LARGE SCALE PRODUCTION OF

RECOMBINANT THERAPEUTIC GRADE

MONOCLONAL ANTIBODIES
(ERYTHROPOIETIN) IN CHO CELL

NAJIHAH BINTI MOHD RAFIDI

1213970
SITI SYAZWANI BINTI MAHAMAD
1211132
NUR HASYIMA BINTI ISMAIL
1215628

To identify the nonmicrobial cell culture

using for recombinant
therapeutic grade
monoclonal antibodies.

To gain further
understanding about
CHO cells works in
the production of
monoclonal
antibodies.

OBJECTIVES

To discuss about the

advantages of using the
non-microbial cell culture
in the production of large
scale monoclonal
antibodies.

To discuss the
downstream processing
involved in the
production of
recombinant
therapeutics for
monoclonal antibodies
from mammalian cell.

INTRODUCTION

Biopharmaceuticals area is currently focusing in the rapid development of high yielding and
long-lasting manufacturing process for monoclonal antibodies to treat many types of illness
Large scale production of monoclonal antibodies needs mammalian cells in order for mAbs to
expand their applications into additional therapeutics area.
Significant advanced have been made in the design of mAbs as therapeutics in order to
improved their quantity and quality of the products.
The use CHO cells has proven by many researchers, is the most preferred production host
as it can improve dramatically the bioavailability, binding specificity, human antibodies
consequences to reduces any immunogenic side effect and optimized affinity.
The most important features that make CHO cells as the primary choice is due to their
ease in genetic manipulation also their adaptability to given condition which they can
produce to very high densities in suspension cultures.
For the products to meet the acceptable levels in large scale production of mAbs, the
utilization of mammalian production systems followed by cell removal and purification
through sequential chromatographic and membrane filtration steps helps to reduce product
and non-product related impurities.

recombinant protein
Recombinant

protein is a protein encoded by recombinant DNA

and can only be produced through chemical synthesis.
The production is conducted in laboratory by molecular cloning
which consists of two basic parts,
I. replication of DNA within living cells
II. replication of DNA in test tubes by polymerase chain reaction
(PCR).
o. Usually, recombinant proteins are majorly derived from rodent
animals and human cells such as BHK, HeLa, HepG2 and CHO
cells.
o. Chinese Hamster Ovary (CHO) cells have become the main
selection of mammalian cell culture process for
biopharmaceuticals productions.

CHO Cells
What is CHO cells?
CHO

cell is a cell line which is

synthesized from ovary of
Chinese hamster that belongs
to a family of rodents in China
and Mongolia.
At first, it is used as cultured
monolayer which employs amino
acid proline in the medium.
Due to its multifunctional
applications and ease of
maintenance, it has been
applied widely particularly to
express recombinant protein.

Advantages of CHO Cells

High

attainable yields.
It is also safe to be used for synthesis of
biologics since it does allow the propagation
of any adventitious pathogenic agents that
can infect humans.
They possess the abilities to allow easy
introduction of foreign DNA and
expression of large amount of desired
protein which is susceptible to genetic
modifications.
The most important features that make
CHO cells as the primary choice is due to
their ease in genetic manipulation also their
adaptability to given condition which they
can produce to very high densities in
suspension cultures.

Erythropoietin (EPOGEN)
Epogen

is recombinant DNA version of human erythropoietin

protein is used to treat anemia especially for patients with
chronic kidney disease, HIV patients due to treatment with
zidovudine and cancer patients who have anemia due to
chemotheraphy. In selected patients epogen may be used to
reduce the need for blood transfusions in surgery.
Recombinant EPO produced in mammalian cells is fully
glycosylated. Two forms of recombinant EPO, Epoietin-alpha and
Epoietin-beta, are commonly used in the clinic. These molecules
are produced in Chinese Hamster Ovary (CHO) cells and exhibit
differences in their carbohydrate structure and
pharmacological properties, but not in their respective clinical
efficacies.

Monoclonal

antibodies are a homogeneous product also a regent secreted by

immortalized B-lymphocytes that are cloned and expanded in continuous cell lines.
The production of mAbs in mammalian cells consists of a long process that
involves steps of transfection of the gene of interest into the cells, selection of
clones, adaptation to different culture conditions (usually suspension and serum
free medium), culture in bioreactors and scale-up to industrial level.
Mammalian cells are currently the main hosts for commercial production of
therapeutic proteins including mAbs.
Chinese Hamster Ovary (CHO) cells are the most preferred production host for
therapeutic monoclonal antibodies based on their capability to perform posttranslational modifications also the successful approval history track record for
expressing heterologous gene products.
CHO cells offer the advantages of being readily transfectable, able to grow in
suspension and serum-free medium, and ability to growth at high densities. CHO
cells own the same function like myeloma cells which primarily used for high level
production of mAbs in the past.

Monoclonal antibodies expression in CHO cells

Downstream Processing of Erythropoietin

(EPO) from Chinese Hamster Ovary (CHO)
Cells
Cell Culture
Harvest
Protein A
Chromatography

Viral Inactivation

Chromatographic
Polishing Steps

Viral Filtration

Ultrafiltration /
Dialfiltration

CELL HARVESTING
Commonly,

centrifugation and filter reactor

effluent approaches has been used to remove
dense cell parts.
This technique will separate the proteins based
on size and mass due to the drag and inertial
forces that are experienced from outward
forces of rotation.
The massive particles with low drag will fall to
the bottom but non compacted particles will
remain as the supernatant in the liquid.

Protein

A chromatography has been proved to be selected for

monoclonal antibodies and able to yield up to 99 % purity
starting from cell culture supernatant.
The cell culture supernatant at neutral pH is directly loaded on
the column and the product will be eluted at low pH. Basically,
between the column load and elution, a wash step that has been
introduced is often at intermediate pH and it will remove host
cell protein and contaminants. Column is stripped pH 2 and
regenerated by high concentrations of chaotropes.
The platform process become possible for Erythropoietin
(EPO) when Protein A chromatography is achieving the high
purities since it is a key volume reduction step.

Protein A chromatography

Viral inactivation
Refers

to low pH incubation step which included since

Protein A column eluate at low pH and most
Erythropoietin (EPO) are stable in solution at low PH.
The protein A elution pool is added with acid solution
(0.5 M phosphoric acid) to adjust the pH into below than
3.8, but strong acids should not be used since it will
denature the product. Under lower pH condition, mostly
monoclonal antibodies will be stable so that complete
inactivation viruses can be selected.
After that, neutralization is needed to move the
product into stable pH. The presence of a buffering
species is important to be considered during
neutralization because this will aid pH to be maintained
around final target pH, thus overshoot pH can be
prevented.

Chromatographic polishing
Polishing

steps may included to reduce host cell

and media derived impurities such as viruses.
Polishing process such as AEX and HIC that are
operated in flow through mode will not allow the
product to bind to the column but impurity species
are retained.
Monoclonal antibodies will die because the
molecules possess high isoelectric points.
AEX is used to remove trace levels of CHO
protein, nucleic acid, endotoxins and any viruses
under conditions where monoclonal antibody is
positively charged and flowthrough the column
whereas negatively charged impurities bind to
positively charged resin.

Viral

filtration is ensure the safety of the

product.
The concept that has been used is all
pathogens could be removed as long as their
size is larger than bio-molecule.
Viral filters are categorized on their pore
sizes into retroviral (<50 nm) and parvoviral
(<20 nm) grade filters which is normally
operated at constant pressure.

Viral filtration

Ultrafiltration

/ Diafiltration with three

column affinity process has been used to
purify monoclonal antibodies.
This three non affinity process has been
streamlined into two column non affinity
process with the integration of High
Performance Tangential Flow Filtration
(HPTFF) that will allow the concentration,
formulation and also the purification.

Ultrafiltration / Diafiltration

As

a conclusion the production of recombinant therapeutics

monoclonal antibodies from CHO cells have promising good
result in term of the quality and quantity of product
obtained after going through downstream processing.
The utilization of mammalian cells has greatly improved the
production of monoclonal antibodies as therapeutics.
However, there are still challenges to be overcome. In order
to achieve goals of mAbs at ultra-high level, some areas
need to be addressed properly. For example, we need to
further investigate the cellular mechanism that limit gene
expression and secretion and also how its influenced the
overall process.

CONCLUSION