Experiment #5: Protein Separation Project

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 Qor 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

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

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

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