Advanced Aminal Cell Culture 2013 2 nd Semester Department of Animal Science Chungbuk National University 3rd Lecture
Advanced Aminal Cell Culture
2013 2nd Semester
Department of Animal Science
Chungbuk National University
3rd Lecture
Syllabus
Date TopicsSeptember 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 INovember 14, 2013 Stem Cell IINovember 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS
December 12, 2013 Final Exam
DateSeptember 26, 2013 Cell Culture For
Antibody / Protein Production
조유미 , Madhusumida
October 17, 2013 Protein Production/Purification 이미진 , 정용호
October 31, 2013 이영 , 윤준호November 14, 2013 Stem Cell I Jia Jia Lin, 염동현
November 28, 2013 Stem Cell II Zhao MingHui,권정우December 5, 2013 Transgenic Animals Lin Zili, 이상배December 12, 2013 Genome
Engineering/NGS 조유진 , 김상욱
Animal Cell Culture for the Protein Productions
• “Protein” Includes..
- Enzyme (use for Research, Chemical..)
- Antibody (Diagonastic, therapeutical..)
- Therapeutical Recombinant Proteins
- Nutrients
Could animal cell culture can be used for ‘Protein’ for the Nutrients?
Not really…
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
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
Biologics
Definitions : Medical products including
- Vaccine- Blood- Gene therapy- Antibody- Recombinant therapeutic protein
* Market size estimation for 2015 : $ 158 billion
Many ….mab. What is ‘mab’?
7 out of 10 best selling drugs in US are Biomolecular Drugs
‘mab’ stands for ‘Monoclonal Antibody’
Animal Cell Culture for the Protein Productions
- Authentic posttranslational modification- Authentic disulfide bonds- Likely to behave more similar with native one.
- Less Cost effectiveness than other expression systems- Difficulties in Scale-Up
Pros
Cons
Antibody
Polyclonal vs Monoclonal Ab
Various Antibodies for the same antigen
Polyclonal antibody
Monoclonal antibody
Monoclonal Antibody & Hybridoma
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?
Antibody producing B cell cannot grow indefinitely
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
Trick : Fuse antibody producing B cell (HGPRT +) with myeloma cell (HGRPT-) and select on HAT medium
MyelomaImmortalCannot grow on HAT medium(HGPRT-)
B-Lymphoc
yte
Limited Life SpanAntibody ProdctionCan grow on HAT medium(HGPRT+)
Cell Fusion using PEG
Selection on HAT medium
Myeloma Hybridoma
B-Lymphoc
yteX XCannot grow on HAT Limited Life SpanImmortal
Antibody Producing
Screening of Hybridomas for specific proteins
- Screening of hybridomas which secret antibody recognizing a desired antigen- Immunoassay etc- Time & Money consuming..
mAb - Purifications
- Hybridoma Culture (In Suspension)
- Affinity Purification (Protein G/A)
* Protein G : Immunoglobulin-binding protein from Bacteria Streptococcus
Magic Bullet : Monoclonal Antibody as Drug
- Targeted Therapies for proteins involved in cancers
- HerceptinTM : Anti-HER2 monoclonal antibody -> Breast cancers
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)
Humanized Antibody
Human Monoclonal Antibody
Therapeutic Mab productions in Animal Cells
- Construction of expression plasmid
- Transfections
Strong PromoterEfficient polyadenylation SignalsModification for the improvement of Antibody
- Generations of Stable Cell Lines
- Culture
- Purification
Insect Cell
- Problems in recombinant protein expression in mammalian cell
Low protein expression levelIt 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 levelSuspension Cultre : Easy to scale up
Baculovirus : Vector for recombinant Protein Expressions
Baculovirus as vector for protein expression in Insect cell
POLYHEDRIN PROMOTER : Strong Promoter for capsid protein in baculovirus (Polyhedrin)
Commonly used Insect cells
- Sf9 : Cell Line established from Spodoptera frugiperda
- High Five : Cell line originated from ovarian cells of cabbage looper
- S2 : Drosophila
Characteristics of Insect Cells
- Grow Optimally at pH 6.2- CO2 exchange IS NOT REQUIRED for insect cell- Adherent & Suspension Culture
CHO Cell
- Generated from Chinese Hamster Ovary- Industry standard for protein productions in mammalian cells- Can adapt in liquid suspension cultres to high volumes
From Gene To Protein
Cell Line Development