cell culture 03
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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
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