Biochemical Engineering CEN 551

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Biochemical EngineeringCEN 551

• Instructor Dr. Christine Kelly
• Animal Cell Cultures (Chapter 12) and
  Glycosylation

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Sources

• Text - Chapter 12
• Peshwa, M. V. Mammalian Cell Culture
• Websites http//www.np.edu.sg/dept-bio/biochemic
  al_engineering/lectures/bioreact1/bioreact3_1.htm

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Animal Cell Characteristics

• 10-30 um larger than bacteria or yeast
• Eukaryotic
• Cell membrane no cell wall shear sensitivity
• Surface is negatively charged grow on
  positively charged surfaces
• Suspension cells or anchorage dependant cells.
• 80-85 water, 10-20 protein, and 1-5
  carbohydrates.
• Lipid bilayer cell membrane that is sensitive to
  shear.
• Optimum growth at 37oC

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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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• Mammalian cell line ? animal cell line.
• Insect, fish, crustacean cell lines are evolving
  technologies.
• Baculovirus virus that infects insect cells.
  Nonpathongenic to humans, has a strong promoter.
• Insect cell lines are naturally continuous.
• Most cell lines derived from ovaries or embryonic
  tissue.
• Hybridoma cells fusing lymphocytes (normal
  blood cells that make antibodies) with myeloma
  (cancer) cells.

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Cell Components

• Endoplasmic reticulum (ER) membrane bound
  channels. Postranslational processing and
  secretion.
• Mitochondria site of respiration where ATP is
  produced.
• Lysosomes organelles responsible for the
  digestion of food. Contain hydrolytic enzymes.
• Golgi body complex glycosylation, protein
  secretion.

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Medium

• Glucose energy source up 55 mmol/L
• Glutamine energy source 2-7 mmol/L
• Aerobically metabolize to CO2 or anaerobically to
  lactic acid.
• Lactate and ammonium toxic byproducts of
  mammalian cell growth.
• Lactic acid and ammonium inhibitory at 30 and 5
  mmol/L, respectively.
• Lactate reduces pH.

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• Oxygen utilized at approximately 0.05-5 pmol
  O2/cell hr. 10-30 DO is non-limiting. Higher
  DO concentrations can be toxic, leading to
  oxidative damage.
• Amino acidscell line dependant, balance is
  critical.
• Growth factors
• Cytokines
• Trace elements

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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.
• by-product of the beef-packing industry, FBS can
  only be obtained where sufficient numbers of
  fetuses become available during the slaughtering
  process.

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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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Serum Component Range

• (mg/ml)Albumin 35-55Immunoglobulins(IgG 75-85
  of all Ig) 8-18Fibrinogen 2-6Alpha-1
  antitrypsin 1-2.5Alpha-2 macroglobulin
  0.5-3.5Transferrin 1.5-3.5Alpha-2ß-lipoproteins
  (LDL) 4-7Alpha-lipoproteins (HDL)
  0.6-1.5Haptoglobin 5Alpha-1 acid glycoprotein
  0.5-1.25hemopexin 1Pre-albumin 0.3-0.4Total
  Protein 62-80

• Ions
• Bicarbonate 25-35 mM
• Chloride 100-108mM
• Sodium 134-143 mM
• Potassium 3.5-4.5 mM
• Calcium 2-2.5 mM pH 7.4

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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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• While global demand for FBS has steadily
  increased over the past years, import of FBS into
  the US and the EU are strictly controlled.
  Whereas the EU allows South American serum for
  the academic research market, the USDA keeps the
  border closed for South American serum. FBS used
  in bioprocessing to manufacture therapeutic
  proteins for a global market has to be either
  Australian/New Zealand or US sourced material.
  Most protocols for FBS in bioprocessing require
  exposure to gamma irradiation.

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Buffer

• Mamallian cells grow best at 37oC and 7.3 pH.
• Bicarbonate based buffer to maintain a constant
  pH coupled with addition of base or acid when
  needed.
• 1-10 CO2 in gas phase is also used to control
  pH.
• CO2 also important in the synthesis of purines
  and pyrimdines.
• CO2 primes energy metabolism.
• Excess CO2 suppresses cell growth and can alter
  intracellular pH.
• Osmolarity increases as pH is adjusted adjusted
  due to the addition of salts, too high osmolarity
  results in cell shrinkage and eventually lysis.

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Typical Mammalian Batch Culture

• Inoculations typically 105 cell/mL.
• Maximum cell concentration 106 cell/mL.
• Typically 3-5 doublings before stationary phase.
• Typical doubling times 12-36 hr, so batch phase
  from 4 to 7 days.

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Traditional Mammalian Cell Culture

• Scale up problematic.

Roller bottles
Tissue culture flasks
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Animal Cell Bioreactors

• Gentle agitation due to shear sensitivity.
• Homogeneous environment (T, pH, DO, CO2).
• Large surface to volume for anchorage dependant
  cells.
• Removal of toxic byproducts
• (lactic acid and ammonium.

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Aeration and agitation in mammalian cell
culture(following material from
http//www.np.edu.sg/dept-bio/biochemical_enginee
ring/lectures/bioreact1/bioreact3_1.htm)

• 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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• Agitation systems used for microbial cells are
  often poorly suited to the use with animal cell
  cultures.The former are generally designed to
  shear bubbles and thus increase kLa. Their high
  shear characteristics however will also tend to
  damage fragile animal cells.
• mammalian cell growth rates are considerably
  slower than those of most aerobic microorganisms
  and oxygen transfer requirements are therefore
  also proportionately lower.

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• As with microbial systems, the successful
  cultivation of animal cells requires that mass
  and heat transfer requirements be met. Agitation
  and aeration are thus critical considerations in
  the large scale cultivation of animal cells.
• Unlike microbial cells, animal cells do not have
  cell walls and are protected from environmental
  forces by only their enriched cell membranes.
  Animal cells are therefore regarded as "shear
  sensitive".
• There are two major physical forces that can
  cause cell damage shear forces and bubble
  energy.

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Shear damage

• Shear forces are created from fluctuating liquid
  velocities which arise during turbulent mixing
  and are visualized as turbulent eddies.
• Shear forces increase with the level of
  turbulence and on the type of agitator used.
• There are two "forms" of shear
• Localized shear which occurs around objects
  moving in the culture media, eg. impellers and
  bubbles.
• Shear in the bulk liquid arising from turbulence
  with the reactor.

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Localized shear

• Localized shear occurs around objects moving in
  the culture media, eg. impellers and bubbles.
• As radial flow impellers move, their blades leave
  a trail of eddies in their wake.
• Under normal operating conditions, the Kolmogorov
  size of these eddies are typically small enough
  to break apart bubbles and to damage animal
  cells
• For this reason axial flow impellers are used in
  the culture of animal cells.

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• Localized shear can also arise around moving
  bubbles either around the bubble or in the wake
  of the bubble.
• Shear arising as a result of bubbles moving
  through the bulk liquid is not considered a major
  cause of cell damage.

Flow lines move fastest near the bubble.
Rising Bubble
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• Shear can also form around solid surfaces around
  which the medium is moving. For example, high
  shear forces can be formed around the surface of
  a poorly finished impeller.

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Shear in the bulk liquid

• In baffled reactors, as the stirrer speed
  increases, turbulent eddies will be formed in the
  bulk liquid As the level turbulence increases,
  the eddy size will decrease and the level shear
  will increase.
• The formation of shear stresses in the bulk
  liquid due to turbulence were once believed to be
  a major cause of cell damage in animal cell
  bioreactors.
• It is now however widely recognized that shear
  forces in the bulk liquid are NOT the major cause
  of cell damage in sparged reactors.

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• Under normal stirring conditions, the average
  size of the turbulent eddies (which are expressed
  in terms of the Kolmogorov eddy size) is
  considerably larger than the average cell
  diameter.
• The cells are able to "ride" between the eddies
  and thus are not affected by shear forces.
• The Kolmogorov eddy size decreases as the
  stirring speed increases.
• Shear damage is maximal when the Kolmogorov eddy
  size reduces to size of the cells. The randomly
  moving liquid lines then produce violent pressure
  oscillations then act to pull the cell apart as
  they enter and leave the turbulent eddies.

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Effect of eddy size
cell
cell
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• The sensitivity of animal cells to liquid shear
  forces varies with the cell line and age.
• Cells have been found to be more fragile during
  stationary and lag phases. Their robustness
  increases during exponential growth.

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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 of the culture
  fluid, cells trapped on the bubble interface or
  in the bubble wake tend to also suffer damage and
  can be literally torn apart.
• The level of 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. Bubble damage is also reduced by
  reducing the bubble velocities near the liquid
  surface. Therefore, the design of the
  disengagement zone is important.

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• Bubble damage can also occur in agitated
  non-sparged bioreactors as a result of the
  entrainment of air through the culture fluid
  surface. In experiments on surface aerated
  cultures, it has been found that cell damage
  begins when cell entrainment is initiated at
  stirring speeds between 150 - 200 rpm.
• Experiments using completely filled reactors in
  which air-entrainment was prevented, have
  demonstrated that stirring speeds up to 800-900
  rpm can be used before cell damage is
  significant. At 800-900 rpm, the Kolmogorov eddy
  size is comparable to the cell size.
• Bubble damage rather than liquid shear forces are
  the major cause of cell damage in sparged animal
  cell bioreactors

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Foam damage

• Foam damage occurs when the bubbles move in
  different directions pulling entrapped cells in
  different directions
• The cells which are attached to the bubbles in
  the foam are thus stretched and eventually pulled
  apart by the moving bubbles.

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Methods of minimizing cell damage

• One method or minimizing cell damage is to
  immobilize the cells eg. in gels, onto
  microcarrier beads or in hollow fiber systems.
• However, not all cells or cell culture processes
  are amenable to immobilization and appropriate
  techniques for growing suspension cells have had
  to be developed.

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• Cell damage in animal cell cultures can occur
  primarily due to
• liquid or hydrodynamic shear damage and
• bubble damage
• The extent of damage caused by these factors is
  dependent upon the
• characteristics of the cell line
• the nutritional state of the cells
• the medium composition
• reactor design and the
• reactor operating conditions.

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Cell lines

• Shear forces in the bulk liquid are generally not
  a problem if the cells are healthy and the
  appropriate cell line is used.
• Cell lines are typically selected for their
  ability to produce a product at desired
  efficiency and productivity. However, for the
  large scale cultivation purposes, cell lines must
  also be selected for their ability to grow in the
  higher shear environment of a bioreactor.

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Media

• When switching from serum-based to serum-free
  media, the cells must also be properly adapted to
  new medium. Failure to do so will lead to the
  cells being unhealthy and more sensitive to
  shear.
• Medium composition is another important
  consideration, particularly with serum free
  media. The higher cell numbers required for
  production scale operations may require a higher
  input of specific medium components such as amino
  acids and sterols.

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• Studies in the 1970's and 1980's on hybridoma
  cell lines found that the media then in use were
  deficient in amino acids. When certain amino acid
  were depleted, then the cells' shear sensitivity
  increased.
• Therefore, an important method of reducing shear
  damage is to ensure that the nutritional
  requirements of the cells are met.

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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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• Stabilizing foams giving cells time to detach
  from the bubbles before they burst.
• Making the bubbles "slippery" so that the cells
  are less likely to be attracted to the bubbles
  and thus less likely to be drawn up to the
  surface by the rising bubbles.
• Albumin and other serum proteins are believed to
  protect cells in a similar manner to Pluronic
  F68. Pluronic F68 is thus an necessary component
  of serum free culture media.

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Impeller design

• The shear sensitivity of animal cells makes
  radial flow impellers unsuitable for use in
  animal cell cultures and shear cannot be used as
  a mechanism for breaking up bubbles.
• The impeller design and its mode of operation are
  critical in the large scale cultivation of animal
  cells.
• As we have seen, axial flow impellers produce
  higher flow per unit power input characteristics
  as compared to radial flow impellers.

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• Another advantage of using axial flow impellers
  is that they are more efficient at lifting cells
  from the base of the reactor.
• Axial flow impellers stirring at relatively low
  stirrer speeds are therefore widely used in the
  culture of animal cells. These impellers are
  operated with the primary objectives of
  optimizing liquid-liquid mass transfer rates and
  heat transfer rates but not increase the surface
  area for oxygen transfer.
• Although axial flow impellers are not designed to
  provide high shear conditions required for
  breaking bubbles, the do prevent the bubbles from
  rising directly to the surface. In this way,
  increase the bubble residence time and thus
  increase the oxygen transfer efficiency.

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Draft tubes

• Airlift reactors have been used to to
  successfully cultivate mammalian and insect cells
  in reactors with liquid volumes of up to 1000 L.
  This is despite the potential problems associated
  with bubble damage.The company Celltech, for
  example uses airlift production as the
  predominant technique for large scale cultivation
  of hybridoma cells.The low shear environment
  provided by airlift reactors, combined with the
  use of appropriate media and shear protectorants
  can compensate for the increased likelihood of
  bubble damage.

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Reducing bubble size

• Bubble damage is recognized as the major cause of
  cell damage in sparged animal cell bioreactors.
• When large bubbles burst, the release more energy
  than small bubbles. Large bubbles are therefore
  more destructive than small bubbles.
• Likewise the degree of 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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• Animal cell bioreactors are not designed to use
  the agitator as a tool for decreasing the bubble
  size diameter.
• The sparger therefore plays a critical role in
  reducing the bubble diameter.
• Specially designed spargers which generate very
  small bubbles have been designed for use in
  animal cell bioreactors.

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Bubble free oxygenation

• Three main techniques by which enhanced oxygen
  transfer rates can be achieved without the need
  for sparging
• headspace oxygenation
• external oxygenation
• direct oxygenation using gas permeable silicone
  tubing or hydrophobic membranes.

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Headspace oxygenation

• The simplest method of bubble free oxygenation is
  the transfer of oxygen from the headspace.
• This method is widely used in small scale systems
  such as T-flasks and spinner flasks. In large
  scale systems, the use of pure oxygen instead of
  air have also tested.
• In headspace aeration, oxygen rich gas is passed
  into the headspace of reactor. The oxygen
  diffuses into the liquid.
• The headspace may be pressurized to increase the
  partial pressure of oxygen in the gas phase

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External oxygenation

• A more commonly used and effective method of
  bubble free oxygenation is to use a separate
  oxygenation chamber
• The medium is oxygenated in a separate unit which
  can either be a stirred tank reactor or a static
  mixer. The oxygenated medium is pumped into the
  bioreactor while the oxygen depleted medium is
  pumped back into the oxygenation unit

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• A cell separation system such as a hollow fiber
  filter, is used to separate the cells from the
  medium before medium passed into the oxygenation
  unit.
• The same principle can be used with immobilized
  cell cultures such as fluidized bed and fiber bed
  reactors.
• New Brunswick's Celligen Bioreactor which uses
  the fiber-bed principle for the culture of animal
  cells.

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Direct bubble free oxygenation

• Various techniques are used to achieve direct
  bubble free oxygenation of animal cell
  bioreactors including the use of gas permeable
• silicone tubing, membranes, sieves
• Oxygen rich gas is passes through tubing or a
  membrane bound capsule. The oxygen diffuses
  through the pores into the liquid medium At the
  same time, carbon dioxide diffuses out of the
  medium into the gas phase.

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• Hyrdophobic membranes are used to physically
  separate the gas from the culture medium. The
  membranes are much thinner than silicone tubing
  and thus offer higher oxygen diffusion rates.
  They are also pleated to increase the surface
  area for oxygen transfer. The membranes are also
  hydrophobic which minimizes the blockage of the
  membrane pores by cells.

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Anchorage Dependant Cells

• Require a large surface area for growth. Primary
  cells will only grow as a monolayer.
• Secondary cells can grow in multilayers.
• Microcarriers.
• Hollow fiber reactors
• Immobilization in gel beads
• Microencapsulation

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Microcarriers

• DEAE-Sephadex beads (non porous), up to 70,000
  cm2/L surface area.
• Can be modified with surface ligands (collagen)
  to increase attachment.
• Homogenous environment.
• Bead to bead abrasion a problem
• Porous beads introduce heterogeneity due to
  diffusional limitations.

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Hollow-fiber Reactors

• Cells grow on external (shell side) surface of
  the fibers.
• Nutrients flow through the tubes and diffuse to
  the cells.
• Microenvironmental conditions vary because there
  is no mixing.
• Have been used for monoclonal antibodies.

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Immobilization or Encapsulation

• To reduce shear on cells.
• Can achieve high cell densities
• Diffusional limitations, microenvironmental
  conditions heterogeneous.

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Products of Animal Cell Cultures

• Monoclonal Antibodies
• Important animal cell product.
• Produced by hybridoma cells.
• Used in diagnostics, therapeutics, and
  separations (affinity chromatography).

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• Immunobiological Regulatiors
• Interferon anticancer glycoprotien
• Produced by animal cells and recombinant
  bacteria.
• Others include lymphokines (immune response
  regulators), interleukines (anticancer), tissue
  plasminogen activator (anti blood clotting)
• Virus Vaccines
• Live or weakened virus grown in cells, then
  harvested and killed.
• Subunit display protein produced in bioreactor.

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• Hormones
• Large, glycosylated molecules, produced using
  cells from the hormones synthesizing organ.
• Enzymes
• Secretion, glycoslyation, posttranslational
  modifications important.
• Insecticides
• Insect viruses
• Whole cells and tissue culture
• Artificial skin and cartilage. Working on more
  complex tissues.

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• Form groups of two make up ten, creative,
  innovative, true/false or multiple choice
  questions about animal cell culture. Be prepared
  to present the questions to the class.

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Glycosylation
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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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http//www.sinica.edu.tw/kkhoo/GlycoProteomics/sl
ide06.htm
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http//www.sinica.edu.tw/kkhoo/GlycoProteomics/im
ages/Slide07.jpg
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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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Chitobiose Core Structure

• All N-linked glycans have the same core
  structure.
• Chitobiose core structure 2 GlcNAc, 3 Mannose,
  sometimes a Fucose.

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Synthesis

• Common core glycan produced on a lipid carrier in
  the cytoplasm and then in the ER.
• The core is attached to the asparagine residue on
  the peptide as it enters the ER.
• The three terminal glucose units of the common
  core are then removed.
• The common core is transported to the Golgi
  apparatus.

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• Three manose residues are removed from the common
  core in the Golgi apparatus
• A GlcNAc is added on one arm of the core
  structure.
• Further varying modifications are performed
• Removal of mannose residues
• Addition of GlcNAc
• Bisecting GlcNAc
• Creation of further branches
• Core fucosylation

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http//opbs.okstate.edu/petracek/Chapter202720F
igures/Fig2027-36.GIF
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O-Linked

• Linked to serine or threonine residues on a
  protein.
• Simpler process.
• Involves only 1-6 sugar residues.
• Occurs in the Golgi Apparatus.
• Amino acid reions with high concentrations of
  serine, threonine, and proline tend to be
  O-glycosylated.

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Final Stage of Glycosylation

• Takes place in the trans-Golgi
• Addition of galactose and sialic acid at the end,
  and fucose residue on the core.

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Glycophosphatidylinositol Residues

• Anchor glycosylation directly bound directly to
  the cell membrane.

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Influence of Host Cell Line

• Bacteria unable to glycosylate.
• Yeast often hyperglycosylate (lots of mannose).
• Yeast different type of O-linked than mammals.
• Plants smaller glycan structures than mammals,
  lacking sialic acid.
• Mouse and Pig NeuGc instead of NeuAc as the
  terminal sialic acid.

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Influence of Cell Culture Conditions

• Adaptation from serum to serum free-medium.
• Glucose concentration.
• Lipid supplement.
• Oxygen concentration.
• pH.
• Ammonium.

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Effects of Glycosylation

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

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Regulatory Issues

• FDA non demands comprehensive carbohydrate
  analysis before licensing glycoproteins.
• Glycan heterogeneity unavoidab but must be
  within prescribed bounds.

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Analysis of Glycosylation

• Gas chromatography
• Liquid chromatography
• Capillary electrophoresis
• Mass spectrometry
• Exoglycoidase arrays
• Sialic acid analysis