Upstream Bioprocessing Flashcards
1
Q
What are the products of traditional bioprocessing?
A
- Antibiotics
- Vaccines / viruses
- Antibodies
- Recombinant therapeutic proteins (i.e. MAb) (Global market worth £30 billion/year)
2
Q
What are the applications for (Stem) Cell bioprocessing?
A
- Cells and Tissues
- Banking/Drug screening programmes
- Larger scale healthcare applications →Stem cell therapies (potential to cure diseases, not just to treat/address symptoms)
3
Q
Describe bioprocessing complexity
A
4
Q
Draw a diagtam of therapeutic agent production
A
5
Q
Draw a table of cell requirements
A
6
Q
Describe the INDUSTRIAL SCALE meaning for biotherapeutics
A
- >10,000 Litres – Monoclonal antibodies (MAbs) and proteins
- 2,000 Litres – Viral vaccines
- 50 Litres – Human mesenchymal stem cells for therapies
(Lawson et al, 2017)
7
Q
Scaled out production
A
8
Q
Scaled Up production
A
9
Q
Advantages of scale-up
A
- Is the current industrial standard.
- It is well established.
- Cost effective.
- Appropriate for Allogeneic approaches
10
Q
Advantages of scale-out
A
- Allows for parallel runs.
- In case of contamination or failure, only minimal loss.
- Appropriate for Autologous approaches.
11
Q
Disadvantages of Scale up
A
- Many engineering challenges (e.g. mixing, gradients, temperature and pH maintenance).
- In case of contamination or failure, maximal loss.
- Equipment scale challenges.
12
Q
Disadvantages of scale-out
A
- Highly laborious unless automation is used.
- Less cost effective.
13
Q
What are the biological and engeineering considerations for scaling up production?
A
- Scale-up is usually the final step of any R&D programme
- Biologists usually don’t fully understand the challenges of a scalable process
- Engineers don’t always understand the biology
- Engineers don’t get involved soon enough
- Scaling up a bioprocess requires important engineering aspects that have to be considered early in the process => DETRIMENTAL effect on the overall manufacturing process
14
Q
Inefficient scale up: Lower economic performance
A
- Lower yield
- Lower capacity
- Lower product quality
15
Q
Inefficient scale up: Operational instability
A
- Mechanical instabilities
- Genetic instability
- Variability
16
Q
Name some planar culture vessels
A
- T-flasks
- Multilayer plates
- Cell factoreies
- Roller bottlesCompacT SelectT– automated cell culture platform
17
Q
What are the advantages of planar culture vessels?
A
- Well accepted and well understood
- Reproducible and standardised
- Option to be completely automated (e.g. TAP CompacT SelecT)
18
Q
What are the disadvantages of planar culture vessels?
A
- Static systems → heterogeneous culture environment (Note: except roller bottles that provide a slightly more homogeneous environment, but with mass and energy transfer gradients)
- Poor surface area-to-volume ratio
- Surface limitation and labour intensive →Unfeasible for the production of large cell lot sizes (109/1012 cells)
19
Q
What is a bioreactor?
A
A vessel in which a bio-based process takes place
20
Q
What is a reactor?
A
A vessel in which a chemical reaction takes place
21
Q
Which vessels are cells grown (e.g. bacteria, yeast)?
A
Fermenter
22
Q
Give a brief history of bioreactors
A
•1918 - Biochemist and former Israeli President Chaim Weizmann developed a bacteria fermenter for the production of acetone.
•1944 - De Beeze and Liebmann used the first large scale (>20 litre capacity) fermenter for yeast production.
•1970s – First commercial fermenters, autoclaves, freeze dryers, colony counters were developed.
- 1980s – First benchtop bioreactor was designed for animal cell culture.
- 1990s – Microprocessor controlled shakers, provide precision control of set-points, alarms, running time, agitation, pCO2 and temperature.
•2001 – Benchtop system can control up to 4 bioreactors.
•2005 – Disposable cell culture flasks.
23
Q
Draw a diagram of bioreactor designs
A
24
Q
Explain how a Pneumatic bioreactor works: Airlift (bubble column)
A
- Aeration and mixing is achieved by gas sparging
- Less energy than mechanical stirring
- Satisfactory heat and mass transfer performance
- Low shear→ suitable for plant or animal cell culture
- Absence of any moving parts →reduced contamination and easy maintenance
- DO and pH control achieved by varying the composition and the rate of the gas flow through the column
- Challenge: foaming
25
Explain how a Hydraulic bioreactor works: Packed (fixed) /fluidised bed
* Used for immobilised cells in high density cultures intended for protein production in long term cultures
* Cells grow on macro-porous beads (e.g. glass, ceramic)
* Mass transfer is achieved by recirculating the medium in a loop for oxygen enrichment
* Low shear rates, simple medium exchange and cell /product separation
* As particles are in constant motion, channelling and clogging is avoided (only Fluidised Bed)
* Non-homogeneous cell distribution (only Fixed Bed), difficult cell harvest.
26
Explain how a Hydraulic bioreactor works: Hollowfibre
* Anchorage-dependent / independent cells
* Cartridge of capillary-like tubules with perfusable membrane walls
* Provides a 3D environment for cell growth
* Enhanced mass transfer
* Used with hepatocytes as an artificial liver (Bio-Artificial Liver-BAL)
* Bio-Artificial Kidney – BAK
27
Explain mechanical bioreactor: Stirred Tank (STR)
* Mixing is achieved by mechanical means using an agitator (e.g. impeller)
* Baffles can be used to reduce vortexing
* Relatively high input of energy
* Heating/cooling mantles
* Gasing options: headspace or purging
* Multiple impellers improve mixing within tall bioreactors
* Impellers with different sizes and shapes are used to produce different flow patterns inside the vessel
28
Mixing in STR: flow patterns
29
STR: impellers
## Footnote
**Rushton**
* Radial flow
* Flat blades
* Suitable for high cell density cultures (e.g. bacteria, yeast, some fungi)
30
STR: impellers
## Footnote
**Pitched-blade**
* Flat blades, 45°
* Simultaneous axial and radial flow
* Suitable for mammalian cell cultures
31
STR: impellers
## Footnote
**Marine**
* Axial flow
* Gentle mixing
* Suitable for mammalian cells
32
What are the characteristics of microcarriers?
**Small:** to maximize cell culture surface area (high area-to-volume ratio)
**Light:** to allow easy suspension in culture medium
**Density:** slightly higher than medium to allow for fast settling when required
**Transparent:** to allow easy observation of cell attachment and growth
**Appropriate surface chemistry:** to promote cell attachment
33
Scalable culture systems - microcarriers
* 1st developed microcarrier in 1967 by Van Wezel (dextran-based)
* Micrometer sized particles made typically from polymers such as polystyrene, modified dextran, gellan
* Commercially available from multiple manufacturers
* Different surface chemistries available that determine physico-chemical properties
* Suitable for adherent-dependent cells
* Allow cell attachment and proliferation in suspension cultures
* Increased surface area-to-volume ratio compared to planar systems
34
Name some scalable culture systems - microcarriers
* Plastic (polystyrene)
* Cytodex 1 (dextran)
* Collagen
* Cytopore (gelatin)
* Hillex II (modifies polystyrene)
* NUNC 2D MiroHex (polystyrene)
35
Bioreactor operation modes: Batch
- Reactants (nutrients and cells) are introduced at the start of the culture
- Products are recovered at the end (batch wise)
36
Bioreactor operation modes: FED BATCH / REPEATED
- Reactants are fed semi-regularly (semi-continuously
- Products are recovered at the end (batch wise)
37
Bioreactor operation modes: CONTINUOUS (PERFUSION):
- Reactor contents is mixed and homogeneous
- Reactants are fed in and product is removed constantly
- Volume of the reactor is constant
38
Single use bioreactors
Solid trend: disposable bags for media storage, process development, for inoculation, actual bioreactor vessels
39
What are the advanatges of single use bioreactors?
Advantage: quicker turn-around times, no steam sterilization or cleaning needed, substantially decreases overall timelines and operational complexity.
40
Single use bioreactors: Two types based on medium agitation
1. Stirrers integrated into the plastic bag
2. Agitated by a rocking motion (e.g. WAVE bioreactor)
* Both up to a scale of 1000 Liters volume
* Both pre-sterilized.
