Lecture 9b scaling up

Lecture 9 Animal Cell BiotechnologyScaling up the production process

pH

• set point pH of 7.4 ± 0.1 common

• without buffering the pH could fluctuate

• for small scale operation, can maintain pH by using gaseous CO2 to control culture pH

Lecture 9 Animal Cell BiotechnologyScaling up the production process:

Controlling the pH with CO2

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P161.


• for larger scale cultures, can directly add acid or base to maintain pH

• insert probe into culture to detect changes in pH

• acid or base pumped in accordingly, under automatic control

→ pH ↓, add base (concentrated sodium bicarbonate)

→ pH ↑, add acid (concentrated HCl) → not normally a problem due to lactic acid production


Oxygen requirements• supply of oxygen to satisfy cell metabolism is one of the

major problems associated with culture scale-up

• O2 consumption rate: 0.06-0.6 mM/hour for 106 cells/ml

• for small volumes (< 1 litre) O2 diffusion from the headspace through the culture surface is sufficient to meet the oxygen demand

• as the volume increases, the surface-volume ratio decreases

Dissolved oxygen polarographic electrode

Dissolved oxygen polarographic electrode (InPro 6050, Metler-Toledo, 2006). The cathode, where the half-reaction with O2 (O2+2H2O+4e-→4OH-) takes place, is in contact with a membrane, that allows the transport of the dissolved oxygen from the external medium. At the anode, the silver oxidation (Ag++Cl-→ AgCl + e-) takes place.

Membrane-covered oxygen electrodeFig. 9.8

Pt cathode: O2 + H2O + 4e → 4OH-

Ag anode: 4Ag + 4Cl- → 4AgCl + 4e

The limitation of O2 supply by diffusion through the head space

Culture volume

(L)

Head space area (cm2)

O2 supply (mmol/h)

O2 demand

(mmol/h)

1 100 0.063 0.063

10 500 0.313 0.625

100 2500 1.56 6.25


OTR = oxygen transfer rateOUR = oxygen uptake rate

To supply sufficient O2 to cells and to avoid O2 depletion:

OTR > OUR


• at > 1 L, the surface-volume ratio is too low to satisfy overall O2 demand

• surface-volume ratio of a fermentor defined by aspect ratio:

aspect ratio = diameter of culture/height of culture

Aspect ratio = width/ height of cultureFig. 9.7

Lecture 9 Animal Cell BiotechnologyScaling up the production process:

Bubble death !


Strategies to prevent cell damage• use of chemical agents to reduce cell damage, such as

0.1% Pluronic F68 → synthetic copolymer of ethylene and propylene oxide, reduces cell:bubble interaction by preventing attachment of cells to bubbles

• cover gas sparger with fine wire mesh to reduce the number of bubbles reaching cells

• use of alternate fermentors (i.e. air lift fermentor)• sparge media in a secondary vessel• use gas permeable tubing (i.e. thin-walled silicone

tubing) within bioreactor

Strategies for controlling dissolved oxygenFig. 9.9

(a) Change in air flow rate(b) Intermittent oxygen sparging

(d) Spin filter isolates cells from sparged gass

(c) Control of gas composition

Dissolved oxygen control

O2 flow rate controller

N2 flow rate controller

C (%)

Q 1

Q 2

PC

C (%)

QT = Q1 + Q2 Q 1

Q 2

PC

DO is controlled by the adjustment of the oxygen fraction in the sparged gas. Flow rate is kept constant and corresponds to the sum of the two controlled gases Q1 and Q2.

Set point

_

+

Modulated feedback control

Fig. 9.11

A typical control loop

controller actuator

sensor

process

+

-

thermometer

PID bioreactor electric resistance

measured value

error

set-point

A set-point is compared to the measured value by the sensor. An error measurement based on a signal to the electric resistance (actuator) is generated by the controller, that will heat up the bioreactor (process).

• On-off controller, in which the action can only assume two states (on or off).

• Controller modulated by pulse width (Pulse-Width Modulation or PWM). In this control type, the action is also on or off but the time that the actuator stays on within a certain cycle can be adjusted continuously. , allowing a final operation of different intensities.

• Cascade controller, composed of one master and one slave loop. This is used when a more rigid control of a process variable is required, for instance, the temperature of the culture medium.

• P-I-D controller, or Proportional-Integral-Derivative. It's based on the principle that the action is taken not only on how large is the error (difference between desired and measured values), but also on the sum of past errors (integral of the error) and to the rate that the error is changing (derivative of the error). where actuation is the controller output, error is the difference between the desired value (set-point) and the one measured by the sensor, t is time and P, I and D are constants that need to be adjusted for each system. The adjustment of the constants for a process, is called P-I-D controller tuning.

dt

error dD. error.dt I. P.error actuation

dt

error dD. error.dt I. P.error actuation

Proportional control.

• The output of the controller is proportional to the error signal.

• = 0 + k.E• where = output signal of the controller• where 0= output signal when the error is zero• where k = controller gain or proportional band• where E = error or deviation from the set point

Integral and derivative control.• Integral control. The output of the controller is a function of

the integral of error and time. Here, the control action increases with time as long as the error is registered.

• = 0 + tI • where tI = integral time constant

• Derivative time. The output of the controller is a function of the rate of change of the error.

• = 0 + td .

• where td = derivative time constant

d Edt

E dt

Feeding flow rate control system based on glucose concentration measured in real time (adapted from Ozturk et al., 1997)

Other bioreactor types

• Airlift fermenter

• Packed bed bioreactor

• Hollow fiber bioreactor

• Single use bioreactor

Airlift fermenter

• Gas mixture sparged into the reactor at the base.

• The gas flow cause the culture medium to rise.

• No mechanical agitators.

The fermenter is 200' high and 25 ft diam. (Chem. Eng. News, 10-Apr-78)

Lecture 10 Animal Cell BiotechnologyOther fermentor Systems:

Air lift bioreactor

Waites et al. 2001. Industrial Microbiology: An Introduction. Oxford: Blackwell Science. P 98

Airlift fermenter

exhaust gases

air inlet

draught tube

Fig. 9.12


Air lift bioreactor

• sparged gas agitates and aerates column

• no mechanical parts, no shear stress

• gas flow through inner tube lifts cells and medium,

• cells and medium spill out over draft tube, circulate down side

• 2-2000 litre reactors available

Hollow-fiber bioreactor• bioreactor consists of a cartridge containing bundles of

synthetic, semi permeable hollow fibers which are similar to the matrix of the vascular system

• good for anchorage-dependent or independent cells

Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p84

Hollow fiber bioreactor

• In fibrous-bed bioreactor, the cells are immobilized on the fibers in the bioreactor.

• Following is scanning electron microscope photos of human osteosarcoma cells in an artificial growth medium, a fibrous-bed bioreactor.

• The cells cling to Dacron fibers

Hollow fiber bioreactorFig. 9.13

Electron micrograph of a cross section of hollow fibresshowing a cell mass in the extracapillary space

Fig. 9.14

gas in

gas outglass column

glass beads

Packed-bed bioreactor

circulation pump

circulating media

Fig. 9.15

Packed - Bed Bioreactor


Packed-bed/fixed-bed bioreactor – glass bead column

• good for anchorage-dependent cells

• 1-100 litre volumes

• cells attach to surface of beads (3-5 mm)

• aerated medium is pumped in from a secondary vessel

• inoculation could be a problem, uneven growth

• heterogenous bioreactor – environment may not be the same throughout the column


Packed-bed/fixed-bed bioreactor – ceramic bioreactor

M.Butler and M.Dawson, eds. 1992. Cell Culture Labfax. Oxford:BIOS Scientific Publishers. p207


Packed-bed/fixed-bed bioreactor – ceramic bioreactor

• ceramic cartridge (30 cm long, 4 cm wide) containing a series of parallel channels (1 mm2 square channels)

→ cells attach and grow on the walls of the channels

• fresh medium is pumped in through the chambers, spent medium is returned to main reservoir

• secreted products can be isolated from the spent medium

Fig. 9.17


Packed-bed/fixed-bed bioreactor – the cell cube

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P170-171.


Packed-bed/fixed-bed bioreactor – the cell cube

• stack of 20 cm2 polystyrene plates separated by 1 mm spacers

• cells attach to either side of plate

• culture medium is “sprayed” over the surface of the plates by multiple inlet ports


Fluidized-bed bioreactor

• similar to packed bed, but particles/ micro-carriers are separated by liquid media

• immobilized cells are held in suspension by an upward flow of liquid medium

M.Butler and M.Dawson. 1992. Cell Culture Labfax. Oxford:BIOS Scientific Publishers. p205


Fluidized-bed bioreactor

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P172.

Cytopilot Mini

2 litres

Fig. 9.19

Cytopilot 100 litres

Single use bioreactor

www.applikon-bio.comMade of STEDIM 71 film

Summary

• Types of bioreactors– Stirred tank– Airlift – Packed-bed– Fluidized-bed

• Parameters to control– Stirring– Temperature– pH– Dissolved oxygen

Technology

Lecture 9b scaling up