This document contains the syllabus for an advanced animal cell culture course offered in the second semester of 2013 at Chungbuk National University's Department of Animal Science. The syllabus lists 8 topics that will be covered across 8 classes between September and December, including introductions to cell culture, stem cells, transgenic animals, and genome engineering. It also provides the dates and instructors for the classes on cell culture for antibody and protein production, and protein production/purification.

1. 3rd Lecture
Advanced Aminal Cell Culture
2013 2nd Semester
Department of Animal Science
Chungbuk National University

2. Syllabus
Date
Topics
September 5, 2013
Introduction : What is Cell Culture?
September 12, 2013
Cell Culture As Model System For Research
September 26, 2013
Cell Culture For Antibody / Protein Production
October 17, 2013
Protein Production/Purification
October 31, 2013
Stem Cell I
November 14, 2013
Stem Cell II
November 28, 2013
TG/KO Animals
December 5, 2013
Genome Engineering/NGS
December 12, 2013
Final Exam

3. Date
September 26, 2013
Cell Culture For
Antibody / Protein
Production
조유미, Madhusumida
October 17, 2013
Protein
Production/Purification
이미진, 정용호
이영, 윤준호
October 31, 2013
November 14, 2013
Stem Cell I
Jia Jia Lin, 염동현
November 28, 2013
Stem Cell II
Zhao MingHui,권정우
December 5, 2013
Transgenic Animals
Lin Zili, 이상배
December 12, 2013
Genome
Engineering/NGS
조유진, 김상욱

4. Animal Cell Culture for the
Protein Productions
• “Protein” Includes..
- Enzyme (use for Research, Chemical..)
- Antibody (Diagonastic, therapeutical..)
- Therapeutical Recombinant Proteins
- Nutrients

5. Could animal cell culture can be used for ‘Protein’
for the Nutrients?
Not really…

6. Animal Cell Culture for the Protein
Productions
- Why we need to express recombinant proteins while we can get them from
natural resources?
• You can get a recombinant protein at the fraction of cost
from natural resources
• Modification of Protein (Truncation, fusion, mutants) is possible
- Expression System for recombinant proteins
•
•
•
•
•
Bacteria
Yeast
Insect Cell
Animal Cell
Transgenic Animal/Plants
Cost/
Technical
Difficulties
Authencity

7. Insulin
- Peptide hormone secreted from b-cell in pancreas
- Before chemical synthesis or recombinant protein production, it was extracted from
pancreas of animal
- Low yields -> High Cost
- Recombinant Insulin

8. Biologics
Definitions : Medical products including
-
Vaccine
Blood
Gene therapy
Antibody
Recombinant therapeutic protein
* Market size estimation for 2015 : $ 158 billion

9. 7 out of 10 best selling drugs in US are Biomolecular Drugs
Many ….mab. What is „mab‟?
„mab‟ stands for „Monoclonal Antibody‟

10. Animal Cell Culture for the Protein
Productions
Pros
- Authentic posttranslational modification
- Authentic disulfide bonds
- Likely to behave more similar with native one.
Cons
- Less Cost effectiveness than other expression systems
- Difficulties in Scale-Up

11. Antibody

13. Polyclonal vs Monoclonal Ab
Monoclonal antibody
Various Antibodies for the same antigen
Polyclonal antibody

15. Monoclonal Antibody & Hybridoma
Antibody producing B cell cannot
grow indefinitely
You can make antibody producing B cell to
grow indefinetly by fusion with a myeloma
(B Cell Cancer)
How you can select the fusion
cell from the unfused cell?

16. HAT medium
HAT medium : Hypoxanthin-aminopterin-thymidine
medim
- Blocks the pathway for nucleotide synthesis
- Cells require Hypoxanthine-guanine phosphoribosyltransferase gene to grow
on HAT medium

17. Trick : Fuse antibody producing B cell (HGPRT +) with
myeloma cell (HGRPT-) and select on HAT medium
Immortal
Cannot grow on
HAT medium
(HGPRT-)
BLymphoc
yte
Myeloma
Limited Life Span
Antibody Prodction
Can grow on HAT medium
(HGPRT+)
Cell Fusion using PEG
Selection on HAT medium
Myeloma
X
Hybridoma
Cannot grow on HAT
Immortal
Antibody Producing
X
BLymphoc
yte
Limited Life Span

18. Screening of Hybridomas for specific
proteins
- Screening of hybridomas which secret antibody recognizing a desired antigen
- Immunoassay etc
- Time & Money consuming..

19. mAb - Purifications
- Hybridoma Culture (In Suspension)
- Affinity Purification (Protein G/A)
* Protein G : Immunoglobulin-binding protein from Bacteria Streptococcus

21. Magic Bullet : Monoclonal Antibody as Drug
- Targeted Therapies for proteins involved in cancers
- HerceptinTM : Anti-HER2 monoclonal antibody -> Breast cancers

22. Huddle for Mab based theraputics
- Most monoclonal antibody was derived from Mouse
- Mouse Antibody are recognized as „foreign material‟ in
human body
- so it would mediate immune response against them
- Then it will be cleared ASAP
How we can overcome this problem?
Answer : Humanized Antibody
(Retain antigenicity, but not inducing immune response in human)

23. Humanized Antibody

25. Human Monoclonal Antibody

26. Therapeutic Mab productions in
Animal Cells
- Construction of expression plasmid
Strong Promoter
Efficient polyadenylation Signals
Modification for the improvement of Antibody
- Transfections
- Generations of Stable Cell Lines
- Culture
- Purification

29. Insect Cell
- Problems in recombinant protein expression in mammalian ce
Low protein expression level
It cause increase in downstream
(Protein Purification and extra) costs
High cost to culture/maintain
- Alternative : Insect Cell and Baculovirus expression system
Relatively high protein expression level
Suspension Cultre : Easy to scale up

30. Baculovirus : Vector for recombinant
Protein Expressions

31. Baculovirus as vector for protein
expression in Insect cell

32. POLYHEDRIN PROMOTER : Strong Promoter for capsid protein in baculovirus
(Polyhedrin)

33. Commonly used Insect cells
- Sf9 : Cell Line established from Spodoptera frugiperda
- High Five : Cell line originated from ovarian cells of cabbage looper
- S2 : Drosophila

34. Characteristics of Insect Cells
- Grow Optimally at pH 6.2
- CO2 exchange IS NOT REQUIRED for insect cell
- Adherent & Suspension Culture

35. CHO Cell
- Generated from Chinese Hamster Ovary
- Industry standard for protein productions in mammalian cells
- Can adapt in liquid suspension cultres to high volumes

36. From Gene To Protein

37. Cell Line Development