Lecture 4:

Cell Line Development

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
Learning Objectives
1. Understand the selection and amplification of cells in development.

2. Describe what clonality is and how it is assured.

3. Explain the fundamentals of plasmid expression.

Topics

What is a cell line?

Genetic Engineering

Selection and Amplification

Clonality and Assurance

Cell Line Selection
Cell Culture
We can culture cells from many different organisms, including:

Bacterial Cell Yeast Cell

Cell Culture
We can culture cells from many different organisms, including:
Mammalian Cell Plant Cell
Cell Line - Definition
Cell Line
A culture developed from a single cell and therefore
consisting of cells with a uniform genetic make-up

Primary Cell Culture

Derived directly from the living tissue – begins with
many cells therefore more variation

Cell lines are often immortalized and grow

continuously in culture

Immortalized cells may be cancerous, arise

spontaneously or be transformed
Why Use Cell Lines?
Need cells to produce protein product

Mammalian cell

If product is to be consistent, then the cells must be genetically identical.

Cell lines arise from a single parent and should be as close as possible to
genetically identical.
Question

What is the evolutionary time between the first single

cell organisms and the first eucaryotic cells?

(Post your answers in the chat – get those participation marks!)

Prokaryotic vs. Eukaryotic Cells
Prokaryote Eukaryote
Small & simple Large & complex
No nucleus Nucleus
No membrane-bound organelles Membrane-bound organelles
Intracellular expression Extracellular expression
Doubling time approx. 20mins Doubling time approx. 24hours
No complex PTMs Complex PTMs
May have cell wall Usually no cell wall - fragile
Easier to grow and shear resistant More difficult to grow and shear/osmotic
stress sensitive
Prokaryote Eukaryote
e.g. E. coli e.g. CHO
Question
Q: What important characteristic of therapeutic
protein drugs is missing on this slide?
(Post your answers in the chat – get those participation marks!)

Prokaryote Eukaryote
Small & simple Large & complex
No nucleus Nucleus
No membrane-bound organelles Membrane-bound organelles
Intracellular expression Extracellular expression
Doubling time approx. 20mins Doubling time approx. 24hours
No complex PTMs Complex PTMs
May have cell wall Usually no cell wall - fragile

Prokaryote Easier to grow and shear resistant More difficult to grow and Eukaryote
e.g. E. coli shear/osmotic stress sensitive e.g. CHO
Cell Line Selection
Before manufacturing ever begins, some questions need to be asked;

Ease of selection of high producers

Simple recombinant protein Adaptation to protein-free
Product Growth &
Type? Monoclonal antibody Productivity suspension culture
Fusion Protein Apoptosis, proliferation rate, max
cell number

Stability of Expression Levels Potential for endogenous viral

Genetic Safety contamination
Stability of transfection
Stability? Issues? Use of animal products in media
Long-term genetic stability (potential contamination)
Mammalian Cell Lines

Abbreviation Full Name Origin History Companies

Genentech, BMS,
Chinese Hamster Fibroblast from Used for mutation
CHO Lilly, Janssen,
Ovary hamster ovary research in 50s
Pfizer

Mouse
Mouse Mouse
NS0 non-secreting Alexion, Biogen
lymphocyte lymphocyte
myeloma

Crucell, DSM,
Per.C6 Human Human retinal cell Fully human PTMs
Merck
Genetic Engineering
Establishing a Cell Bank
Gene for
Candidate clones
therapeutic Small Scale
protein Cell Pool
Studies

Transfection Selection

Host cell Master cell

selected

x 500 x 200 Culture

Expanded
Large Scale Working cell Master cell
Production bank (WCB) bank (MCB)
Walsh, Gary. Pharmaceutical biotechnology: concepts and applications. John Wiley & Sons, 2007.
Poll

Do you know what a palindrome is?

Yes

or

No
Recombinant DNA Plasmid
3. Restriction enzymes cut plasmid
at specific points to form sticky ends.
Insert will be positioned here to
complete construct.

2. A circular DNA
plasmid is designed

1. Gene of interest that codes for

desired protein is copied using
polymerase chain reaction (PCR).
This forms the INSERT. The insert
contains the genetic code for the
protein of interest.
Getting the Gene into the Cell
Human cell

Gene the code

protein of interest
Genetically modified CHO
cells which now produce
protein of interest
Transfection Methods
How do we get the recombinant DNA vector into the cells?

Method Mechanism
Precipitates of the + charged calcium and the - charged phosphate
Calcium Phosphate
bind to DNA and enter nucleus

DEAE-Dextran Binds and interacts with negatively charged DNA molecules

Lipofection Liposomes transport genetic material across the membrane

Electroporation Electric field opens pores in membrane
Biolistic Particle Bombardment, direct entry via “gene gun”
PEI Polyethylenimine forming polyplexes
Question
What important gene transfer method is missing on this slide?
(Post your answers in the chat – get those participation marks!)

Method Mechanism
Precipitates of the + charged calcium and the - charged phosphate
Calcium Phosphate
bind to DNA and enter nucleus

DEAE-Dextran Binds and interacts with negatively charged DNA molecules

Lipofection Liposomes transport genetic material across the membrane

Electroporation Electric field opens pores in membrane
Biolistic Particle Bombardment, direct entry via “gene gun”
Question

Biological Methods

Method Mechanism

Bactofection Bacteria-mediated transfer of eukaryotic host cells using plasmid DNA

Transduction Viral vectors introduce genetic material (GOI) into host cell
 CRISPR/Cas9

Ishino, Yoshizumi & Krupovic, Mart & Forterre, Patrick. (2018). History of
CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to
Genome Editing Technology. Journal of Bacteriology. 200. JB.00580-17.
10.1128/JB.00580-17
Selection and Amplification
Genetic Engineering - Selection of Cells

2nd+ Rounds of Selection:

Select cell which produces
1st Round Selection: the most protein
Select cell which produces
the most protein
Biopharmaceuticals Poll

What are the characteristics used to select the clone?

A. Selection of a clone that is robust

B. Selection of a clone that produces a protein with the desired post

translational modifications

C. Selection of a clone with a high growth rate (generally 18 to 24 hours)

D. All of the above

Cell Line Selection and
Amplification Methods
Dihydrofolate Reductase (DHFR) gene Vector + Insert
DHFR-deficient cells require specific nucleotides for
growth
Link GOI to the DHFR gene and transfect cells
Grow cells in nucleotide-free medium Insert
Only cells which have taken up the GOI/DHFR vector
will grow
Amplify with MTX

Glutamine Synthetase (GS) gene

Grow cells in glutamine-free media
Amplify with MSX We link our gene of interest (GOI)
Faster but more expensive to another gene which gives our
cells a competitive advantage
Clonality and Assurance
Clonality
“For recombinant products, the cells substrate is the transfected cell containing the
desired sequences, which has been cloned from a single cell progenitor.”
- ICH Q5D

How do we know our cell line all originated from a single parent cell?

Two paths to assurance:

Exact procedures during cell line development
Dilution, Imaging and Cell Sorting
Testing for clonality after cell line generation
Limited Dilution
Limited Dilution Cloning (LDC) Decreasing Cell Conc.

Serial dilutions are performed until it is

Increasing Selection Agent Conc.

probable that there is a single cell/well;
We usually calculate to have less than 1
cell/well on average
The FDA requires two rounds of limiting
dilution to ensure clonality
Cells may be exposed to selection agent
Single cells expressing the selection gene
will grow into a colony and then the clonal
population will express the gene of interest
Imaging Clone Select Imager

Automated clone picking using imaging.

Sophisticated instrumentation such as
the ClonePix technology from Molecular
Devices.
This technology uses a low density
semi-solid media to reduce cell
movement and promote individual
colony growth.
Imaging can be used in conjunction with
dilution to verify that colony originated
with single cell.
Cells are plated and growth is recorded
from Day 0
Molecular devices. Molecular Devices. (n.d.).
Retrieved May 17, 2022, from http://www.moleculardevices.com/
Cell Sorting
Cell Sorting can be carried out using a flow
cytometer.

Fluorescence Activated Cell Sorting (FACS)

allows cells to be separated according to
fluorescent properties or light scattering

Can be used in conjunction with limited dilution

to reduce the number of dilution runs required.

Sabban, S. (2011). Development of an in vitro model system for studying the interaction
of Equus caballus lgE with its high-affinity Fc receptor (thesis).
Robotic Clone Selection with Imaging
Modern approach to cell line development
Objective is to quickly isolate high producer clones
and to provide a record demonstrating clonality with a
single cloning step
This can save 12 weeks of cell line development time

Clonepix

Berkley lights

CelSelector
ALS Automated Lab Solutions GmbH. (n.d.). Als cellcelector. ALS
Automated Lab Solutions GmbH. Retrieved May 17, 2022, from
https://www.als-jena.com/cellcelector-cell-and-colony-picking-
system.html
Clonality of Banked Cells
Two methods of checking clonality of cell line:

Fluorescence in situ hybridization

Is the plasmid present in the cell’s genome and is it in the same location as in the original cell(s)?

Sub-clone analysis:
Is the phenotype of a sample of single-cell cultures comparable to the original cell culture?
TLA-based Transgene and Integration
Site Sequencing
Target locus amplification (TLA)
PCR using transgene specific primers
NGS

Technology developed in the Netherlands by de Vree et al. (Nature Biotechnology, 2014)

https://www.nature.com/articles/nbt.2959
https://www.cergentis.com/

Replaces FISH and allows for rapid statistical determination of clonality

Offered by numerous CROs, 70-100K$ to analyze approx. 100 clones

Poll

What do regulators require for Master Cell Bank analysis?

A. Tests showing genetic stability

B. Tests showing stability of expression over the manufacturing process
C. Tests showing the absence of known viruses
D. Tests showing the absence of bacteria and yeast, i.e. sterility tests
E. All of the above
Sub-Clone Analysis
Cells thawed from cell bank

Phenotype of cultures characterized for

extended periods of time.
Doubling rate
Specific productivity
Product quality

Compared to historical measures from

original culture
Assurance of Clonality
If there are questions over clonality, an augmented control strategy must be in place to
guarantee that product is uniform

The more assurance you have of clonality the less rigorous the control strategy has to be

Controls may include:

Reducing number of generations of cells in culture
fewer generations = less chance of mutation
End of process characterization of cells
Additional testing on protein product quality
Cell Line Qualification
Adventitious agents
Mycoplasma
Endogenous viruses
Exogenous viruses

Genetic stability
GMP stability testing programs

Media sourcing
Materials of animal origin
Topics Recap

What is a cell line?

Genetic Engineering

Selection and Amplification

Clonality and Assurance

Summary
Assurance of clonality provides assurance that the cells will react consistently in culture

This should lead to less variation in product quality and yield

Lack of assurance can be overcome but requires additional control and testing
Upcoming

Assignment #1 Due Date:

Wednesday June 12, 2024

Assignment instructions can be found under Assignments tab on Canvas.

Mid-Term: June 19, 2024

Questions?
References
ALS Automated Lab Solutions GmbH. (n.d.). Als cellcelector. ALS Automated Lab Solutions GmbH. Retrieved May 17, 2022, from https://www.als-jena.com/cellcelector-cell-
and-colony-picking-system.html
Molecular devices. Molecular Devices. (n.d.). Retrieved May 17, 2022, from http://www.moleculardevices.com/
Sabban, S. (2011). Development of an in vitro model system for studying the interaction of Equus caballus lgE with its high-affinity Fc receptor (thesis)
https://www.synthego.com/guide/how-to-use-crispr/pam-sequence
Ishino, Yoshizumi & Krupovic, Mart & Forterre, Patrick. (2018). History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing
Technology. Journal of Bacteriology. 200. JB.00580-17. 10.1128/JB.00580-17