GENETICALLY MODIFIED ORGANISMS
FOR
MEDICINES PRODUCTION
Antonio Moreira
University of Maryland Baltimore County
III SYMPOSIUM
SINDUSFARMA-IPS/FIP-ANVISA
New frontiers in manufacturing
technology, regulatory sciences and
pharmaceutical quality system
Brasilia
August 5, 2014
Presentation Outline
• Primer on genetic engineering and biotherapeutics
• Main genetically modified organisms currently used in
biomanufacturing
• Biomanufacturing processes
• Examples of bioprocess development studies
• Status of the biotech industry for therapeutic products
How Do We Deliver & Maintain Our Protein
Coding Sequences?
Plasmids: Autonomously replicating,
circular DNA molecules
Circular plasmids can be transfected
into cells directly or plasmids can be
linearized.
Selections used include antibiotics,
puromycin, hygromycin, neomycin,
presence of DHFR gene (MTX r)
Drug level can be used to select
for increased numbers, or copies
of gene.
4
Clark & Pazdernik.
Biotechnology: Applying the
Genetic Revolution. 2009.
Biologics As Therapeutics
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•
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Natural Products
Blood Products - Transfusions
Vaccines
Purified Proteins from natural
sources / tissues
• Recombinant Proteins
– Microbial
– Cell Culture
• Monoclonal Antibodies
• Combination Products
• Cell Therapy – Bone Marrow
Replacement / Reconstruction
• Stem Cell Therapy
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Figure 1. Total sales in the US and Europe of traditional pharmaceuticals (blue) and
biopharmaceuticals (green) are shown by year for the past decade. Sales information was
obtained from company annual reports and other publically available sources.
Jones & Ecker. Pharmaceutical Processing, October 2013.
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Figure 2: List of FDA-approved antibody therapeutics.
BioPharm International, February 2013
8
Figure 1: Primary mechanism of action of antibody-drug conjugates: targeted
delivery of a potent cytotoxic agent to cause cell death.
BioPharm International, February 2013
9
Bioprocesses Mirror the Complexity of
Biological Products
•
•
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•
•
•
Classical Pharmaceutical Drugs
– Defined Structure and Characteristics
Basics of biopharmaceuticals
– Complex chemistry and structures
– Proteins, nucleic acids, lipids,
polysaccharides
– Cells
Inherently unstable (until purified or
formulated)
– Degradative enzymes co-produced
– Temperature, pH, concentration
Bio-safety constraints - complexity and purity
Process design - control for productivity
Process design for GMP vs. flexibility
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NCE Versus Biopharm Comparison
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Figure 1: Global biosimilar approvals, 2006-2012 (in Europe, unless
otherwise indicated.
Emerton, Duncan. Supplement to BioProcess International. June 2013.
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KEY TARGETS
Developers are trying to create functional replicas of leading biologic
drugs.SOURCES: Company data, Biotechnology Information Institute
Thayer, C&EN Houston. cen.acs.org. October 7, 2013.
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Biomanufacturing
• Biomanufacturing involves three key processes:
– Controlled growth of microorganisms, cells, tissues or
organisms
– Conversion of simple raw materials or complex
molecules to desired product
– Isolation & purification of the product from complex
mixtures
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Major Production Systems
• Bacterial Cells
– E. coli
• Yeast Cells
– Saccharomyces cerevisiae
– Pichia pastoris
• Mammalian Cells
– Chinese Hamster Ovary – CHO
– Baby Hamster Kidney –BHK
– NS0 or Sp2/0 (Mouse Myeloma)
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Subcellular Structure of Escherichia coli: Scanning electron micrograph of E. coli. The rod-shaped
bacteria are approximately 0.6 microns by 1-2 microns. Courtesy of Rocky Mountain Laboratories,
NIAID, NIH.
Clark, D. & Pazdernik, N. Biotechnology; Applying the Genetic Revolution. 2009
16
Major Protein Expression Platforms
• Yeast Cells/Fungi
Pichia, Saccharomyces, Kluyveromyces, Aspergillus
Pichia spp.
Saccharomyces spp.
Aspergilus spp.
Clark, D. & Pazdernik, N. Biotechnology; Applying the Genetic Revolution. 2009
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Clark & Pazdernik. Biotechnology: Applying the Genetic Revolution. 2009.
Figure 1: Industrial cell-free biology.
Swartz, AIChE Journal, January 2012. Vol 58, No. 1.
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Fig. 2. Cartoon comparison of in vivo recombinant DNA protein expression with cell-free protein synthesis (CFPS). CFPS systems
provide a more rapid process/product development timeline. Example proteins shown include a virus-like particle (VLP), single-chain
antibody variable fragment (scFv), and a membrane bound protein (MBP).
Carlson, ED, et al, Cell-Free Protein Synthesis: Applications Come of Age. Biotechnol Adv. (2011).
Swartz, AIChE Journal, January 2012. Vol 58, No. 1.
Mammalian Antibody Production –
Cell Culture
Media
Prep
HEAT
COOL
Vial Thaw /
Inoculum
Expansion
50-Liter
Media
Pasteurizer
500-Liter
5000-Liter
Upstream Processes
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20,000-Liter
Mammalian Antibody Production –
Harvest
Harvest from
Production Bioreactor
Transfer to
Purification
Suite
Depth Filter
Virus
Inactivation
rProtein A affinity
chromatography
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Disc-Stack
Centrifuge
Mammalian Antibody Production –
Downstream Processing
Ion (Cation)
Exchange
Chromatography
Intermediate
Storage
Ion (Anion)
Exchange
Chromatography-
Intermediate
Storage
Viral
Filtration
20m2
API
I.B.I
CryoPreservation
System
Bulk
Filtration
(BDS)
UF/DF Step
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Intermediate
Storage
Hydrophobic
Interaction
Chromatography
Stirred-tank Reactor
• Most common type
• Cylindrical tank
• Agitator motor, shaft &
impellors
• Air/gas inlet & exhaust
• Sampling & harvest ports
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Example of a Commercial Bioreactor
•Sandoz cell culture
manufacturing facility
•Located in Schaftenau,
Austria
•Facility contains two
13,000 L bioreactors
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Supplement to Pharmaceutical Engineering, 2013, p.10-16
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Bioreactor Operating Modes
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Batch
Fed-Batch
Continuous
Perfusion
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Table 1: Selected Perfusion Operations Issues:
PRODUCT
MANUFACTURER
IL-12/23 Mab (Stelara)**
Janssen/J&J
TNF Mab (Simponi)
Janssen/J&J
Glucosidase alfa (Myozyme)**
Genzyme/Sanofi
Galactosidase alfa (Fabrazyme)**
Genzyme/Sanofi
Protein C (Xigris)
Lonza for Eli Lilly
Factor VIII (Kogenate-FS)**
Bayer
Interferon beta (Rebif)**
Merck-Serono
IL-2 receptor Mab (Simulect)
Novartis
TNF mAb (Remicade)**
Janssen/J&J
FSH (Gonal-F)
Merck-Serono
Galactosidase. beta (Cerezyme)**
Genzyme/Sanofi
Platelet Mab Fab (Reopro)
Janssen/J&J
**Annual sales over 500 million
Source: 11th Annual Report and Survey on Biomanufacturing Capacity
and Production, April 2014
Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August
2014, pg. 13.
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Selected Continuous Bioprocessing
Benefits
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Reduction in facility size, manufacturing footprint, etc.
Significant costs savings, particularly investment in facilities
Increases in flexibility
No scale-up of bioprocesses
Increased process robustness
Less manual interactions
Less bulk fluid input
Less sensor insertions and other incursions into the process
Increased automation
PAT and upfront bioprocess design using QbD can be easier to implement
Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August
2014, pg. 13.
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Typical Downstream Processes
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Cell Lysis/Disruption (if needed)
Chromatographic purification
Product Concentration
Sterile Filtration
Formulation
Fill & Finish
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Purification
• Sequence of steps, generally 3 to 4
• Selective removal of contaminants
• Isolates molecules by physical or chemical
characteristics
• Volume reduced at each step, ideally
• Initial coarse cuts
• Polishing steps
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Production Scale Units
Purity Goals
• Residual host & contaminant proteins
– ppm level
• Nucleic acids
– 100 pg/dose
• Viruses
– Below detection limits
• Endotoxins
– < 5EU/kg/hr for parenteral use; <0.25 EU/kg/hr for
intrathecal use for drug products w/o a compendial limit
• Microorganisms
– None
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Regulation of New Technology
Protein Production Issues
•
Expression Technology
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–
Cell line selection and optimization
New production systems to increase yield
• Bacteria – newer strains 10 g/L
• Yeast – over 5 g/L
• Mammalian Cells – achieving 2-5 g/L
•
Recovery & purification
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Characterization of complex structures
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Product aggregation
Solubility
Stability
Glycosylation
Pegylation
Process and Facility design
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–
Automation
Cleaning
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Product Realization
Design Space
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•
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Determining
What and How
the Process
Affects Product
Characteristics
Past Experience
Preclinical Studies
Clinical Studies
Identification of
Key Product
Characteristics
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Past Experience
Process Development
Product Characterization
Target Product Profile
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Process Control
Strategy
Testing
Quality Systems
Ensuring
Patient
Receives the
Expected
Product
Quality Assurance
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Discovery to Therapeutic Delivery Pathway
and the Process Information Gap
Phase
Discovery
Issues
Product Characterization
Non-Instrumented
Fully Instrumented
Process
optimization
Production
Define
Design Space
Scale
Quality Control
Therapeutic
delivery
Fundamental question: Can we predict manufacturing behavior of cell lines as early as
in non-instrumented devices?
Discovery phase is not integrated with following phases. It lacks of the “knowwhy” process perspective.
Based on Kirouac and Zandstra 2008, Cell Stem Cell 3:369-381
PSDs Applications in Upstream Bioprocess
Development and the Process Information Gap
SCALE-UP
STRAIN/CLONE
SCREENING
EARLY R&D
STUDIES
Non-instrumented
PROCESS
OPTIMIZATION
PRODUCTION
Fully instrumented
Static + Stir
MCB
vial
Stir
Shake
Roller
Wide variety of PSDs.
SCALE-DOWN
Application of Process Analytical Technology for Extended Cell
Passaging: A Proof-of-Concept Revealing Study
MCB
vial
Expression system:
Non-adherent SP2/0-based myeloma/
mouse (2055.5)
Protein:
IgG3 antibody specific for the
Nisseria meningitides capsular-polysaccharide
(MCPS).
Media:
CD Hybridoma GTTM
20mL
CO2 Incubator
5% CO2
37OC
3-Day passage scheme
Pi+1,j
Pi+3,j+1
P1,1, P2,1, P3,1
P4,2, P5,2, P6,2
Pi+n,j+n’
i: Number of passages
j: Number of T-flasks
i+n: Total number of passages
j+n’: Total number of T-flasks
Vallejos et al. (2010) “Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask” Biotechnol & Bioeng. 105(6):1040-1047
Application of Process Analytical Technology for Extended Cell
Passaging: A Proof-of-Concept Revealing Study
 Cells are exposed periodically
to sub-optimal DO levels (0%).
 What happened at passages
18-20 and passages 27-29?
Vallejos et al. (2010) “Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask” Biotechnol & Bioeng. 105(6):1040-1047
Application of Process Analytical Technology for Extended Cell
Passaging: A Proof-of-Concept Revealing Study
Dissolved Oxygen (% Air saturation)
Improving cell passaging techniques in T-flask at low cost!
100
80
60
40
20
0
T75-Static
T75-Rocking
8.5
pH
8.0
Vallejos et al. (2012)
Biotechnol & Bioeng.
109 (9):2295-2305
Sub-optimal DO and pH levels are avoided
in rocking T-flasks.
7.5
7.0
6.5
6.0
0
20
40
60
80
Cell Culture time (h)
Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305
100
120
A Novel Scale-Down Paradigm for the Wave
Bioreactor
Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305
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A Novel Scale-Down Paradigm for the
Wave Bioreactor
At matched kLa both systems (rocking T-flasks and wave bioreactor) perform similar
except for specific productivity
Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305
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Comparability Study 5L Vs. MB
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
Same seed in both systems; Passage 6-10
DO 30%
pH 7.2, Control w/o base addition
Matched Kla
3 replicates
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H/D
Impeller type
Number of impellers
Overlay rate
Base line comparability study
5L
MB
1.5
2.2
pitched blade pitched blade
2
3
0.03vvm
0.03vvm
Kla hour-1
DO set point
pH set point
2
30%
7.2
2
30%
7.2
Temp.
Rpm
370C
220
370C
220
Difference in the Antibody Product Titer
The antibody titer in 5L was about 50% higher than that in
the minibioreactor
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Glutamine Profile in 5L Vs. Minibioreactor
 Glutamine degradation was not significant
 L- Glutamine levels reach zero when minibioreactors reach
their stationary /peak cell density phase, while the 5L has
sufficient amount of glutamine
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Glutamine Supplement Experiment
Glut MB control
Glut MB Supplemented
Titer MB Control
Titer MB Supplemented
Titer 5L
 Protein titers were similar to 5L when glutamine was supplemented
 Identical amount of starting glutamine concentration
Difference in the glutamine consumption
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Comparability Study With CO2
Monitoring
( Source: Ge et al., 2005)
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Comparability at Similar CO2 Stripping
Rates
The CO2 profiles of minibioreactor (MB) and bench
scale bioreactors (5L) were found to be similar
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Result of CO2 Profile Matching
H/D
Impeller type
Mixing time
overlay rate
mixing time
Kla per hour
CO2 Stripping rate
ppm/min
Peak VCD
(X10^6cells/ml)
Peak titers (mg/L)
G0F
G1F
G2F
Base line study
5L
MB
1.5
2.2
pitched blade
pitched blade
4S
4S
0.03vvm
0.03vvm
4S
4S
2
2
After providing similar CO2
stripping rates
5L
MB
1.5
2.2
pitched blade
pitched blade
4S
4S
0.03vvm
1.1 vvm
4S
4S
2
2
298 ±4
145 ±4
298±4
223 ±4
4±0.8
178±12
42.5±5.5
48.3±2.4
7.4±2.5
4.5±0.8
122±8
48.3±0.9
44.3±0.8
7.4±0.2
4.4
163±12
45.2±1.4
45±4.2
9.9±2.9
5.1±0.8
164±8
46.5±0.9
42.1 ±1.6
11.2±0.8
 Protein titers in MB with similar CO2 stripping rate were
found to be similar to that in 5L
 A difference in the CO2 profile could be a reason for a
difference in the titers
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Productivity of IgG3 at varying %DO. Concentration of IgG3 samples was measured by A280nm.
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Comparison of averaged relative protein carbonyl concentration measured by ELISA in HTBRs at various %DO (10%,
40%, 60% and 80%). Error bars represent standard deviation of protein carbonyl content.
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Current and Novel Inputs to Bioprocess
Scale-down and Evaluation
New Approach Sentinel genes
showing cell
physiological
response
Real-time
process setpoints for %DO,
pH, T, pCO2
Pre-culture
tunable
bioprocess
parameters P/V,
kLa, tmix
Culture growth
parameters - XV,
µ, qoxygen, qp,
Glu, Gln, Lac,
Ammonia
Scale-up/
Scaledown
5L bench-scale
to Minibioreactors
 New approach to bioreactor Scale-change
Lack of measurable attributes at cellular level
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Post-culture
product quality Titer, Purity,
Glycosylation
Recent Advances in the
Biotechnology Industry
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•
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Mammalian Cells
• Significant increase in manufacturing scales
• Major product titer improvement (up to 8-10 g/l)
• Large gains in recovery yields
• API batch sizes of up to 50 to 100 KG
• Perfusion processes up to 1000L scale with high perfusion rates and lasting up to 200
days
Monoclonal Antibodies
• Fully humanized products
• Exploitation of antibody fragments with favorable characteristics
• Novel scaffolds introduced
• Glyco-engineering of antibodies and fusion proteins
• First antibody-drug conjugates approved
Innovation in Process and Product Development
• Maturation of technologies and processes
• Higher degree of automation
• Process robustness driven by science and process management
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Recent Advances in the
Biotechnology Industry (Cont.)
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•
Process Development
• Improved cell lines
• Enhanced media compositions and media optimization
• Improved chromatographic media
• Improved expression vectors
• Use of “omics” approaches
Implementation of Disposable Technologies
• Many components available
• Smart integration of mature technologies
• Fully disposable facilities
Increased Efficiencies and Risk Mitigation
• Elimination of animal-derived raw materials
• Implementation of virus clearance
• Implementation of platform bioprocess technologies
• Pursuit of PAT and QbD initiatives
Pharmaceutical R&D
• Formulation, manufacture and delivery of biological drug products
• Development of stable high concentration formulations
• Use of pre-filled syringes
• Development of auto-injectors
• Development of combination products
• Aseptic processing technologies
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Expectations for the Next Decade
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Development of combination products
Increases in automation and paperless manufacturing
Biopharmaceutical manufacturing facilities
• Platform based large scale for large market regions
• Disposable plants in emerging markets
Focused attention on QbD and PAT objectives
Increased downstream process efficiency
Enhanced physical characterization of bioreactors
• Mass transfer (especially CO2)
• Mixing
• Shear forces
Renewed focus on traditional engineering principles
• Technology transfer
• Process scale-up
• Modeling unit operations (i.e., CFD) including Artificial Neural Networks and Statistical Online
Control
Continuous processing
New molecular formats
Evolving regulatory requirements
Global access to high quality biopharmaceuticals
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Acknowledgements
Academic/Industry Colleagues:
Dr. Marcia Federici
Dr. Debra Barngrover
Dr. Govind Rao
Former Graduate Students:
Dr. Bhargavi Kondragunta
Dr. Jose Vallejos
Dr. Shaunak Uplekar
Ms. Nacole Lee
Executive Administrative Assistant:
Ms. Susan Mocko
Antonio Moreira, Ph.D.
Vice Provost for Academic Affairs
University of Maryland Baltimore County
1000 Hilltop Circle
Administration Building, Room 1001
Baltimore, MD 21250
Tel: (001) 410-455-6576
Fax: (001) 410-455-1107
Mobile: (001) 443-254-3696
Email: moreira@umbc.edu
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