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Cell culture 03


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Cell culture 03

  1. 1. 3rd Lecture Advanced Aminal Cell Culture 2013 2nd Semester Department of Animal Science Chungbuk National University
  2. 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. 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. 4. Animal Cell Culture for the Protein Productions • “Protein” Includes.. - Enzyme (use for Research, Chemical..) - Antibody (Diagonastic, therapeutical..) - Therapeutical Recombinant Proteins - Nutrients
  5. 5. Could animal cell culture can be used for ‘Protein’ for the Nutrients? Not really…
  6. 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. 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. 8. Biologics Definitions : Medical products including - Vaccine Blood Gene therapy Antibody Recombinant therapeutic protein * Market size estimation for 2015 : $ 158 billion
  9. 9. 7 out of 10 best selling drugs in US are Biomolecular Drugs Many ….mab. What is „mab‟? „mab‟ stands for „Monoclonal Antibody‟
  10. 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. 11. Antibody
  12. 12. Polyclonal vs Monoclonal Ab Monoclonal antibody Various Antibodies for the same antigen Polyclonal antibody
  13. 13. 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?
  14. 14. 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
  15. 15. 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
  16. 16. Screening of Hybridomas for specific proteins - Screening of hybridomas which secret antibody recognizing a desired antigen - Immunoassay etc - Time & Money consuming..
  17. 17. mAb - Purifications - Hybridoma Culture (In Suspension) - Affinity Purification (Protein G/A) * Protein G : Immunoglobulin-binding protein from Bacteria Streptococcus
  18. 18. Magic Bullet : Monoclonal Antibody as Drug - Targeted Therapies for proteins involved in cancers - HerceptinTM : Anti-HER2 monoclonal antibody -> Breast cancers
  19. 19. 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)
  20. 20. Humanized Antibody
  21. 21. Human Monoclonal Antibody
  22. 22. 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
  23. 23. 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
  24. 24. Baculovirus : Vector for recombinant Protein Expressions
  25. 25. Baculovirus as vector for protein expression in Insect cell
  26. 26. POLYHEDRIN PROMOTER : Strong Promoter for capsid protein in baculovirus (Polyhedrin)
  27. 27. Commonly used Insect cells - Sf9 : Cell Line established from Spodoptera frugiperda - High Five : Cell line originated from ovarian cells of cabbage looper - S2 : Drosophila
  28. 28. Characteristics of Insect Cells - Grow Optimally at pH 6.2 - CO2 exchange IS NOT REQUIRED for insect cell - Adherent & Suspension Culture
  29. 29. CHO Cell - Generated from Chinese Hamster Ovary - Industry standard for protein productions in mammalian cells - Can adapt in liquid suspension cultres to high volumes
  30. 30. From Gene To Protein
  31. 31. Cell Line Development