Biochemical Engineering CEN 551

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Biochemical EngineeringCEN 551

• Instructor Dr. Christine Kelly
• Exam 3 Review

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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, 100 pm 200 pm Poster
  Presentations
• Tuesday, April 20 Presentations
• Thursday, April 22 Exam 3
• Tuesday, April 27 Presentations

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

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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.

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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.

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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).

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• 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.

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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

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• 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.

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• 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.

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• 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.

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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.

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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.

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Effect of eddy size
cell
cell
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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.

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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.

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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.

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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.

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• 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.

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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).

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Three Types of Glycosylation

• N-Linked
• O-Linked
• Membrane anchor

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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.

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Effects of Glycosylation

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

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• 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

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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.

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• 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.

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Host Organisms

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

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

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Genetic Instability

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

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• 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.

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

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• 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.

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

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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.

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• 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 hosts
  native protein to prevent destruction. Can fuse
  handle or tail for affinity chromatography. Can
  fuse hosts secretion signal to direct out of the
  cell.

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• 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.

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

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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 wouldnt
  normally be turned on. Example to degrade
  hazardous waste to lower concentrations than
  would normally induce the pathway.

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• 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.

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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.

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• 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.

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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).

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• 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.

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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.

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• 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.

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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.