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Cell Culture in Process
Development
Andrea Cortez
What to expect from this
Presentation?

Overview of Cell Culture Process

Cell Culture Maintenance and Monitoring
with process development considerations

Insights for Future Technicians
What are Cells?

Cells are the smallest discrete elements
of living organisms.

Composition : 90% water, the remainder
is half protein, 15% each carbohydrates
and nucleic acids, and 10% lipids, with
the last tenth representing trace
compounds and elements such as
vitamins and minerals.
What is Cell Culture?
Basic : It’s like farming or animal husbandry on a
very small scale. It’s about the feeding and
care of living things so that they will provide
valuable products.
Technical : Propagation of cells outside the
organism, in-vitro, (microorganisms, plant or
animal)
Biotechnology : Process utilizing mammalian
(e.g., CHO) cells to make proteins that are
similar to those made in humans for
therapeutic or research purposes
What is Fermentation?
Basic :A chemical process in which an agent
causes an organic substance to break down
into simpler substances; the anaerobic
breakdown of sugar to CO2 and alcohol by
yeast; the breakdown of carbohydrates by
microorganisms to produce energy.
Biotechnology : Large scale cultivation of
microorganisms, i.e., manufacturing or
production scale. In some cases, it is a
process utilizing bacterial (e.g., E. coli) cells to
produce proteins.
What is Process Development?
Improvement or change in any process
towards an ideal point of maximum
capability, potential, quality or usefulness
Propagation of Cell Cultures
Adherent Monolayer-Cells adhere to the
substrate in order to survive and proliferate;
hard to scale-up; used for R&D purposes only.
Suspension Cultures – cells grow floating in
medium; cells survive and proliferate without
attachment, e.g., transformed cell lines, or
cells from malignant tumors (hybridoma cells);
Preferred method for scaling up cultures, i.e.,
large-scale manufacturing.
Propagation of Cell Culture . . .
In order to maintain cell culture, the cells must be
sub-cultured (splitting or passaging).
 Subculture by dilution – the process of
transferring the required number of cells in
media suspension from an old flask (source)
into another, at a new, lower density, then
topping off spinner with new fresh medium.
 Medium Exchange – cells are separated
from the media by centrifugation, old media
are removed and new fresh media are added
back to the cell pellets.
Cell Metabolism
To stay alive, an organism or cell needs a continuous
supply of energy in usable form.
Metabolism is a catch-all word to describe the energyrelated chemical processes occurring within an
organism or cell.
Cellular Respiration is the process by which the chemical
bonds of energy-rich molecules such as glucose are
exploited for energy.
Some of that energy is trapped in the form of a
nucleotide called adenosine 5’-triphosphate (ATP).
CO2 and water are the waste products.
Glycolysis is the breakdown of glucose to pyruvic acid
(pyruvate), does not require oxygen and is performed
by all living things.
Cell Metabolism
In cell culture, it is literally the medium
through which we provide a benign
physical environment for the cells not
only to thrive, but also to produce.
We want to provide them the kind of
environmental conditions that will make
them happy and free of stress and mostcost effective.
Phases of Cell Growth
Phases of Cell Growth
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Lag Phase. Immediately after inoculation of the cells into
fresh medium, the population remains temporarily
unchanged. Although there is no apparent cell division
occurring, the cells may be growing in volume or mass,
synthesizing enzymes, proteins, RNA, etc., and
increasing in metabolic activity.
Exponential (log) Phase. The exponential phase of
growth is a pattern of balanced growth wherein all the
cells are dividing regularly. The cells divide at a constant
rate depending upon the composition of the growth
medium and the conditions of incubation. This is the
ideal phase for experiments.
Phases of Cell Growth
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Stationary Phase. Exponential growth cannot be
continued forever in a batch culture (e.g. a closed
system such as a test tube or flask). Population growth is
limited by one of three factors: 1. exhaustion of available
nutrients; 2. accumulation of inhibitory metabolites or
end products; 3. exhaustion of space, in this case called
a lack of "biological space".
Death Phase. If incubation continues after the
population reaches stationary phase, a death phase
follows, in which the viable cell population declines.
During the death phase, the number of viable cells
decreases geometrically (exponentially), essentially the
reverse of growth during the log phase.
Microbial Nutrients Requirements
Carbohydrates: For bacteria and CHO, glucose is
the most readily metabolized sugar; most fungi
can use disaccharides
Lipids: Some bacteria and fungi can use longchain fatty acids such as linoleic and oleic acids.
Steroids other than cholesterol are generally
unnecessary. Ergosterol is a nutritional
requirement for all fungi
Purines and Pyrimidines: Generally only in
bacteria have cases of purine and pyrimidine
metabolism been reported. Algae do not use
them at all.
Microbial Nutrients Requirements
Vitamins and Growth Factors: There is a considerable
species variation in the requirements of vitamins and
related factors by microorganisms. Generally vitamins
A, C, D, & K are unnecessary for growth.
Amino Acids: Useful to most industrial microorganisms,
w/ the exception that yeasts have no use for critrulline.
Some bacteria can metabolize forms of amino acids
that eukaryotes cannot.
Nitrogen Sources: Few popular species require or use
N2 compounds, but some species have been shown
to use them. Fungi requires ammonia, nitrate, and
nitrite.
Microbial Nutrient Requirements
Sulfur Sources: Some yeasts can use elemental
sulfur and sulfate but not want sulfur-containing
organic compounds. Bacteria require
glutathione and thioacetic acid, whereas yeasts
will take sulphonic acid amides, thioacetate,
thiocarbonate, thioglycolate, and glutathione.
Trace Elements and Inorganic Ions: Mineral
nutrients required by microorganisms are
species dependent, but consist generally of Fe,
K, Mg, and Mn. Sometimes S, N, Ca, Co, Cu, P,
and/or Zn are required.
Basic Steps in Cell Culture
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Formulation of Media
Sterilization of Media (use of filter)
Note: Pre-warm media prior to use (inoculation)
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Sterilization of Equipment (use of autoclave)
Creation (inoculation) of an active culture
Growth of the organisms
Daily monitoring of cell culture (morphology,
physical environment, color of medium, cell
density, feed/glucose requirement)
 Induction/extraction of the product
 Disposal of effluent
Cells Physical Environment
pH (7.2 – 7.4)

Use buffers: bicarbonate when culture is acidic, CO2 when too
alkaline
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Use pH probe measurement (on-line/off-line)
Temperature (37.00C)

Use heating blanket

Use RTD for measurement (on-line)
Osmotic Pressure (260-320 mOsm/kg)

Salt may need to be added or removed to accommodate cell’s
needs

Use Osmometer for measurement (on-line/off-line)
Dissolved Oxygen (Physiologic 20-80% air sat)

Air sparge/O2 supply, agitator

Use dO2 probe (on-line/off-line)
Measurement of Cell Growth and
Viability

% PCV (Packed Cell Volume)-use a special
graduated centrifuge tube to measure cell
density (alive and dead cells)
 VCD (Viable Cell Density) – use
hemocytometer to determine cell density
based on live cells/mL
 % Viability – use hemocytometer or trypan
blue staining method is used to count dead
cells that are stained blue
Other Parameters Measured
Nutrients – Glucose and Glutamine
Waste Product
Lactate: produced by the oxidation of pyruvate;
inefficient use of glucose; decreases pH and
increases osmolality of medium; inhibits cell
growth above critical concentration
Ammonia : by-product of glutamine; can inhibit
cell growth and production
Physical Environment : pH, Temperature, dO2,
osmolarity, Na.
Purpose of Cell Culture in Process
Development
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Improve product quality (safe drugs)
Increase the amount of final product
Robust process before transfer to
manufacturing
Validation studies
Optimization studies
Assessment of new technology
Assessment of media sources
FDA approval
Purpose of Cell Culture in Process
Development

Majority of the experiments are for data
generation
-one experiment is about 18 x 2.0L cell
cultures

Non-GMP (Good Manufacturing
Practices) Environment
Overview of a Cell Production
Process
Inoculum Train
(Non-Selective
Medium)
Production
(Non-Selective
Medium + Feed)
Harvest
Increasing Cell Density
Cell Source
Cell Mass
“Maintenance”
“Accumulation”
Process
Manipulations
Cell Growth and
“Productivity”
E. Coli vs. CHO Cells
Doubling times :
E.Coli @ 37 0C 1 hour
E.Coli @ 33 0C 2 hours
CHO @ 37 0C 16-24 hours
CHO @ 330C 30 hours
Final Fermentation/Cell Culture Times:
E. Coli
1-2 days
CHO
1-2 weeks
Growing E. Coli vs. CHO Cells
E. Coli
 Flask in Shaker
 Rapid growth rate
 OD for counts
 Constant air supply
 Generates heat
 Continuous Glucose
Feed
 pH control with NH3
 Produces Acetic acid
CHO
 Spinner w/ agitator in a
spinner base
 Slower growth rate
 PCV for counts
 dO2 control air/O2
 No heat built up
 Glucose in medium or
feed (may be added in
the entire experiment
depending on need)
 pH control with HCO3
 Produces Lactic acid
Standard cell culture configuration
for a 3 Liter BioNet Bioreactor
Sensor Package:
pH Electrode (220 mm
Length)
pH Cable (6 foot Length)
Dissolved Oxygen Sensor
(220 mm Length)
Dissolved Oxygen Cable
(6 foot Length)
Temperature Sensor (220
mm Length)
Source : Broadley James Corporation (www.broadleyjames.com)
Bioreactor Configuration Continuation . . .
Standard Configuration for Cell Culture:
Headplate (18 ports)
Autoclavable Glass Vessel
Lipseal Stirrer Assembly
Scoping Marine Impeller
pH Electrode Holder
Dissolved Oxygen Sensor Holder
Triple Port
Thermowell
Sparger Pipe
Air Overlay Pipe
Septum Holder
Adjustable Sample Pipe
Blind Caps (8 each)
Mill Nuts
Stand
Source : Broadley James Corporation (www.broadleyjames.com)
Bioreactor Configuration Continuation . . .
Motor Package:
40 mm Brushless Motor
Digital Motor Controller
Motor Interface Cable
Additional Options:
Level/Foam Sensor and Cable
Air Outlet Condenser
Assorted Impellers
Consult Factory for Custom Configurations
Heating Blanket
Source : Broadley James Corporation (www.broadleyjames.com)
Headplate Configuration
Sparger Pipe Line
pH Electrode
dO2 Sensor
Baffle
Agitator
Baffle
Thermowell
Blind Cap
Air Overlay
Triple Port (for base, vent)
Harvest Tube
Septum Holder
Feed Line Port
Sample line
Source : Broadley James Corporation (www.broadleyjames.com)
Typical Operations of Technicians
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Prepping the bioreactors (sometimes fast
turnarounds)
Set-up and DCU programming
Daily Counts
Inoculation of 2.0L/10.0L/20.0L
Harvest of Cell Cultures
Passaging of spinners
Solera or passaging of 20L
Support Tasks (maintenance of machines, etc.
Main Goal : To execute the experiments and produce good data.
Technician Skills Necessary in Cell
Culture Laboratory
Technical Skills:
Aseptic technique – Contamination is a major problem-happens even to the
best!!! Sterile techniques and practices are very important when performing
cell culture.
 Use of laminar Flow Hood
 Use of filter or sterilizer
 Use of disinfectant
 Single use flask
Basic Math – Conversions and measurements
-Mass Balance Equation
Input = Output
C1V1 = C2V2
Good Documentation - Must be permanent, clear, timely, correct, traceable,
complete
 Certifies that written procedures have been followed
 Provides complete history of product
 Provides means to investigate problems that may arise
Technician Skills Continuation . . .
General Skills:
Communication – Good verbal and written
communication
Teamwork –Participates in and contribute to a
team environment of mutual respect and high
performance
Accountability – Takes accountability, responsibility
and initiatives in building commitment and
driving results.
Detail-Oriented – Follow procedure/SOP in detail.
Always thinking about safety on the floor,
improvement of operation, success of each
project
Acknowledgement
Paul Bezy
Tara Yokomizo
Gokhan Abali
Hardat Prashad
Scott Broadley