Biochemical Engineering CEN 551

1. Biochemical EngineeringCEN 551 Instructor: Dr. Christine Kelly Animal Cell Cultures (Chapter 12) and Glycosylation

2. 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

3. 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

4. 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).

5. 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.

6. 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.

7. 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.

8. 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

9. 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.

10. 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.

11. Serum Component Range • 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 (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

12. 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.

13. 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.

14. 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.

15. 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.

16. Traditional Mammalian Cell Culture • Scale up problematic. Roller bottles Tissue culture flasks

17. 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.

18. Aeration and agitation in mammalian cell culture(following material from http://www.np.edu.sg/~dept-bio/biochemical_engineering/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.

19. 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.

20. 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.

21. 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.

22. 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.

23. Rising Bubble Vortices form in the wake of the rising bubble. • 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

24. 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.

25. 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.

26. 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.

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

28. 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.

29. 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.

30. 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.

31. 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

32. 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.

33. 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.

34. 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.

35. 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.

36. 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.

37. 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.

38. 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.

39. 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.

40. 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.

41. 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.

42. 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.

43. 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.

44. 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.

45. 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.

46. 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

47. 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

48. 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.

50. 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.