Lecture 9 Animal Cell BiotechnologyScaling up the production process

Scaling up animal cell/microbial processes

• optimal physiological conditions obtained via small scale processes maintained for large scale operation

• must consider:

1. fermentor/bioreactor system

2. control of agitation, pH, temperature, dissolved oxygen

Lecture 9 Animal Cell BiotechnologyScaling up the production process

T-flasks

Lecture 9 Animal Cell BiotechnologyScaling up the production process

Spinner bottle

Lecture 9 Animal Cell BiotechnologyScaling up the production process

Multiple process vs. unit process• can have 1000 100 mL flasks or a single 100 litre

fermentor (same volume)

1. Multiple process• easy to replicate samples and conditions, large surface

area/volume ratio, tedious to take care of all samples, labor intensive

2. Unit process• good for large scale production of biological products,

ease of handling, decreased possibility of contamination

Lecture 9 Animal Cell BiotechnologyScaling up the production process

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P203.

Lecture 9 Animal Cell BiotechnologyScaling up the production process

Suspension vs. Anchorage-dependence

• suspension cells are readily scaled up to production level using similar fermentation vessels as microbial cells (with modifications)

• anchorage-dependent cells require a surface for attachment in order to grow

→ grow as static cultures (i.e. hollow fibres)

→ grow on microcarriers, in a stirred tank reactor (fermentor)

Cell culture processes• Anchorage-dependent systems

Microcarrier (dextran, collagen or plastic) : 200 µm

Advantages: -Higher cell yields - increased productivity. - ↑ surface ratio (100 -200 cells). - suspension cultures • Suspension systems: > Industrial culture

Cytopore microcarriers

Cell Culture system• Vero cells

• Why? Regulated by WHO

• already licensed for vaccine

production such as polio, influenza,

rabies, others

• Serum-free medium

• Why? Safety and reproducibility

• Microcarriers

• Why? Scalability and ease of infection

•

Macroporous microcarrier

Lecture 9 Animal Cell BiotechnologyScaling up the production process

Usually scale up 10x at a time:

1 → 10 → 100 → 1000 → 10,000 L

Factors limiting scale-up• supply of oxygen

• shear damage from mixing

• build up of toxic metabolites

Lecture 9 Animal Cell BiotechnologyScaling up the production process

during scale up decreased product yields may be experienced due to the following:

1. inoculation – must consider quality and quantity of inoculum used to start process

2. choice of medium – cheaper materials often used for large scale production due to cost limitations - omission / reduction of serum, antibiotics

3. large scale sterilization – longer sterilization times may result in degradation of heat labile compounds, reducing quality of media

4. development of gradients - larger fermentors/bioreactors may be subject to development of nutrient, temperature, pH, and oxygen gradients

5. other factors -scaling up may also alter the generation of foam, shear forces

$ $$

Lecture 9 Animal Cell BiotechnologyScaling up the production process:

The Stirred Tank Reactor

• bioreactor is another term for fermentor

• stirred tank reactor simplest and most widely used system (pot and paddle)

→ small vessels (<20 litres) made of glass

→ large vessels (>20 litres) made of stainless steel

Bioreactor (Fermenter)

• Vessel that allows the growth of cells.

• Stirred tank bioreactor (STR). Growth: Bacteria, yeast, mammalian cells.• Homogeneous solution mixed by an impeller: -Laminar stirred: Just one dimension to lift the liquid.

www.corning.com

Cultures >1L: Uneven oxygen exchange and poor nutrient distribution

Stirred Tank Reactor

• Shape: -curve bottom mammalian cells -square bottom poor mixing

• Materials used: Vessel up to 10 L (glass) Head plate steel stainless

• Heat control: (heat pad or jacket water) - Constant To -mammalian cell culture (37o C)

Lecture 9 Animal Cell BiotechnologyScaling up the production process:

The Stirred Tank Reactor

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P156.

Fig. 9.2

Fig. 9.3