Advanced Bioprocess

Engineering
Prof. Sakis Mantalaris

Department of Chemical Engineering & Chemical

Technology

Imperial College London

Lecturers
Prof. A. Mantalaris
Dr. E. Veliou
Dr. P. DiMaggio

Course Outline

Overview & Introduction

Cellular Systems & Metabolism
Monoclonal Antibody Technology & Genetic Engineering
Cellular & Bioprocess Characterisation
Proteomics, Genomics & Metabolomics
Analysis of Data
Material Balances
Fluid Flow & Mixing
Mass Transfer
Cell Growth Kinetics
Reactor Engineering & Scale-up

What is a bioprocess?
Any process that uses living cells or their
components to obtain desired products.
Bioprocess engineering is the application
of engineering principles to design,
develop and analyse processes using
biocatalysts.
What type of cells are used?
What cell components can be used?
What are the applications?

Course textbooks
Principles of Fermentation Technology
by Stanbury, Whitaker and Hall
Bioprocess Engineering, Basic Concepts
by Shuler and Kargi
Biochemical Engineering
by Blanch and Clark
Bioprocess Engineering Principles
by Doran PM
Bioreaction Engineering Principles
by Nielsen and Villadsen

Steps in Bioprocess Development

Discovery

Cell line construction

DNA

Clinical trials

product

supply

Industrial production

Product formulation

Purification

Industrial processes: Heparinase I production from E-coli

(a) Production by insoluble expression; (b) Production by soluble expression

Biotechnol. Bioeng. 2007, 53 (6): 575-582

Heparinase I production from E-coli

Start with a volume of several
thousand litres
Large-scale fermentation
requires substantial inoculum
volume
Derive approximately 100L of
product
Several purification steps
Including 2 large-scale affinity
chromatography steps: binding
agents, such as proteins A and
G, very expensive

Penicillin industrial production process

Valuable products made
at very low levels
Fragile, often unstable,
products
Temperature control
Sterilisation
Fermentation scale-up
Long growing cycles

Homogeneity in large
volumes (agitation)
Oxygen supply
Heat removal

Insulin industrial production process

Biotechnol. Bioeng. 1995, 48: 529-541

Bioprocess considerations
Choice of
host cell line
reactor system
operation mode
Design of
agitation mechanism
medium and feed
Control system
Measurements, data
acquisition and processing

Host cell line

Insulin produced in E-coli (bacteria)

Intracellular product that forms inclusion bodies
IBs need to be retrieved by cell disruption and
solubilised
In mammalian & yeast cells, products are
usually secreted

Host cell line: product structure

Non-glycosylated products, e.g. Insulin can be
produced by any host
Opt for easy to cultivate cells, such as bacteria or yeast

Glycosylated products
Yeast cells can perform some glycosylation reactions
Have been engineered to perform mammalian-like
glycosylation more recently
Mammalian cells the only available option currently

Examples of glycosylated products

Recombinant proteins: interferon, erythropoietin
Monoclonal antibodies: brain, kidney ovarian and prostate
cancer, autoimmune disorders: Crohns disease and
psoriasis

Why is glycosylation important?

Antibody
Complement

NK cells/neutrophils

ADCC

Cancer cell
Both mechanisms act through glycosylation site of the antibody

Mammalian cell line construction process

Volume
1 ml

Transfection: 96 well plates

2 ml

24 well plates

200 cell lines

5 ml

Tissue culture 25cm2

100 cell lines

25 ml

Erlenmeyer flasks: suspension

50-70 cell lines

Erlenmeyer flasks: growth curves

20-30 cell lines

50-200 ml

400 ml

Erlenmeyer flasks: fed-batch evaluation

Transfection: Insertion of product DNA into cells through

electroporation or with chemical agents

2000-3000 cell lines

10 cell lines

If results are satisfactory...

400 ml

Stability study

5-10 L

Fermenter study

10 cell lines

If product goes ahead to

production
1ml to 10L

Cloning and scale up

1 cell line

Cloning: Start with one cell and allow a colony to form

Why? Ensure all cells have similar growth patterns and

product synthesis rate

Timeline and decision-making

Timeline
4-6
weeks

Transfection: 96 well plates

1-2
weeks

24 well plates

1 week

Tissue culture 25cm2

2 weeks
1 week

2 weeks

Decisionmaking

confluence
confluence &
product concentration

Erlenmeyer flasks: suspension

Cell & product

concentration

Erlenmeyer flasks: growth curves

Cell & product

concentration

Erlenmeyer flasks: fed-batch evaluation

Cell & product

concentration;
metabolism

Top cell lines

Ranked according to
Cell growth and metabolism characteristics
Final product concentration
Product structure
Avoid high mannose structures due to immunogenicity
Match (as much as possible) structure of molecule
identified during drug discovery

Degree of product aggregation

Looking for less than 10%

Result: select cell line that can be used on

industrial scale
Can move on to clinical trials

Reactor system
Reactor design
hold-up
stirrer speed & power
consumption
oxygen transfer
coefficient
mixing time

Operation mode
Agitation mechanism
sheer damage
aeration
homogeneity

Fluid flow & reactor design

Fluid flow & reactor design

Reactor design: Immobilised cells

Packed bed
Minimal cell damage
Used commercially with
immobilised cells and
enzymes
Aspartate, fumarate
production
Resolution of amino acid
isomers

Mass transfer facilitated

by high liquid flow rates
What do you notice
about air supply?

Heterogeneous systems
Enzyme reactors

Enzyme
basket

Medium and feed design

Hexose (glucose/fructose)
Carbohydrates

Complex
Lipids

Lipids

Nucleotides

Amino acids

Amino acids

Energy

Cofactors &
Vitamins

Why can we not vary one

experimental parameter at a time to
study biological systems?
Need for:
Data analysis
Systematic design of experiments

Cellular characterisation & process control

Continuous
monitoring

Control
- pH using CO2

- pH

- O2

- dO2

- Foaming using
antifoams

Measurements

- Stirring speed

- Cell concentration
or biomass

But also wish to

control:

- Nutrient
concentration

- Cell growth

- Product
accumulation

via feed addition

- Cell death

via minimisation of
toxic metabolites

Case study

Group 1
Microbial system
Scale-up of a laboratory
production system
Growth inhibition by
product
Consider:

Fluid flow & mixing

Mass transfer
Reactor design
Process monitoring
Design of control loops
for continuous operation
Aseptic conditions

Group 2
Mammalian system
Scale-up of a laboratory
production system
Secreted product
Multi-product facility
Consider:

Fluid flow & mixing

Mass transfer
Reactor design
Process monitoring
Design of control loops
for fed-batch operation
Aseptic conditions