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