ChainNR: Non-reducedR: Reduced
Stable Clone Stability Study for 60 Generations
18 days2 weeks30 days30 days6 weeks
Cell Bank 1
Cell Bank 2
Cell Bank 3
EP
Cell Line Development Banking
Thaw & Passage
Production Cultures
Consistent Quality: 60 Gen Stable Cell Line
96
52
35
M 1 2 3 4 5 6
Cultivation Days:
Non-reduced
Reduced
188
0 30 60
Kda
Consistent Glycosylation: 60 Gen Stable Cell Line
Summary
• Flexibility in Application: – Cell therapy, Cell-based Assays, Bioproduction
• Transfection Attributes– Consistent and Reproducible– High efficiency, viability and cell recovery– Cell type diversity– Outperforms other methods
• Seamless Scalability– Streamlines downstream processing– Reduce production volumes– Eliminates need for extraneous agent removal
• Transient and Stable Cell Line Generation– Preservation of essential product attributes – cGMP compliant production
Accelerating Biotherapeutic Development
• More relevant characterization assays indicative of biomanufacturability
Start Development in the Host Manufacturing
Background
• More extensive characterization earlier in development; faster to fail
• Decreased # of stable clones needed
High Titer
Transient Production
• Reduce down-time in development pipeline due to the time needed to generate a stable cell line
Simultaneous Transient Protein Production &
Stable Cell Line Generation
