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PhD Thesis Defense 6.12.12

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Chris Warner

on 24 May 2018

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Transcript of PhD Thesis Defense 6.12.12

Rapid, Large-Scale Production of Full-Length, Human-Like Glycosylated Monoclonal Antibodies

Christopher Warner
Personalized Medicine
Therefore, require novel system utilizing CHO with reduced cell development time
Our approach to increasing titers involve maximizing Qp through optimizing transfection conditions (DNA delivery rate) and maximizing cell viability for high volumetric productivity
Requires Proper Glycosylation
(such as provided by CHO cells)
Part of the Adaptive Immune System
(Passive Administration)

Proven Efficacy/safety

Large discovery pipeline

Wealth of manufacturing knowledge
Christopher M Warner, MBS MBA,
PhD candidate and SMART Scholar

Dr. Matthew Croughan
KiriLynn Svay
Delyan Rusev
Dr. Nathaniel Freund

Dr. Edward Perkins
Dr. Ali Nadim
Dr. Tina Etcheverry

Ralitsa Kiskinova
1-300 mg
Very High
1-10 gm
Product Quality
Cost of Production
Speed to Production
Future Studies
Scale Up of transient systems
Scale Down 3 phase systems
Utilizing Cell Retention systems
Filter Studies- Opti-Pro-Opti
Wash experiment
Perfusion - Opti Pro Opti
Tools to study MOA
GFP expression
Experiments to date

SEAP v Il-8
Media Time v temp
Agitation Study
Chitosan Experiments
Perfussion Concentration
Density Study
Wash Study
Bioreactor POC Studies
Manuscripts in Preparation
Challenges and Opportunities in the Manufacture of Medical Countermeasures
J of Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science
Diffusion Limitations of Transient Transfection
J of Biotechnology and Bioengineering
Perfussion Cultures for Maximizing Transient Transfection Productivity
J of Biotechnology and Bioengineering
Plasmid Copy Number
mRNA Transcript Level
Flow Cytometry
Population Dynamics
Apoptosis Analysis
Cell Cycle Analysis
Product Characterization Techniques
Glycosylation Analysis
Potency Assays
Titer = Qp * IVCD
Cells are grown in shaker flasks with either OptiCHO (red) or ProCHO media (yellow), passaged every few days (less than 5*10^6 cells/ml)

Once cell density is reached, pull out cells and centrifuge down

Resuspend into 6 ml ProCHO media at 20*10^6 cells/ml in transfection media (ProCHO) at a shaker at 130 rpm, 37C

Add 350 ug DNA into 700 ug PEI (25 kDa linear) allow to incubate for 10 minutes exactly

Add DNA-Transfection Complex into concentrated culture

Incubate for 3 hours then add 24 ml (5x dilution) of Growth Media (+ 3.8 mM Valproic acid) and temperature shift to 32◦C

Maintain culture up to 10 days
Baseline Transfection Protocol
Grow cells in 3-L Bioreactor maintaining exponential phase
Pull out cells
Spin down in Sorval J-21 (Cregg Lab)
Re-suspend to 200 ml
Transfer to 1-L Bioreactor
Sample, pull out satellite for control
Incubate DNA and PEI
Add DNA/PEI to reactor
Feed and temperature drop, pull out satellite for control
Commercialized/ In development for 1500-5000 L capacity
Expanding market, funding by DoD
Can be easily & inexpensively installed in ware houses/ mobile DoD units (Deployed overseas)
Don’t work as well for microbial systems because of cooling needs
Availability of Large Scale installed capacity
Particularly for non-viral systems
Rapid capacity expansion through use of disposables
Short production timeline
Effector function if needed
Need titers 100- 1000 mg/L
Immunotherapeutic medical countermeasures through cell culture
Disposable bioreactors
Small Scale (<15 L)
Batch centrifugation (Table top, floor model)
Small to Moderately large (<1500 L)
Acoustic Filtration
Inclined settler
Spin Filters
Alternating tangential flow
Centritech Centrifuge
Small to Large Scale (>2400 L)
Cross flow filtration
Continuous centrifugation with gentle discharge
Cell Concentration Techniques
Accelerated Manufacture of Pharmaceuticals (AMP)
Sepulveda J et al. Infect. Immun. 2010;78:756-763

High Affinity Molecule

Long Circulating Half Life

Clearance Mechanism
64% Utilization rate of bioreactor capacity (Levine 2010)
Stable mAb platform
Operational flow of TGE
Transient mAb platform
Develop transient production process at KGI and increase titers through rational process optimization.
Process alternatives for TGE
Baseline 1 process relies on high cell densities, large concentrations of both DNA and PEI for transfection, and both cell concentration and medium exchange operations.

Baseline 2 process has a lower titer, uses less DNA, requires medium exchange but not cell concentration.
Backliwal et al. 2008; Rajendra et al. 2011
Baseline 1 with HEK
Baseline 2 with CHO
Experimental verification of process parameters
Cross Flow Filtration
Cells are retained, media is not.
Cross flow filtration system (TFF) is compatible with transient transfection. Can implement cell concentration step to very-large scale.
Transient Transfection with scalable unit operations
Figure 2. CHO cultures were concentrated using a bench top centrifuge or hollow fiber system then transfected using standard PEI method. Cultures were cultivated for 8 days and IgG titer were assayed through standard ELISA. Error bars represent one standard deviation of the mean (N=3)
Thank You!
Dr. Matthew Croughan
George B. and Joy Rathmann Professor, Director of the Amgen Bioprocessing Center
Dr. Tina Etcheverry
Senior Consultant at EtchWave Consulting
Member of Board of Directors at Biotech Partners

Dr. Ali Nadim
Professor & Joseph H. Pengilly Chair in Mathematics School of Mathematical Sciences, Claremont Graduate University
Dr. Edward Perkins
Senior Research Scientist at the Environmental lab
of the Engineering Research and Development Center in Vicksburg, MS
Acoustic Filtration
Acoustic Filtration had less than 50% separation yield, resulting in decrease in culture concentration over time. Could potentially get to work, but still limited scale up (<200- 1,000L)
(In days)
Table 1.2. Transient versus Stable Gene Expression in mammalian cells

Transient expression Stable expression
Expression of transfected genes are usually within 16-96 h, but expression is not propagated through subsequent generations

Transfection requires a large amount of plasmid DNA

Expression level usually is relatively low
(5-50 mg/L)

Expression product is good for pre-clinical assessment, but no clear guidelines for clinical production

It takes only a short period to expression a product for “Proof of Principal” demonstration Heterologous DNA is integrated into the host genome and is stable throughout many generations

Cell line development and testing requires an extensive period (4-12 months)

After amplification and high producer selection, expression level is relatively high (500-5000 mg/L)

Stable cells lines can be used for large scale manufacturing leading to clinical trials and commercialization
Equipment Costs:
Super Designer Pro database
Operating Conditions
Industry experience
Super Designer Pro pre-sets
Process Layout
Capital Costs:
Process Simulation
Calibrating the Model
Equipment Needs
Equipment Requirements
Consumable Requirements
pDNA COGS- Reference
MAbs. 2009 Sep-Oct; 1(5): 443–452
mAb Drug Substance COGS at this scale are between $100-300/gm
Recipe Cycle Time
Individual Unit Operations
Recipe Time
Material Flows Time Metrics
Urthaler et al. 2005
Economic scale up
(Hewitt et al. 2007; Onyeaka et al. 2003;).
Upstream Calibration
Downstream Calibration
Sensitivity Analysis
Adapted from DARPA 2006
Williams et al. 2010
Bioproduction can fulfill Bio-defense needs
Bio-defense antibodies require complex assembly
Available capacity could supply bioproduction requirements
TGE processes are necessary for bio-defense
Rough Estimation of production capability
Large biopharmaceutical plant currently idle with over 200,000 L capacity
Roche/Genentech Cell Culture Plant in Vacaville
Transient mAb platform
Transient mAb platform
Low Agitation
High Agitation
High Agitation
In Water
Medical Countermeasures
Gene Expression
Pre-Clinical Material
Non-human glycosylated
Low Titers
Available Capacity
High Cost of DNA
Scalability Concerns
Upstream Calibration
Downstream Calibration
Volumetric Productivity
Final OD
Specific Yield
Plant Up Time
Cost of Capital
Process Failure Rate
Final Product Configuration
Processing Rates
Misc. Calibration
pDNA production
Innoculum Expansion
Phase 1 15 mg/L

Phase 2 100 mg/L
Phase 3 1300 mg/L
* Paired Ttest with 1 tail pvalue<0.05
Levine 2010
Process Simulation

Process simulation
pDNA production 1 Kg/day at $253/g

Transfection Method Development:
Mass transport consideration for optimal polyplex delivery
Process optimization for highest reported titers to date
Phase 1 15 mg/L

Phase 2 100 mg/L
Phase 3 1300 mg/L
Evaluate LS-TGE for Bio-defense
Future Work
Enhance titers to 1300 mg/L using strain development and process development approaches

Novel disposable bioreactor as a TGE production platform

Develop Novel transfection agents exploiting mass transport phenomena
Evaluate the possibility of LS-TGE through process simulation

Develop transfection methods to achieve titers that will meet DoD objectives

Transfection optimization through engineering analysis

Process Optimization
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