Transcript BIOMAN 2011 CHO-tPA Production System Upstream Processing

BIOMAN 2011
CHO-tPA Production System
Upstream Processing
Mike Fino
MiraCosta College
Program Concentrations
Research
Development
Production
• Discovery
• Preclinical
studies
• Clinical studies
• Scale-up
• Quality
• Compliance
MiraCosta
R&D
MiraCosta
Bioprocessing
Program Coursework
100-level courses
• Basic Techniques in
Biotechnology (4-units)
– Introductory experience working in
a technical environment
• Business and Regulatory
Practices in Biotechnology
(3-units)
– Fully online
– Overview of the industry from
discovery to GMPs
• Biostatistics (4-units)
– Lecture and lab
– UC/CSU transferrable
Program Coursework
200-level courses
Hybrid Lab courses
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Advanced Cell Culture
Isolation and Purification of DNA
Techniques in DNA Amplification
Recombinant DNA
Principles of Separation and HPLC
Techniques in Immunochemistry
and ELISA
Qualification and Validation in
Biotechnology
Bioprocessing: Cell Culture and
Scale-up
Bioprocessing: Large Scale
Purifications
Biofuels Production and Analysis
Fully online Courses
• Data Analysis with Excel
• Technical Writing for
Regulated Environments
CAREER EXPERIENCE
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Internship
Work Co-op
Program Certificates
Laboratory
Skills
Bioprocess
Technology
Research and
Development
1-2 semesters
1-2 semesters
3-4 semesters
11 to 12.5units
12 to 13.5units
40 to 43.5units
All BTEC
classes
All BTEC
classes
BTEC and GE
classes
Biopharmaceuticals or Biofuels
Career &
Professional Development
Transfer / LongRange
Biopharmaceutical Production
• Upstream
– Product formation
• Downstream
– Product purification
• Quality Control
– Product safety and efficacy
A Time of Transition and Translation
• The lead candidate coming out of discovery
research will now be subjected to a
development process that sees it change
through a prism of
– Scale
– Process Control
– Compliance
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From Art to Science
Scale-up
• Development is the
confluence of
business, engineering,
manufacturing, quality
assurance, quality
control, and regulatory
affairs
• The end goal is process
understanding so that
a commercial process
and product are in a
state of control
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Process Control & Compliance
Materials
Machine
Methods
Controlled
process/
product
Man
• Say what you do
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Do what you say
Environment
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Document it
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The Upstream Process
• The confluence of inter-related efforts in:
– Cell line development
– Media development
– Bioreactor process design
– Bioreactor process control
Cell Line Engineering
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Expression Systems
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Bacteria
Yeast
Mammalian
Insect
Transgenic Plants
Transgenic Animals
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End game of cell line engineering?
• PCD = picograms per cell per day
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cont’d
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Back of the envelope calcs
• If we have a cell line that produces our product at
100 pg/cell-day and we want to produce material
for a Phase I clinical trial in a single batch then…
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Our product is dosed at 1-mg/kg
Our process takes about 1 week
Typical cell densities are 1e6 cells/mL
Assume 20 people in the trial
Assume average person is 75-kg
• …how big does my culture need to be?
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Large scale mammalian cell culture
• We use the CHO-tPA system as a model
culture system for several reasons
– Seeding densities as a way to show why a stepwise
– Aseptic technique and processing to underscore
the slower growth rates vs microbes
– Currently the main choice for any glycosylated
protein
Inherent Variability: Erythropoietin
• Contains 40% carbohydrate, only 2 disulfide bonds
• 3, N-linked ASN (24, 38, 83), 1 O-link (SER126) glycosylation
sites
– Glycosylation at these sites may be responsible for resistance to
denaturing conditions
• O-linked site not essential for in-vitro or in-vivo activity
• Sialic acid residues (avg 10 moles/mole EPO) responsible for
preserving phamacokinetic behaviour
– Muteins lacking 2 or 3 N-linked sites are poorly secreted
• N-linked glycosylation and sialylation is critical to optimal
secretion, structure, in-vivo potency
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The Upstream Process
• The confluence of inter-related efforts in:
– Cell line development
– Media development
– Bioreactor process design
– Bioreactor process control
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The Upstream Process
• The confluence of inter-related efforts in:
– Cell line development
– Media development
– Bioreactor process design
– Bioreactor process control
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Reactor Selection: Many factors to consider
Developing higher yields
• In 1986, the industry standard from stable CHO-derived
cell lines was a specific productivity of 10 pg/cell/day, 50
mg/L titers, 2e6 cells/mL and processes lasted 7 days
• Today, we are seeing 90 pg/cell/day, 10 g/L, 10e6
cells/mL, and processes lasting up to 3 weeks
1. Generation of recombinant cell lines with high specific
productivities
2. Formulation of media to support high density cell
cultivation
3. Understanding of bioprocess conditions for cell cultivation
4. Sustained viability of cell lines in high-density batch and
fed-batch cultures
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Stirred Vessel Bioreactor
• Process control of the
physiologic environment
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Disssolved oxygen
pH
Temperature
Medium components
(feed: glucose, glutamine;
hormones, growth factors)
– Waste products (CO2,
ammonium, lactate)
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Batching Types (Process Modes)
• Single Batch
• Fed Batch
• Continuous Batch
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Batching Types
• Single Batch
– 7-14 days
– Run is over when there are no
more nutrients
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Batching Types
• Fed-Batch
– Addition of concentrated nutrients =>
higher product concentration
– 2-3 weeks
– Intermittent common for viral vaccine
– rProtein production is largely
fedbatch
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Batching Types
• Continuous
– Continuous addition
of feed and collection
of harvest with cell
retention device =>
higher product
concentration
– Especially for
process-labile
products
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Parameters measured or controlled in
bioreactors
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The Upstream Process
• The confluence of inter-related efforts in:
– Cell line development
– Media development
– Bioreactor process design
– Bioreactor process control
Essentials to Control a Process
 Process Variables
 Identify which variables are important to what you’re doing
 Bioreactors: pH, DO, Temperature, cell count, nutrient levels, waste levels,
CO2 levels
 Process Probes
 You need some instrument that can convert the chemical or physical
phenomenon into an electrical one
 Controller
 The basic function of a controller is to make a comparison between the
current reading of a Process Variable and the desired Setpoint
 Based on that comparison, the controller produces an output
 Process Manipulation
 Based on the Output, you then attempt to manipulate the Process Variable
to bring it closer to the Setpoint
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Bioreactor Control System
• Yellow number is the Process Variable, PV
• White number is the Set Point, SP
• Blue number is the Controller Output
PV
SP
Controller
Output
Depending on the level of the output, something will be changed (e.g. valve
opened/closed) to bring the PV closer to the SP
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A Control System
• A simple error calculation will be made:
– SP - PV = Error
• Depending on the nature of the Error an
Output is calculated
– Magnitude
– History
– Current slope
• The Output will do something to bring the PV
closer to the SP
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Our BioNet Systems
• Bioreactor
– Probes
– Heating blanket
– Agitator and motor
• Control tower
– Collects, stores, and processes information
– Controls gases and pump additions
– Primary interface
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Bioreactor Control System
• MANual mode
– Controller not utilized, full operator instruction
– Example is agitation – we set it and forget it
• AUTO mode
– Controller will try to bring the PV in line with the SP
– The Output of the AUTO mode will be looking to do
something, based on what’s defined in the Setup Page
• CAScade mode
– These are the devices that are looking for direction from the
AUTO output
– This is for all the instruments (pumps, valves, MFCs) that will
be controlled in order to maintain the SP
– Their response is defined in the Setup Page
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Priming the Base
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Gas Controls
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The main controlled process variables
• pH
– Cues from animal physiology dictate starting
points for this
• Most normal cell cultures around 7.4
• Transformed cells around 7.2
– Our cultures do not aggressively change pH so we
will be using gases to maintain our pH
• The Output of the controller will change gas mixtures to
affect pH
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• Agitation
– This one, while it is technically controlled to a
setpoint, is not influenced by our cultures – we set
it and forget it
– Common ranges are 80 to 120 RPM
– For cell cultures we need to be concerned about
shear damage
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Controlled process variables
• DO – Dissolved Oxygen
– For a given temperature and barometric pressure
and solution composition, water can only “hold”
so much oxygen.
– Example: if you were to keep adding sugar when
you’re making Kool-aid.
• Eventually, for the given temperature of the kool-aid,
there’s some critical amount of sugar it can hold. After
that it just precipitates out.
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• The same is true for DO
– Water (given pressure, temp, and composition)
can only hold so much oxygen
– After that, it’s saturated
– This is about 5-10 parts of O2 per 1 million part of
water
• Common setpoints for O2 are 30-50% of
saturation
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Controlled process variables
• Temperature
– Temperature takes its cues from animal physiology
for animal cells: 37oC
– We keep ours at 37oC because though we’re
always cautious because cells are much more
tolerant to deviations on the low side than the
high.
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Instrumentation Calibration
• Most lab instruments contain what can be called a transducer.
• It’s some component that is transducing a physical or chemical
phenomena into an electrical phenomena.
– We need to let the computer (or whatever is
monitoring the probe) know what the electrical
signals mean
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Calibration example
• For pH probes, an electrical signal is generated
at about 60mV per pH unit. This is a slope.
– We need to locate this line, with the intercept.
– Three points is best to define a line and locate the
calibration curve; two is the minimum
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Dissolved Oxygen example
• For the DO we also want to establish a line
(standard/calibration curve)
– We don’t have nice standards though like for pH
– We therefore set a zero and a span (i.e. 100%)
• Expose the probe to deoxygenated water (or a
simulator)
• Expose the probe to fully saturated water
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Temperature example
• The same idea continues, at least two points,
more is better to define the calibration curve
– What can be used for temperature?
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Process instrumentation
• The previous examples are controlling the process environment
with real-time, online measurements.
– A setpoint is defined and the control system works to make sure the
process variable equals the setpoint
• There is also offline instrumentation, where you need to take a
sample and use an instrument not connected to the system
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Process instrumentation (cont’d)
• The data from these instruments can be used
to make adjustments to your culture variables,
e.g.
– Cell growth rate
– Metabolite consumption
– Toxic substance build-up
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