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Bioreactor Design & Control

Continued…
Module 2: Upstream Processing
Work Plan
Week Date Topic
Introduction to Biopharmaceutical Manufacturing: Overview and
1 May 1, 2024
Emerging Trends in the Industry Part I
Introduction to Biopharmaceutical Manufacturing: Drug Product
2 May 8, 2024
Devlopment
3 May 15, 2024 Upstream Processing (USP) - Overview
4 May 22, 2024 USP – Cell Line Development
USP – Bioreactor Design and Control
5 May 29, 2024
Industry Guest Presentation: StemCell Technologies
6 June 5, 2024 USP – Introduction to Environmental Monitoring
7 June 12, 2024 Downstream Processing (DSP) – Introduction and Harvesting
Midterm Exam
8 June 19, 2024
DSP – Chromatography and Column Packing
Topics
Scale-up considerations
Reactor types
Reactor operating systems
Agitation and Aeration
Reactor Design & Construction
What all bioreactors have to do…
Control the environment for cell growth and production

Prevent contamination

‘Protect the product’ (direct product contact)

Contamination
Optimizing the environment for mammalian cell culture growth also promotes
growth of viable contaminants*.
Contamination in upstream processing usually results in loss of batch.
At small scale, single-use flasks provide a low-risk option. Manipulations are
conducted in biological safety cabinets using aseptic techniques.
At large scale, stainless steel bioreactors and any connections/additions that
need to be made must be contamination-free.

*Note: Contamination does not just mean viable organisms. A

contaminant is any physical, chemical or biological substance/
residue that is found where it is not supposed to be.
Materials of Construction
316L quality or greater
Biocompatible
Passive layer
Strong

Polished stainless steel

Mechanical polishing
Electro polishing

Components fused in an oxygen-free

environment

Specific roughness measures (ASME-BPE)

 Bag Inspection Bag seam
Key areas to inspect:
Seams Delicate
Particularly triple seams on shoulders cable-tied
Manually heat-sealed tubing
Defects are sometimes seen – may be a fold but
bag can still be integral
Keep ties/ bubble wrap on to help

Cable tied connections

Hand-tied connections (especially smaller tubing)
Film laser cut but tubing hand-cut – may not be
even

Agitator/sparger rim Agitator/ sparger rim

Rare defect location but still possible
NOTE: Always inspect and do any leak testing before you perform any welds or other augmentations. Otherwise,
manufacturer will not stand over the bag integrity.
Instrumentation
Intrusion Proof

Food Grade Elastomers

Non-leachable
Stainless Steel Hard-piping
Clean Steam In stainless steel systems, each of
In stainless
Cleaning steel systems, these utilities must be hard piped
Solution directly to the bioreactor
each of these utilities Plant
must be
Steam
hard piped directly
WFI to the
This makes the plant very inflexible –
bioreactor Gas Supply one plant, one process!

This makes the plant very

inflexible
Glycol – one plant, one
Water
process!
Single Use Hard-piping

In single use systems, clean steam, WFI Gas Supply

and cleaning solution requirements have
been eliminated

All of this makes the facility more flexible – Glycol

one plant, multiple processes! Water
Single-Use Bioreactor Design
Cytiva (Xcellerex XDR) Sartorius (Biostat STR)
Single-Use Bioreactor Design
Pall (Allegro)
ThermoScientific (HyPerforma DynaDrive)
 Material Compatibility
Shake flasks are often made from polycarbonate
which is rigid and compatible with cell culture

Rocker bioreactors and stirred-tank reactors usually

made from layers of low-density polyethylene with
other materials such as ethyl-vinyl alcohol in between

Cell contact material must be USP class VI compliant

Source: http://www.bioprocessintl.com/manufacturing/supply-
chain/enhanced-assurance-supply-single-use-bags-based-
material-science-quality-design-partnership-suppliers/

Q: what are key properties of the material layers?

“The compatibility of materials used for product contact surfaces with the products
should be ensured under the process conditions by evaluation e.g. adsorption and
reactivity to the product”
- EU GMP Annex 1 (draft)
Source:
http://www.maquinsa.info/pdf/UnSoloUso/
bolsas/Pl-01026_PL-01077(2D).pdf
Sampling (SUBs)
Luer lock connectors with an internal septum:
Sterile syringe is aseptically attached to
sample line causing the septum to be pushed
back to allow liquid flow

Liquid only ever drawn out of line to reduce

likelihood of contamination entry

Line must be flushed between samples (10x

hold-up volume)

Not a true “aseptic” sample – welding of bags

still often performed
Reactor Operating Systems
Modes of Operation - Batch
Primarily the mode of operation during seed train expansion

Medium added once, prior to start of culture

Cells are inoculated into fixed volume – they consume nutrients and metabolites accumulate
Cells division stops when all nutrients are consumed and toxic by-products build up

Volume remains
constant in each vessel
Batch Culture
This is
great for
Advantages scale-up Disadvantages for Production Bioreactors
Well characterized Lower productivity
Ease of operation and maintenance Nutrient level depletes fast
Low cost Lower maximum cell densities
Low risk of contamination Accumulation of waste by-products
Easy to validate

What about batch

mode for a
production
bioreactor?
Fed-Batch Culture
Fed batch can prolong life by feeding with fresh medium
1. Inoculate cells into a small volume of media
2. Additional medium is added periodically to increase or maintain culture
3. Alternatively, extend the batch by feeding in various substrates (glucose, amino acids, etc.)
4. Duration and cell density of culture can be extended

Growth Feed Production

Modes of Operation – Fed-Batch
This is the most popular mode for
production bioreactors Fed-batch
Advantages
Increased control of cell culture
environment
Growth rate and/or production rate
extended
Higher titers
Fed-Batch Culture
Disadvantages
High demand products still require larger bioreactor (>10,000L)

Higher capital costs and increased medium costs

Large-scale bioreactors can create scale-up challenges

Different age profiles for the protein product produced over an extended run

Product in contact with proteolytic enzymes at 37°C throughout run

 Batch vs. Fed-Batch vs.
Perfusion Culture
This seems like the
Batch Fed-Batch Perfusion
ideal environment
for cells
Concentration

Concentration

Concentration
Time Time Time

Nutrient
Let’s explain how it
Waste Metabolites is achieved
Perfusion Culture
Suspension cells can now be used
The culture is maintained at constant growth rates and/or cell density for an extended
period of time

Separation device is not

1. Initial volume of medium added
where cells grow
and cells inoculated

2. Cell concentration increases and

more media may be added until
steady state is reached

3. Perfusion of fresh media as well

as removal of harvested
material/waste media begun
Modes of Operation - Perfusion
Agitation & Aeration
Bioreactor Agitation
A stirred tank reactor achieves mixing through
mechanical agitation.

Mechanical agitation achieves the following:

Keeps cells in suspension

Ensures homogeneity
pH, nutrient, heat transfer

Aids in gas transfer

Disperses gas bubbles throughout the bioreactor
Increases the residence time of gas bubbles
Shears large bubbles to form smaller bubbles
Agitation system
Drive motor Usually consist of an agitator and
baffles

Seal
Agitator consists of:
drive motor
Baffle seals
shaft
Shaft impeller

Impeller

Mayo, J. (n.d.). Glass-lined reactor model comparison. De Dietrich Process

Systems Chemical & Pharma Process Solutions. Retrieved May 24, 2022,
from https://www.ddpsinc.com/blog-0/glass-lined-reactor-model-comparison
The Agitator May Be Either Top Or
Bottom Mounted
Allegro STR (PALL) With top mounted agitators, the seals
are not submersed in culture, resulting
in reduced risk of contamination.

Bottom mounted agitators require a

shorter shaft, thus use less energy but
require higher maintenance due to seal
damage in drive shaft.

Eibl, R., & Eibl, D. (2019). 4. In Single-use technology in Sartorius Biostat

biopharmaceutical manufacture. essay, Wiley.
How Do We Measure Agitation?

Revolutions Per Minute (RPM) and Power Consumption

For scale up power unit volume or computational fluid dynamics

Does ?
Aeration
Gas is added to the bioreactor below the liquid surface through a sparger

When designing/choosing a
sparger you need to consider:
Gas flow rate capacity (L/hr)
Bubble diameter
Location Ring
Cleaning ease Sintered

Disc
SU Aeration

Sparger design can be open pipe, ring,

fritted/ micro or a combination of these
designs

Pre-installed at base of bag

Gas supply is required

Purpose is to provide O2 / CO2 and possibly

to strip out CO2 if levels are too high
Choosing a Sparger
Different spargers are suitable for different culture applications.
Drilled Hole / Macro Sparger Sintered / Micro Sparger
Topics Recap
Scale-up considerations
Reactor types
Reactor operating systems
Agitation and Aeration
Bioreactor Control
Module 2: Upstream Processing
Learning Objectives
Describe critical control in bioreactors for:
pH
Temperature
Dissolved oxygen (DO)
Foaming

Explain pH and DO probe design and function

Outline the concept of oxygen mass transfer and factors that

may affect it
Topics
Introduction to Bioreactor Critical Control
pH
Temperature
Dissolved Oxygen
Oxygen Mass Transfer
Foam Control
 Introduction to
Bioreactor Control
Scale Up
As systems become larger and more complex, the level of control
required to maintain a healthy culture increases.

Volume

Complexity

Critical Process Parameters (CPPs) such as pH, dO2, temperature and

agitation, must be more tightly monitored and controlled.
pH
Importance of pH
pH may affect: pH can be affected by:
Cell viability Media composition
Enzymatic activity Cell metabolism
Bioactivity of protein Gas flow
Glycosylation
Protein product degradation
pH 6.0 pH 5.0

+ H+ + + H+
H+H + H H+H +H+H+H+
H + H H
Decrease H+ H++H++H+ HH++ H H +
+ +

H+ H+ H+ H H H+ H+ H
pH by 1.0 H+ H+ H+ HH++
H+ H++H+ H+
H+ H+H H
+ +
H+H H++
H+ H + H+ + +H
H+ +
H H +H + +HH
+ +
H HH+H
pH Control in Mammalian Cells
High pH may be caused by over aerating the culture:
CO2 may be driven out of solution

Low pH may be caused by under aerating the culture

Oxygen limitation in animal cells may lead to lactic acid production

pH control requires a
balance between aeration
and acid/base control
Temperature
Temperature Control
Temperature for optimal cell growth and temperature for
optimal product formation may differ

Higher temperatures (above 40°C) may cause:

- Cell viability issues
- Protein denaturation and aggregation

Lower temperatures slow enzymatic reactions:

- Growth may decrease and stop altogether

Genetically engineered cells may grow at one

temperature and be induced to produce protein with a
lower temperature.
Temperature Control
In general, mammalian cells will grow best at approximately 37°C.
Dissolved Oxygen
(DO)
Dissolved Oxygen (DO) control
Oxygen is necessary to provide energy for growth
and product formation via respiration.

Low solubility

Lack of results in poor growth and protein synthesis

Excess can result in harmful by-products (reactive oxygen species)

Replacing dissolved oxygen as it is consumed can be

a major limiting factor.
Bioreactor Gas Control
Gas is provided to the bioreactor via filtered overlay and sparger line

Supply gas can be a mixture of air, oxygen,

nitrogen and carbon dioxide

Gas flow monitors and solenoid valves can

alter the gas mixture dynamically in response
to culture conditions

DO probes give real-time feedback to the

automated system for control
Oxygen Mass Transfer
OTR and OUR

OUR = Oxygen Uptake Rate

OTR = Oxygen
Cell line dependent rate at which
Transfer Rate
oxygen is consumed in a bioreactor for

https://www.gelifesciences.com/en/ie/solutions/bioprocessing/knowledge-center/7-factors-that-affect-
The rate at which
a given set of operating conditions.
oxygen can be delivered
to a bioreactor for a
given set of operating
conditions.

oxygen-transfer-to-cells-in-bioreactors
Foam Control
Foam in a Reactor

Cell culture may foam for a variety of reasons:

Vessel volume
Proteins and amino acids in media
Cell lysis promotes foaming
Agitation and aeration Foam

This can result in the blockage of hydrophobic

gas filters!

Bench-top Bioreactor
Foam Control
Foam control – antifoam and shear protectant:
Reduce surface tension of bubbles and/or prevent cells
sticking to bubbles.
E.g., Poloxamer 188, Polyethlyene glycol

Excess antifoam can impact in downstream processing by

fouling filtration membranes.
Bioreactors operated at a working volume of 75% of the
total volume, to prevent foam-over and to allow for volume
increase.

The effect of antifoam on kLa should be assessed for each

particular culture condition.
Contamination Control
Learning Objectives
1. Understand how personnel can contribute to contamination in manufacturing areas.

2. Describe the correct cleanroom behaviour and disciplines.

3. Be familiar with cleanroom features.

What is Contamination?
Dictionary definition of contamination:
to pollute, to infect, to defile by touching or mixing with, to corrupt.

To biopharmaceutical manufacturers contamination is a physical, chemical or

biological substance or residue present in a product or area where it is not
supposed to be.

Contamination must be controlled to ensure the “safety, quality and efficacy of

sterile products”.

Ref: EU GGMP Annex 1

Contamination Control Strategy

Cleaning & Raw

Sanitization Material
Regime Control

Facility &
Environmental Process
Monitoring
Design

Personnel
Training &
Qualification
Keeping Product and Patients Safe

’clean’

Clean Room Example of operations

Grade
The higher A Open aseptic handling of product (highest risk)
the grade,
B Closed processing of product, transport to or
the ’closer’ to
background zone for grade A
the patient
C Preparation of solutions prior to filtration, weighing
D Dirtiest area (e.g. packaging, cleaning) – lowest risk

‘dirty’
Types of Cleanrooms
Non-Unidirectional Uni-directional
Airflow Cleanrooms Airflow Cleanrooms
dilution of dirty air displacement of dirty air

Lower Grade Cleanrooms Higher Grade Cleanrooms

Grade A (inside isolators or
Grade C and Grade D RABS) and Grade B,
(background to isolator) background to RABS

0.36 – 0.54 m/s (guidance value)

Annex 1 EU GGMP

https://en.wikipedia.org/wiki/Cleanroom
Air Quality Control
Cleanroom air quality
is controlled and
optimised by Air-
Handling Units (AHUs)

HEPA filters will reduce the Return air ducts in cleanroom

introduction of particles, with walls and air changes will reduce
an efficiency of 99.97%. the retention of particles

BUT…the generation of particles is largely attributed to

the behaviour of personnel in the cleanroom!
Environmental Monitoring
We look at contaminants from various sources…

Air Supply Surfaces Personnel

…and we look at different contaminant types

Drug Discovery Characterization Pre-Clinical Clinical Phases Marketing
Studies I, II & III Authorization
Skin flakes, hairs, fibres, lint, Bacteria, Yeast, Moulds
Non-viable dust, particles generated by Viable
Spread from surfaces and through
Contaminants machinery Contaminants
the air, by touch transfer, aerosols,
(Particulates) Spread by movement, transfer of (Microbes)
equipment and material transfer.
equipment and personnel, AHUs.
Viable Particle Counts
Non-viable Particle Limits
Process Validation of Aseptic Filling

Culture Media

Equipment Setup

Culture Media injection:

Incubation & Microbiological Process validation of aseptic filling with

analysis: culture media aims to ensure that
pharmaceutical or biotechnological products
are produced under sterile conditions and
Results interpretation & comply with quality and safety standards.
Documentation and Reporting
Cleanroom Contamination
Cleanroom personnel are main source of contamination - we account
for >80% of all contamination.
Types of personnel- borne contaminants include:

Particulates • Skin flakes, hairs, clothing fibres

• Coughs, sneezes
Aerosols
• Excessive talking / shouting

Microbiological • Skin, hair, mucosal membranes

Behavioural disciplines and gowning practices are the main ways in

which particulate and microbial levels can be controlled!
Cleanroom Contamination
Our movements within the cleanroom will affect how many
particles are generated and how far they will spread.
Not Permitted in Cleanrooms

Individuals who suffer from the following conditions may be prevented from
entering the cleanroom either temporarily, or permanently

Skin conditions – psoriasis, eczema, sunburn, open sores etc.

Respiratory conditions – chesty coughs, COPD etc.
Allergies – to materials, chemicals or the product.
Not Permitted in Cleanrooms
Certain items are not permitted in a cleanroom…
Cosmetics
Nail polish or false nails
Hair products
Aerosols and perfumes
Jewellery (wedding bands may be accepted)
Watches
Contact Lenses
Mobile phones (designated phones may be provided)
Pencils and erasers
Paper items (in critical areas)
Cardboard
Wood
Topics Recap
Bioreactor Types, Design, Operations
Bioreactor Critical Control
Environmental Monitoring
Contamination Control
Questions?