Introduction of Biotechnology

and Manufacturing
Suh, Chang Woo PhD.
April 01, 2014
PT. Daewoong Infion
Lecturers
<Education Background>
- 1998.03 ~ 2002.02 : PhD in Biochemical Engineering, University of Hanyang
- 2002.03 ~ 2004.02 : Post Doc. in Microbiologics, Microbiochip Center, Korea
<Career Information/Research Background>
- 2004.02 ~ 2012.6 : Daewoong Bioresearch center for development of
EGF, EPO, hGH, BMP-2, and BTA
- 2012.07 ~ Present : Global business development for biologics.
Establish new biological GMP in Indonesia.
Name: Suh, Chang Woo

Department: PT. Daewoong Infion
Telephone : +62-813-3324-2224
E-mail: cwsuh@daewoong.co.kr
Biotechnology & Future
1) What is biological products?
2) History and future of Biotechnology

How to make Biological Products
2) Manufacturing process
3) Quality Control of Biologics

Introduction of PT. Daewoong Infion
1) Mission / Goal / History
2) Product Information of EPOSIS
3) Progress of construction

CONTENTS
PART 1.
BIOTECHNOLOGY & FUTURE
WHAT IS BIOLOGICAL PRODUCTS?
HISTORY AND FUTURE OF BIOTECH.
Biological is a medicinal product such as a vaccine, blood or blood
component, allergenic, somatic cell, gene therapy, tissue, recombinant
therapeutic protein or living cells that are used as therapeutics to treat diseases.





Cultured cartilage
Heparin
Growth Hormone
Albumin
rh-EPO
Human blood
Red blood cell
Cord blood
Kidney
Bone
Heart,
cornea
Steam Cell
Cultured organ
Vaccinie
mAb
Tissue
Gene therapy
Definition of biological Products
Modern Biotechnology from DNA
James Watson & Francis
Crick (1953)
Englishmen responsible
for the discovery of the
double helix structure of
DNA using X-ray
photographs
Wow!! Recombinant Technology
Paul Berg (1972)
Stanford University scientist who first developed recombinant DNA
technology, a method for insertion of genetic material from one
organism into another.
Stem Cell!! Magic but Dangerous
Stem cells are undifferentiated biological cells that can differentiate into
specialized cells
Clones will come to our world!
Ian Wilmut (1996)
Dolly is the first animal cloned from diploid cells is
produced in Scotland
Are you origin or copy?
Dolly gave birth to four
lambs
Dolly the sheep, the first mammal to be cloned from an
adult cell
How do you think about Human Clone?
Bush says cloning human embryos is 'morally
wrong.'
Movie: The Island (2005)
Are you agree with it?
Photo of mouse growing a "human ear"
- a shape made of cartilage
Human can join with Machine.
knowledge from the joints, muscles, and nerve
PART 2.
BIOLOGICAL PRODUCTS
FIVE FEATURES FOR A BIOTECH. DRUG
MANUFACTURING PROCESS
QUALITY CONTROL OF BIOLOGICS
What is recombinant protein?
Recombinant DNA technology, joining together of DNA molecules
from two different species that are inserted into a host organism to
produce new genetic combinations that are of value
to science, medicine, agriculture, and industry.
API Original Name Company in Korea
Nepidermin Easyef DW
Erythopoietin Eprex DA, CJ, LG, DW
Interferon alpa Intron A DA, CJ, GC, LG
Interferon gamma Immukine LG
Somatropin Genotropin DA, LG, DW
Filgrastim Neupogen DA, GC
BMP-2 Infuse DW
Neurotoxin Botox MT, HG, DW
Abciximab ReoPro Isu
Infliximab Remicade Celltrion
Korean Recombinant Protein Drugs
Choice of Production Cell Line
Manufacturing Process of r-Protein
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Purification/Concentration
(Ultrafiltration/ion exchange or
precipitation)
Sealing/Labelling
/packaging
Generation of Cell Substrate
Preparation of Expression
Construct
Cell Banking
Cell Culture/Fermentation
Purification/Isolation
Drug Substance
Formulation
Drug Product
Propagation of WCB
Starter culture
Production-scale
culture/bioreactor
Recovery
(Centrifugation or filtration)
Chromatographic purification
Addition of excipients
Sterile filtration/
Aseptic filling
Adjustment potency
Key Point of Biological Products
Expression system: new cell lines
Mamalina cell lines: regulation of syclin-dependent kinase, anti-apoptic
protein BCl2, PRC-6
Yeast strain humanized for N-linked glycosylation
Other alternative expression systems:
Insect cells (SF9, Hi5)
Transgenic plants (Maize, tobacco, potatoes)
Transgenic animals (goat, sheep, rabbit, )

Production system: Serum free media
Cell-culture systems
Structure and post-processing modifications
Impact on yield and consistency of production

DNA plasmid for hEGF
Recombinant E.coli
for production of hEGF
E.coli
E.coli
EGF
EGF
EGF
EGF
Recombinant hEGF
Cloning
Fermentation
DNA synthesis
by chemical reaction
E.coli
10 NH3 2CH4
4C2H6
2 H2O
Production Process for rhEGF
Cell Culture
Fluid
Fermentation
Cell Removal by microfiltration
Filtration
Chromatography
Filtration
Chromatograpahy
Chromatograpahy Filtration
Lyophilization
rhEGF
E.coli
Protein Purification
1. Molecular weight
Ultracentrifugation
Dialysis
Gel filtration
SDS PAGE

2. Charge
Ion Exchange Chromatography
Native gel electrophoresis
Isoelectric focusing

3. Affinity
Exploited with cloning
His-tagged proteins purified on Nickel columns.
GST fusion proteins purified on glutathione columns.

4. Solubility

SDS-PAGE
1. Catalase, cytochrome C,
a-lactalbumin
2. Hemoglobin, Cytochrome
C, a-lactalbumin
3. BSA, cytochrome C,
a-lactalbumin
4. Hemoglobin, myoglobin,
a-lactalbumin
5. Ferritin, cytochrome C,
a-lactalbumin
6. Ferritin, myoglobin,
a-lactalbumin

1 2 4 3 5 6
lighter
Gel Filtration (SEC)
The largest molecules are eluted from the column first and the smallest are eluted
last. Molecular weight range is dependent upon the pore size of gel matrix
Ion Exchange Chromatography
U
V

a
b
s
o
r
b
a
n
c
e

Increasing salt
concentration
Affinity

Immunoaffinity chromatography - antibody
Metal affinity chromatography - chelation
Dye affinity chromatography - dye tendency

The most expensive, the most
various, the most excellent
The affinity between a ligand
with a high specificity and the
protein of interests. (or a
particular impurity)

Use early in a purification
scheme

Ex, Protein A affinity :
Mab - capture stage

Quality Control for Biological Products
Genetic Stability
Cell Substrate
Viral Safety
Preclinical Safety
Generation of Cell Substrate
Preparation of Expression
Construct
Cell Banking
Cell Culture/Fermentation
Purification/Isolation
Drug Substance
Formulation
Drug Product
Product Stability
Specifications
Methodology of Characterization
Safety
LAL test, rabbit pyrogen test, bacterial culture methods
Purity & Characterization including but not limited to:
Reversed-phase HPLC, Peptide mapping, MS
SDS-PAGE, Western analysis, capillary electrophoresis
SEC, AUC, FFF, light scattering
Ion Exchange Chromatography
Carbohydrate analysis (capillary electrophoresis, HPAEC = high-pH anion-
exchange chromatography, IEF for sialic acid)
Identity
N-terminal sequencing
Peptide mapping
Immunoassays (ELISA, Western blotting)
Potency
Animal-based assays, cell-based assays, reporter gene, biochemical (e.g.,
enzyme activity)
Protein content
RIA, ELISA, UV absorbance, Bradford
29
1. Expression System
Identify, isolate and clone the gene coding for the
desired protein

Construct a vector containing:
The gene
The expression controls (promoter, secretion signal)

Insert the vector into the selected micro-organism
Escherichia coli
Saccharomyces cerevisiae
Mammalian cells
Genetically modified plants
30
2. The Production System
Purpose
Optimize survival conditions for the genetically modif
ied microorganism
So that it produces the desired protein
With acceptable yield

Materials & Methods
Selection of cell culture medium
Selection of culture conditions
Selection of culture equipments:fermentor, cytoculto
r

3. The Purification System
Purpose
extract protein from a complex growth medium
Achieve close to 100% purity
Without altering the protein

Materials & Methods
Sequence of purification steps
Filtration/ultrafiltration
Precipitation/resolubilization
Chromatography (ion-exchange, affinity, etc.)
32
4. Nature of Active Product
Proteins
Chains of amino-acids
Sequence of amino-acids encoded by genes
Folded into 3-D conformation
To obtain biological activity
Host-dependent post-translational modifications (su
gars)
5. Pharmaceutical Formulation
33
Purpose
Maintain biological activity
By maintaining active protein conformation in sol
ution

Materials & Methods
Stabilization (albumin, glycerol)
Storage at low temperature
Part 3.

Introduction of PT. Daewoong Infion

Mission / Goal / History
Product Information of EPOSIS
Progress of construction
(Utility, AHU, Panel, Paint, Pipe, Electric)
Mission of PT. Daewoong Infion
1. Contribute to the development of the Indonesian Biologics
Technical transfer of Biologics to Indonesian Pharmaceutical company
Co-development with governments and hospitals of Biosimilar and new biologics

2. Contribute to the health of the Indonesian people.
Medical technology & skill transfer through Doctor to Doctor and Doctor training
education
Medical industry Development

3. Respected company to all Employees, Partners, Customers
Everyone Win Win
Provide the right information & optimized solution to the customers
Priority to the growth of employees than the development of the company
Goal of Manufacturing Biologics
To be the first and the best Bio GMP factory in
Indonesia
Provide more affordable biological products for the
Indonesian people
Provide training program development,
standardization and bio-GMP expert education
program for contributing to the biotechnology industry
in Indonesia
Prototype of Biologicals manufacturing in Indonesia
Contribute to Indonesian government income (devisa)
History of PT. Daewoong Infion
1. Progress:
April 2012: Contract of JVC.
July 2012: Approval of investment from BKPM.
Feb. 2013: Approval of DOE, Domicile and NPWP.
May. 2013: Approval of GMP Drawing from BPOM.

2. Concept & Basic Design
Total area (1st+2nd): 2,550 m
2

API (630 m
2
), Product (620 m
2
), QC (230 m
2
), non-control area (1,070 m
2
).

3. Initial Total Investment : USD 20 MIO
EPOSIS

Recombinant Human Erythropoietin
Pre-filled Syringe Inj.
Can we see EPO?
EPO is a Protein which is a hormone in human body.
Other names include:
Erythropoetin
Epoetin
Hematopoietin
Hemopoietin
Erythropoietin
Signal and Action
Mechanism of action
The feed-back regulation of EPO synthesis

Ref.) Holes human anatomy and
physiology 10
th
edition
EPO regulates the
differentiation and
proliferation
of erythroblast
precursor cells
Indications & Complications
Indications
Acute & chronic renal failure
Drug poisoning
Refractory edema
Metabolic Disturbances
Complications
Low blood pressure
Fatigue
Nausea
Iron-deficiency Anemia : blood loss from the tech
nique of hemodialysis

Where is my blood?
Chronic Kidney
Disease
CRF, Cancer, Dialysis, HIV,
Infection and other cause
Anemia
Transfusion
EPO
Treatment
Infecti
on
Best Choice
Progress of Construction
(a) Frame for Panel
(c) Floor & Painting
(b) Ducting & Piping
(d) Mushola
Progress of Construction
(e) pure water generator
(g) electric generator
(f) WFI generator
(h) chiller
Education of Human Resource
Director of Factory
Manuf. QA QC Eng. Admin.
Head
Culture
Purification
Formulation
Filling
Packaging
Head
Lot release
Validation
AQR
SMF/SOP
Regulation
Document
CAPA/OOS
Changing
Process
Education
Head
Chemical test
Biological test
Raw materials
Microbial
Sterile test
Animal test
Stability
Equipment
Head
Machinery
Equipment
AHU/HVAC
Clean room
Electric/pipe
Water/WFI
Head
Ware house
Production
Accounting
HR manager
BD/Export
GMP Committee
Tech. Transfer of Erythropoietin
1. Technical Transfer of Manufacturing
1) Establish of Cell Line in Korea
WCB (working cell bank) from RCB (research cell bank)
Qualification of WCB
2) Media Fill in Daewoong Infion
Conducting Media Fill Test before PV production
3) PV production in Daewoong Infion
PV 3 batch
Long term stability test on going for two years
Tech. Transfer of Erythropoietin
2. Technical Transfer of Analytical Method
SOP training
Calibration Training for Equipment
Method Validation
Animal Management and Testing Training
Formulation research
Global First/Advanced generics
R&D out-sourcing
Hyderabad, India
Product development
for domestic market
Botanical Medicine
Beijing, China
Yongin, Korea
150 Scientists
Well-balanced research areas;
- NCE/Biologics
- Generics/Incrementally modified drugs
- Botanical Medicines
Active scouting of external innovation
Daewoong R&D Centers
Pharmtech Dec. 2002
Annual salaries as job function
Terima Kasih !
Always Smile with Daewoong Infion