1. Experiment #5: Protein Separation Project Michael Eatmon Sarah Katen Nell Keith Monica Sanders

2. Introduction

3. Project Description and Goals (1) Scale-up of chromatography column used to separate a mixture of bovine hemoglobin and bovine albumin Batch process – specified amount of protein loaded on to column for each run Required to process 50,000 liters/year of a mixture of 6.0 g/liter albumin and 4.0 g/liter hemoglobin

4. Project Description and Goals (2) Total Capital Investment (TCI), cash flow, and profit of project will be evaluated Selling price of hemoglobin is $700 per kg Selling price of albumin is $500 per kg

5. Methods Considered Method #1: Purchase a single large column and run batches only on this column – lowest initial investment, but greater up-time and operating costs. Method #2: Purchase multiple columns and only run one batch per day per column – highest initial investment, but lowest operating costs. Optimum method between these extremes

6. Theory

7. Theory (1) Anion exchange chromatography – resin is a weak base and attracts negative ions when it is mixed when mixed with a solution at a pH below its pK (~10) TRIS-chloride buffer used b/c TRIS is neutral and will not interact with the resin Salt dissolved in TRIS buffer to cause proteins to desorb Components in stationary phase move down the column more slowly than those that dissolve in the mobile phase

8. Theory (2) Image courtesy of http://www.cartage.org.

9. Theory (3) Isoelectric point = pH at which net charge per amino acid is zero Isoelectric point bovine hemoglobin = 6.8 Isoelectric point bovine albumin = 4.7 Bovine albumin will come out of the column first because it is less charged in the buffer solution and has a lower affinity for the resin.

10. Theory (4) Column regenerated between runs by washing it with NaOH at pH of 14 to remove biological compounds adsorbed to resin. Resin stored in 20% ethanol to prevent drying

11. Theory (5) Pressure drop monitored to determine if column is appropriate for scale up Column length limited by pressure drop Calculated with the Darcy Equation

12. Theory (6) Need superficial velocity and Blake- Kozeny constant to get the pressure drop.

13. Design Plan

14. Design Plan (1) For scale-up, resolution is held constant Resolution of proteins in linear gradient elution ion exchange chromatography:

15. Design Plan (2) To remove the volume terms in this expression, we define

16. Design Plan (3) Substituting into previous equation gives

17. Design Plan (4) For scale-up purposes, the diffusion coefficient and column void fraction will be the same for the pilot and scaled-up design

18. Design Plan (5) As particle size increases, either Q or dp must decrease while the other is held constant The same resin and particle size will be used for the scaled-up design This gives:

19. Pressure Drop Restrictions The column height can be changed along with the linear velocity using the normalization between the two in order to meet pressure drop restrictions. When the flow rate is kept constant, the pressure drop decreases as column diameter increases

20. Scale-up Plan (1) In this experiment, desired elution flow rate is not given; the desired production is given in throughput The scale-up relationship can still be used as we know that throughput is proportional to elution flow rate

21. Scale-up Plan (2) The desired throughput is divided by the pilot throughput to get a scale-up factor (SUF) that will be used to determine the scaled-up column volume With the new volume, a scaled-up column height (L) may be determined using prefabricated column diameters as specified by manufacturers

22. Optimization In order to optimize the system, several diameters may be considered to find the most favorable system that will meet pressure drop restrictions. Also, the number of columns and batches per day will be variables in scale-up optimization.

23. Experimental Plan

24. Experimental Plan (1) The major variables in the system are: –the rate of change of the salt gradient –the final salt concentration within the column –the gradient elution velocity –the amount of protein loaded into the column over a given amount of time.

25. Experimental Variables (1) First variable - rate of change of the salt gradient Depends on elution volumes used Lower volume gives more rapid rate of change Different proteins elute at different salt concentrations Too rapid (low volume) – poor or no separation Too slow (high volume) – high separation time

26. Experimental Variables (2) Second variable – final salt concentration Changing the concentration of NaCl introduced into the system; a higher concentration gives a more rapid rate of change. Different proteins elute at different salt concentrations Too high – poor or no separation Too low – high separation time

27. Experimental Variables (3) Third variable – gradient elution velocity Too fast – proteins wash out in bulk; poor separation Too slow –separation time too slow for industrial processes

28. Experimental Variables (4) Fourth variable – amount of protein loaded into the column over a period of time Too much – not enough absorbent surfaces to hold protein – poor separation Too little – requires more time to separate desired throughput – inefficient

29. Apparatus

30. Operating Equipment Bovine albumin and bovine hemoglobin Bio-Rad econo column –DEAE Sepharose Fast Flow Bio-Rad econo UV monitor Bio-Rad gradient former

31. Operating Procedures Protein separation column created Protein loaded onto column Salt gradient added at feed rate Salt gradient added at elution rate Protein separation measured on chart recorder Regenerate column for next run

32. Operating Variables Elution rate and salt gradient varied Salt gradient volume: 75-100 mL Elution rate: 3.0-4.0 mL/min pH ≈ 8.2 Total cycle time recorded

33. Safety Hazards Should be handled in a well ventilated area The skin should be rinsed 15 minutes upon exposure to the skin Bovine hemoglobin can irritate the upper respiratory tract causing allergic reactions and asthma

34. Experimental Results

35. Protein Separation Trial Runs 9 different trials Maximize protein separation as measured by protein peak area ratios Minimize time between protein peaks Consistency

36. Successful Trial Run Conditions Bed Height14.25 cm Gradient Mixer Volume 75 mL in each side Gradient0 to 0.5 M NaCl in buffer Feed2.5 mL loaded at 0.5 mL/min (5min) Elution3 mL/min Detector2.0 AUFS Chart Speed30cm/hr Elution Time50 min

37. Trial Results Trial 1: Some separation, protein peaks spread very far apart Trial 2: Better separation, but peaks too close together Trial 3: Best separation, very small time between elapsed between peaks Trial 4-6: Little or no separation, indistinct peaks Trial 7-9: Incorrect peak formation, no peaks

38. Trial #1

39. Trial #2

40. Trial #3 (run to be scaled-up)

41. Hemoglobin/Albumin Peak Ratio peak area of bovine albumin, cm 2 peak area of bovine hemoglobin, cm 2 hemoglobin/albumin ratio Trial 1 2551190.467 Trial 2 1982051.035 Trial 3 88326.53.710 Trial 4 1829616.444 Trial 5 423859.5 Trial 7 1617210.75

42. Scaled-Up Design

43. Scale-up Procedure Scale-up factor (SUF) determined by dividing desired protein throughput per column by pilot throughput Trial #3 (10/5/04) scaled-up Assumption of 8-hour workday, 5-day workweek Assume 20-year project life; construction in 2004, production begins in 2005

44. Parameters to be Scaled-Up Experimental VariablePilot Value Bed height (cm)14.75 Column inner diameter (cm)0.8 Column volume (cm 3 )7.41 Protein throughput (g)0.025 Final salt concentration (M)0.5 Gradient volumes (mL)75 Elution rate (mL/min)3.0

45. Process to be Scaled-up Equilibration with 20 mM TRIS buffer (pH 8.2), 5 bed volumes in 20 minutes. Load protein Elute protein to final column concentration of 0.5 M NaCl in 20 mM TRIS, assuming same residence time as pilot scale Regenerate bed by flooding with: a) 3 bed volumes 1.0 M NaCl in 15 minutes b) 5 bed volumes 1.0 NaOH in 20 minutes c) 3 bed volumes deionized water in 15 minutes d) 3 bed volumes 20 mM TRIS in 15 minutes Repeat steps 3 and 4 for each run. Flood column with 20 % ethanol (1 bed volume) in 20 minutes for storage.

46. Scale-up Procedure (1) Desired daily throughput divided by arbitrary number of runs per day Column SUF determined from throughput and applied to pilot column volume to determine scaled- up volume Prefabricated column specifications used to determine bed height for given diameters

47. Scale-up Procedure (2) Scaled-up specs. used to determine superficial velocity and then pressure drop across column, to ensure compliance with column pressure rating Total cycle time used to determine the number of possible cycles per day Cycles per day divided by cycles per column per day to determine number of columns for scaled-up design

48. Scale-up Procedure (3) Arbitrary number of runs from initial step varied iteratively in order to minimize number of columns for each set of specs. Economic optimum at point of lowest equipment cost, as further calculations are based on total delivered equipment costs

49. Scaled-up Process 4 columns - 35 cm x 50 cm - P<2 bar Bed volume of 48 L Three runs per column per day Daily throughput of 2 kg protein Gradient volumes per run of 490 L Elution rate of 20 L/min

50. Process Equipment Equipment sized based on scaled-up specifications Components include chromatography columns, gradient and buffer tanks, fluid pumps, mixers, and tubing Equipment priced at lowest possible cost while meeting design requirements

51. Purchased Equipment Cost and Total Capital Investment Equipment prices determined from up-to-date online catalogues Total purchased equipment cost: $65,000 Total Capital Investment (TCI) determined by method of percentage of delivered equipment cost TCI: $380,000

52. Product Costs and Profitability Annual Product Cost (APC) is summation of all yearly raw materials, utilities, labor, and fixed production charges APC (2005): $480,000 APC per kg/feed: $960 Revenue from sales less APC is annual cash flow Return on Investment (ROI): 390%; above standard minimum of 10% (depreciation neglected)

53. Sources of Error Approximately 5% error inherent in calculation of SUF Scale-up parameters determined by arbitrary assumptions—assigned an approximate 10% error Accuracy in equipment pricing lends itself to TCI accuracy within 10% Error in APC neglected

54. Comparison to Literature Values

55. Comparison to Literature Values (1) Ratio of peak areas of experimental data

56. Comparison to Literature Values (2) Ratio of absorbance of hemoglobin to that of albumin times the concentration ratio

57. Comparison to Literature Values (3) Experimental Error Calculation

58. References Laboratory Operating Manual. Design Lab, CH E 4262, 2004. Harrison, Roger G., Paul Todd, Scott R. Rudge, Demetri Petrides. Bioseparations Science and Engineering. New York: Oxford University Press, 2003. Peters, Max S., Klaus D Timmerhaus, and Ronald E. West. Plant Design and Economics for Chemical Engineers, 5th ed. New York: McGraw- Hill Companies, Inc., 2003 Welty, James R., Charles E. Wicks, Robert E. Wilson, and Gregory Rorrer. Fundamentals of Momentum, Mass, and Heat Transfer, 4th ed. New York: John Wiley & Sons, Inc., 2001.