Bioprocess cell line development strategies for EPO production

1. CHO Cell Line Development
for EPO Production
- Claudyo L. Mande
- Lui Tandra Jonathan
- Muhammad Ilham Fahri
- Amudra Kurnian Meghantara
- Anak Agung Freda
- Vidi Miranda P.
- Asti Aprilia P.

2. Overview: Why CHO?
Cell Line Development Industrial Manufacturing
- The presence of very effective
transgene amplification methods
→ dihydrofolate reductase
(DHFR) and glutamine
synthetase (GS) → to establish
stable, high-yield recombinant
CHO cell lines
- CHO cells exhibit similar
glycosylation profiles to original
human proteins,
- CHO are less susceptible to
infections by human viruses
- Stable high product
yields
- Stable product quality
- Process reproducibility
- Reduced timelines
- Approved by regulatory
agencies: FDA
- Lower costs

3. General Strategies in CHO Cell Line Development

4. General Strategies in CHO Cell Line Development

5. Transfection in CHO Cell Line Development
1. vector plasmid
2. gene of interest (epo)
3. expression host
4. transfected using lipofectamine 2000
+
CHO

6. Gene of Interest Amplification and Selection (EPO)
Generate gene amplification and isolate the higher-yield clones using the selectable markers
DHFR : catalyze the conversion folic acid → tetrahydrofolate → produce glycine, purine, thymidylate
(GHT)
DHFR system → have high efficiency in gene amplification
Selection
markers
Screening
agents
Selection principle
Concentration
range
DHFR
(Dihydrofolate
reductase)
MTX
(Methotrexate)
Folic acid antagonist that inhibits the DHFR activity
and DNA synthesis
usually
between
25 - 1000 nM
GS (Glutamine
Synthetase)
MSX (L-
methionine
Sulfoximine)
Inhibits the glutamine synthetase gene
usually
between
25 - 500 nM

7. Gene of Interest Amplification and Selection (EPO)
The transfected cells (CHO DXB11/DG44) are cultured in the medium
without GHT
]
The surviving cells contains EPO and DHFR genes
The surviving cells cultured in the medium contains methotrexate (MTX)
MTX will try to inhibit the DHFR activity
MTX will stimulate the cells to produce DHFR copy number to cover
inhibition by MTX
Since gene of interest (EPO gene) in the same locus of DHFR, then it
will get amplified as well → increase production of protein target (EPO)

8. Gene of Interest Amplification and Selection (EPO)
Higher MTX concentration → higher EPO protein expressed (Santoso, et. al, 2020)
The sample number 2. (200 nM) has the lowest titer of EPO protein : 15.0 mg/L and
sample number 1.1.1.1. (4000 nM) has the highest titer of EPO protein : 170.0
mg/L.

9. Single Cell Cloning
Flow cytometry used to select cells with desirable phenotypes.
In particular, fluorescence-activated cell sorting (FACS)
classifies cells based on the determined fluorescence level by
multiple factors, such as the size and density of cells.
FACS assay based on heavy- and light- chain assemblies provided insights into
the optimal antibody expression in CHO cells by first performing a two-color
sorting of green fluorescent protein and yellow fluorescent genes that fused
with recombinant antibody heavy- and light-chain genes, respectively (Sleiman
et al., 2010).
(Biomedical & Biological Sciences, 2018)
Flow cytometry, capability to analyze millions of cells per minute (can save time, human energy, and
money/resources (Koughpuumar and Borth, 2012).
(+) flow cytometry can also use to analyze cell growth and metabolism (Hinterkörner et al., 2007).

10. (2) Clonal fluorescence microscopy (Clonepix) is a method to analyze the individual immobilized cells by grow
in semi-solid medium, provide nutrients for cell growth, and add fluorescently on surface of the semi-solid
medium. The cells must be characterized by (1) flow cytometry in the early cloning selection stage to identify
cell lines with high productivity potential and help eliminate unstable cell lines.
The unique combination of clonographic fluorescence screening and flow cytometry methods contributes to the
efficient isolation of clone cell lines at high productivity within 15 weeks and their possible application to CHO
cells.(Roy et al., 2017)
(+) The Clonepix system detects fluorescence by adding fluorescence-bound “clone detection” reagents

11. Analysis of EPO protein titer
The target protein can be characterized qualitatively by SDS-PAGE and
Western Blotting
● blocking solution (Skim Milk)
● Epo detecting use rabbit polyclonal anti-EPO antibody
● conjugated with goat anti-rabbit IgG-HRP and TMB substrate
Glycosylted EPO: 25-28
kDa
Unglycocylated Epo: 20 kDa
Identity & purity

12. Analysis of protein titer (Cons)
The expression level of the amount target protein must be evaluated
quantitatively by ELISA
Standard recombinant EPO ranging
from 0-200 mIU/mL
potency/activity

13. Analysis of protein titer (Cons)
the hEPO have a glycosylation site → Lectin based assay (present/absent
sugar on EPO)
measure with absorbance: 610/525 nm
Impurities
Binding of Wheat Germ Agglutinin
(WGA) to N-acetylglucosamine
and sialic acid demonstrated the
presence of N-acetylglucosamine

14. Bioreactor Culture
● After establishing subclones and
targeted protein production,
bioreactor culture are performed to
scale up the protein production.
● Fed-batch culture are chosen
because it provide simple cultivation
system rather than perfusion but still
produce high yield.

15. Bioreactor Culture
● Culture are scaled up
slowly (volume wise) to
avoid long lag phase
● For Adherent cell lines,
microcarriers are used for
attachment place of cells
in scaled up cultivation

16. Stability Evaluation
● DHFR gene amplification
location are determined
using this method
● Telomeric position of
DHFR gene amplification
provides higher gene
copy number and stability
after long duration
cultivation
(Yoshikawa,2000).

17. Cell Banking
● Validated cell clones then will be banked as
MCB in some vials to provide Master Stock.
● Working Cell Bank for future high scale
cultivation will be derived from Master Stock.
● Cell Bank are preserved in -70 C to maintain
cell viability when cultivated.

18. Thank You