Biochemical Engineering CEN 551

1. Biochemical EngineeringCEN 551 Instructor: Dr. Christine Kelly Exam 3 Review

2. Schedule • Today April 8: Review for exam 3 (Chap. 12, 14 and 15). • Tuesday, April 13: No class, but posters due. • Thursday, April 15: Homework Due and Presentations • Saturday, April 17, 1:00 pm – 2:00 pm: Poster Presentations • Tuesday, April 20: Presentations • Thursday, April 22: Exam 3 • Tuesday, April 27: Presentations

3. April 15: Mittal, Sameer, Xu, Anitescu April 20: Meka, Chapeaux, Chang, Sayut, Pasenello April 22: Exam 3 April 27 Price, Reis, Prantil, Lu, Menon

4. Learning Objectives for Exam 3 Characteristics of animal cells • List the characteristics of animal cells compared to bacterial cells. • Describe the optimum growth conditions (pH and temperature) for animal cells. • Define the types of animal cell lines: primary, secondary, and transformed. • Cite typical doubling time of mammalian cells.

5. Animal Cell Characteristics • 10-30 um  making them larger than bacteria or yeast • Cell membrane – no cell wall: shear sensitivity • Optimum growth at 37oC and 7.3 pH • Typical doubling times = 12-36 hr, so batch phase from 4 to 7 days.

6. Cell Lines • Primary culture: cell recently excised from specific organs of animals. • Secondary culture: cell line obtained from the primary culture. Can be adapted to grow in suspension and are non-anchorage dependant. Will only grow for about 30 generations. • Continuous, immortal, transformed cell lines: cells that can be propagated indefinitely (cancer cell lines are all continuous).

7. Serum, medium and endotoxins • Describe the major components of animal cell medium. • Describe serum, it importance and liabilities in animal cell culture medium • Describe the two major animal cell byproducts and their affect on the culture. • Define enodotoxins.

8. Serum: The clear liquid that separates from the blood when it is allowed to clot. • Fetal Bovine Serum (FBS; also named as 'FCS') • widely used in animal cell culture as an essential supplement. • serum and protein free media have only been established for selected protocols. • Growth hormones

9. During harvesting, and centrifugation of fetal blood, serum may become contaminated by bacteria and mycoplasma. Sterile filtration and strict sterile control of the end-product is therefore one of the key responsibilities of serum suppliers. Mad cow disease important factor in pressure to use serum free media.

10. Cell wall residues of gram negative bacteria, commonly named 'endotoxins', are another thread in the serum manufacturing process. Sloppy collecting and processing methods of the raw serum, may result in a higher endotoxin burden of the respective serum lot. Endotoxins are very hard to remove from the serum, and are even capable to pass the different filtration steps. Endotoxins can influence cell growth, but may also be passed to the end-product, intended for human therapy.

11. Aeration and Agitation • Describe the major cause of shear damage in sparged mammalian cell reactors. • Describe what type of impellor used in animal cell culture and why. • Describe what Pluronic F68 is used for in animal cell medium.

12. Aeration and agitation in mammalian cell culture • In microbial cultures, oxygen transfer rates can be improved with smaller bubble size, higher stirring speeds and higher gas hold-up. • Mammalian cells damaged (sheared) by turbulence and by the action of bursting bubbles.

13. Shear in the bulk liquid • As turbulence increases, eddy size will decrease and the level shear will increase. • shear forces in the bulk liquid are NOT the major cause of cell damage in sparged reactors. • Under normal stirring conditions, the average size of the turbulent eddies is larger than the average cell diameter.

14. High stirrer speed Low stirrer speed Effect of eddy size cell cell

15. Bubble damage • Bubble damage is often the major cause of cell damage animal cell culture, particularly in sparged reactors. • Bubble damage occurs in two forms: • damage due to the bursting of bubbles at the surface of the fluid. • damage due to shearing of cells trapped in the foam.

16. Bubble burst damage • As bubbles burst at the surface, cells trapped on the bubble interface or in the bubble wake can be literally torn apart. • Damage is dependent on the physical properties of the culture fluid and on the bubble size and velocity. • Large bubbles cause more cell damage than small bubbles.

17. Pluronic F68 • Pluronic F68 (a mixture of polyoxyethylene and polyoxypropylene) is a non-ionic surfactant that is used to protect animal cells from damage caused by shear and the effects of sparging. • Pluronic F68, like all surfactants, acts at the surface of objects immersed in the liquid medium.

18. Reducing bubble size • When large bubbles burst, the release more energy than small bubbles. Large bubbles are therefore more destructive than small bubbles. • Damage will increase with the rate of energy release from the bubble burst process. Thus the level of damage tends to increase with the air flow rate.

19. Glycosylation • Define glycosylation. • List the two organelles involved in glycosylation. • List the three types of glycosylation, and indicate which type is more complex. • Describe consequence of not having the sialic acid end cap on glycosylation of therapeutic recombinant proteins. • Describe one way to measure glycosylation patterns.

20. Glycosylation • The addition of sugar residues to the protein backbone. • Most extensive posttranslational modification. • Carried out in the ER and Golgi apparatus prior to secretion or surface display. • All mammalian cell surface proteins of glycoproteins. • Most secreted proteins are glycoproteins (notable exceptions include insulin, growth hormone).

21. Three Types of Glycosylation • N-Linked • O-Linked • Membrane anchor

22. N-Linked • Bonded to the R group of an asparagine residue. • Consensus peptide sequence is… Asn – X – Ser or Thr • Consensus sequence is not always glycosylated. • Three types of N-linked: complex, high mannose, hybrid.

23. Effects of Glycosylation • Pharmacokinetics and clearance (especially the degree of sialylation). • Immunogenicity. • Solubility and protease resistance.

24. Products and Recombinant Hosts • Describe the relative required purity, cost, and volumes for pharmaceutical verses industrial verses food products. • Describe the most common 7 types of host systems for recombinant proteins, and cite the major strengths and weaknesses of each

25. Constraints based on product type • Pharmaceutical • Objective is safety and efficacy. • Purity, authenticity, posttranslational possessing. • Cost result of research and clinical trials – not manufacturing. Cost of manufacturing not as an important issue. • Animal feed supplements or pharmaceuticals • Purity is requirement. • Cost important also.

26. Industrial • Low manufacturing cost critical. • Can tolerate lower levels of purity. • Food Processing • Safety important. • Purity requirements less stringent than pharmaceuticals. • Volume is large. • Cost important for penetrating the market.

27. Host Organisms • E. coli • Gram positive bacteria • Lower eukaryotic cells • Mammalian cells • Insect-baculovirus system • Transgenic animals • Plants and plant cell culture

29. Genetic Instability • List and define the types of genetic instability. • Use analytical solutions to predict genetic instability.

30. Genetic Instability • Maximum target-protein production vs. well growing culture. • Production of lots of recombinant protein is always detrimental to the cell.

31. Cells lose the capacity to make the target protein – they often grow more quickly that the original strain. • Segregational loss. • Structural instability. • Host cell regulatory mutations. • Growth rate ratio.

32. Regulatory Standards • Describe the factors that determine regulatory standards for recombinant organisms.

33. Containment required depends on • The ability of the host to survive in the environment • The ability of the vector to cross species lines or the DNA to be transformed into another species. • Nature of the recombinant genes.

34. Plasmid Design Describe the function and importance of the components that should be considered for plasmid design for production of recombinant proteins.

35. Plasmid Design • Origin of replication. Regulates reproduction of plasmid and copy number of plasmid. Different origins for different host types. • Number of gene copies. Higher levels of production with more copies of the gene. Multiple plasmids or multiple copies on the same plasmid. E. coli typically has 25-250 plasmids per cell.

36. Promoter/Inducer. Strong promoter means higher rate of transcription  faster production. Promoter should be tightly regulated – off = very little transcription, on = lots of transcription. Inducer should not be toxic or expensive, easy to manipulate. • Terminator. Strong promoters need strong terminator to prevent read through (transcription) of the DNA after the gene. • Fusion proteins. Can fuse small part of host’s native protein to prevent destruction. Can fuse handle or tail for affinity chromatography. Can fuse host’s secretion signal to direct out of the cell.

37. Selective pressure. Antibiotic resistance or necessary metabolite gene on plasmid to ensure only the plasmid containing cells will grow in the bioreactor environment. Can leak complimenting factor to medium and cells that lose the plasmid will still have some complementing factor for several generations. • Par and cer loci. Sections of DNA on the plasmid that promote even distribution of plasmids to daughter cells.

38. Metabolic and Protein Engineering • Describe metabolic and protein engineering and their objectives.

39. Metabolic Engineering: Why not just use the natural strain? Put a pathway under the control of a regulated promoter – turn on the pathway when it wouldn’t normally be turned on. Example: to degrade hazardous waste to lower concentrations than would normally induce the pathway.

40. Increase the concentration of enzymes with a strong promoter. • Produce the product in an easier to grow host. • Combining several pathways. • Patent the organism – cannot patent ‘unengineered’ organisms.

41. Protein Engineering • New proteins or altering the amino acid sequence of existing proteins. • Can require crystal structure of the protein to examine modifications that may have benefit. • Driving force for computer modeling of protein structure from amino acid sequence.

42. Medical Applications • Define tissue engineering. • Describe the current commercial and near commercial tissue engineering products. • Discuss what aspects of this course can be applied to tissue engineering products. • List the steps involved in gene therapy. • Discuss what aspects of this course can be applied to gene therapy.

43. What is Tissue Engineering? “The application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function” (Whitaker Foundation “Tissue engineering”).

44. Developing in vitro tissues based on cells derived from donor tissue. • Used in transplants. • Commercial examples: skin and cartilage. • Artificial liver outside the body is in trials. Uses hollow fiber reactor and pig liver cells. • Under development: liver, pancreas, kidney, fat, blood vessel, bone marrow, bone, neurotransmitter secreting constructs.

45. Gene Therapy • Transfer of genes into cells for a therapeutic effect. • Patient has faulty gene that does not encode for a correctly functioning protein. • Genes can be delivered ex vivo (outside the body) or in vivo (inside the body). • If ex vivo, the organ is removed, then transplanted back in. • Genes are delivered to the cells with a virus. • Clinic trials have been problematic.

46. A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common. • An abnormal gene could be swapped for a normal gene through homologous recombination. • The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. • The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

47. Mass Production of Retrovirus • Two part system: cell line and recombinant vector (virus). • Cell line engineered to produce essential viral genes that have been deleted from the viral genome. • Virus incapable of causing disease – carriers of therapeutic genes. • Retrovirus can only be used with dividing cells for integration of therapeutic genes. • Require high titer of highly active viruses.