Biochemical EngineeringCEN 551

Instructor: Dr. Christine Kelly

Animal Cell Cultures (Chapter 12) and Glycosylation

Sources

• Text - Chapter 12

• Peshwa, M. V. Mammalian Cell Culture

• Websites: http://www.np.edu.sg/~dept-bio/biochemical_engineering/lectures/bioreact1/bioreact3_1.htm

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

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).

• 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.

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.

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.

• 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 acids:cell line dependant, balance is critical.

• Growth factors

• Cytokines

• Trace elements

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.

• 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.

Serum Component Range(mg/ml)

Albumin 35-55Immunoglobulins(IgG 75-85% of all Ig) 8-18Fibrinogen 2-6*Alpha-1 antitrypsin 1-2.5Alpha-2 macroglobulin 0.5-3.5Transferrin 1.5-3.5Alpha-2+ß-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

• 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.

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.

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.

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.

Traditional Mammalian Cell Culture

• Scale up problematic.

Roller bottles Tissue culture flasks

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.

Aeration and agitation in mammalian cell culture

(following material from http://www.np.edu.sg/~dept-bio/biochemical_engineering/lectures/b

ioreact1/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.

• 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.

• 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.

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.

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.

• 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.

Rising Bubble

Vortices form in the wake of the rising bubble.

Rising Bubble

Flow lines move fastest near the bubble.

• 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.

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.

• 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.

Effect of eddy size

High stirrer speedLow stirrer

speed

cell cell

• 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.

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.

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.

• 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

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.

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.

Cell damage in animal cell cultures can occur primarily due to:

1. liquid or hydrodynamic shear damage and

2. 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.

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.

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.

• 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.

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.

• 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.

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.

• 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.

Draft tubesAirlift 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.

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.

• 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.

Bubble free oxygenationThree 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.

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

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

• 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.

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.

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.

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 beadsMicroencapsulation

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.

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.

Immobilization or Encapsulation

• To reduce shear on cells.

• Can achieve high cell densities

• Diffusional limitations, microenvironmental conditions heterogeneous.

Products of Animal Cell Cultures

Monoclonal Antibodies• Important animal cell product.• Produced by hybridoma cells.• Used in diagnostics, therapeutics,

and separations (affinity chromatography).

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.

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.

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.

Glycosylation

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).

http://www.sinica.edu.tw/~kkhoo/GlycoProteomics/slide06.htm

http://www.sinica.edu.tw/~kkhoo/GlycoProteomics/images/Slide07.jpg

Three Types of Glycosylation

•N-Linked

•O-Linked

•Membrane anchor

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.

Chitobiose Core Structure

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

sometimes a Fucose.

N

MannoseN-acetylglucosamine

(GlcNAc)

Fucose

Asparagine

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.

• 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

http://opbs.okstate.edu/~petracek/Chapter%2027%20Figures/Fig%2027-36.GIF

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.

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.

Glycophosphatidylinositol Residues

Anchor glycosylation directly bound directly to the cell membrane.

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.

Influence of Cell Culture Conditions

• Adaptation from serum to serum free-medium.

• Glucose concentration.

• Lipid supplement.

• Oxygen concentration.

• pH.

• Ammonium.

Effects of Glycosylation

• Pharmacokinetics and clearance (especially the degree of sialylation).

• Immunogenicity.

• Solubility and protease resistance.

Regulatory Issues

• FDA non demands comprehensive carbohydrate analysis before licensing glycoproteins.

• Glycan heterogeneity unavoidab – but must be within prescribed bounds.

Analysis of Glycosylation

• Gas chromatography

• Liquid chromatography

• Capillary electrophoresis

• Mass spectrometry

• Exoglycoidase arrays

• Sialic acid analysis