1. Simulated Moving Bed Chromatography in the Pharmaceutical Industry Ron Bates Bristol-Myers Squibb April 19, 2004

2. Outline Short Biography What is Bristol-Myers Squibb Chromatography –Batch vs continuous HPLC, LC, SMB, P-CAC Simulated Moving Bed Chromatography –Introduction –Theory (brief) –Operation –Applications in the Pharmaceutical Industry

3. B.S. Chemical Engineering, RPI, 1993 Ph.D. Biochemical Engineering, University of Maryland, Baltimore County, 1999 –Focus: ion-exchange chromatography Pfizer, Groton, CT, 1999-2003 –Focus: small molecule chromatography, HPLC, LC, SFC, SMB, FLASH, extraction, crystallization, precipitation Bristol-Myers Squibb, Syracuse, NY, 2003-present –Focus: protein chromatography

4. Bristol-Myers Squibb Top-ten pharmaceutical company Products in numerous therapeutic areas Cardiovascular & Metabolic DiseasesMental Health Pravachol, CoumadinAbilify Headache and MigraneInfectious Diseases ExcedrinReyataz, Sustiva Oncology Erbitux, Taxol Strong pipeline focused in 10 therapeutic areas –Oncology, Cardiovascular, Infectious Diseases, Inflammation, etc. Sites around the world –U.S. Research/Manufacturing sites MA, NY, NJ, CT, IL, Puerto Rico

5. Bristol-Myers Squibb Syracuse, NY Clinical and Commercial Manufacturing Plant –Small-molecule pilot plants Process development and optimization Clinical manufacturing –Penicillin-based products Last US-based Penicillin manufacturer –Bio-synthetic products –Biotechnology Development, Manufacturing, Analytical Biosciences, Quality Control / Assurance

6. Bristol-Myers Squibb Syracuse, NY - Biotechnology Two lead protein therapeutics –Abatacept: commericial in 2005 Commercial-scale manufacturing Commercial launch out of Syracuse Facility BLA filing – Dec. 2004 –LEA29Y: Phase III clinical trials in 2005 Development for next generation process Clinical production in 2004 Expansion in analytical and quality groups to support processes

7. Batch vs. Continuous Chromatography

8. Discrete starting and ending points –Example: 10 minute HPLC cycle –Types: GC, HPLC, FLASH, FPLC, LC, etc. –Can be run in many modes: Linear, overloaded, frontal, etc. Batch Chromatography

9. (Raffinate) Feed Desorbent Effluent to Waste Load Elution Effluent to Waste DesorbentElution (Extract) (To Waste) Strong Solvent Regeneration Reference: Linda Wang, Perdue University

10. Batch Chromatography Empty zone

11. Continuous Chromatography Feed is loaded onto column and product is collected continuously Annular (P-CAC) –Preparative continuous annular chromatography Countercurrent –Simulated moving bed chromatography (SMB) Feed column

12. P-CAC Reference: Genetic Engineering News, Oct. 1, 1999

13. P-CAC Reference: Genetic Engineering News, Oct. 1, 1999

14. P-CAC Reference: Genetic Engineering News, Oct. 1, 1999

15. P-CAC Reference: Genetic Engineering News, Oct. 1, 1999

16. Simulated Moving Bed Chromatography (SMB)

17. What is SMB SMB is Simulated Moving Bed Chromatography. SMB is continuous countercurrent chromatography. The feed is pumped into the system and two (or more) product streams are continuously collected. SMB has been used for the production of millions of tons of bulk commodities (p-xylene, high fructose corn syrup, etc...) for the past four decades. Due to improvements in column and equipment technology, SMB has recently been used in the pharmaceutical industry (Sandoz, SmithKline, UCB, Pfizer). –HPLC costs: $100/kg to $5000/kg –SMB costs: $50/kg to $200/kg

18. SMB versus HPLC Advantages of SMB: –Lower solvent utilization (up to 10 times less than batch HPLC) –Generally can use less expensive, larger stationary phases –Able to get high recovery and high purity –Sometimes better productivity –Lower labor and QC costs –Only partial separation of solutes is required to obtain high purity. –Higher yield than batch – 10% more than batch. –High throughput – 5 to 10 fold increase. –Lower solvent consumption – An order of magnitude lower. –Continuous process. Disadvantage of SMB: –Binary separation only –Complexity

19. Commercial Applications of SMB Hydrocarbons Sugars Agrochemicals Antibiotics Peptides Chiral Drugs –Gaining tremendous momentum – FDA approves of the technology –Chiral resin manufacturers sell resins specifically made for SMB Proteins? –Useful as polishing step? SEC: remove aggregated form of product –Multicomponent separations more difficult than traditional uses 8, 12, even 16 zone systems being examined

20. Basic Principle Mobile Phase Feed Continuous Countercurrent Chromatography stationary column A sample is injected in the centre of a stationary column The two components move at different speeds and are separated If we now move the column from right to left, at a speed halfway between that of the solutes, they now move in different directions...

21. Basic Principle Mobile Phase Feed column The two solutes now move in different directions relative to a stationary observer. If the column is very long, the bands will continue to separate. Continuous Countercurrent Chromatography

22. Basic Principle Mobile Phase Feed column If we continue to add sample at the center, the components will continue to separate Continuous Countercurrent Chromatography

23. This is clearly a continuous system, but there are problems. The column needs to be of infinite length, the actual moving of solids is very difficult and some way to introduce and remove the sample and the products are needed. We solve this by cutting the column into small segments and simulating the moving of them Basic Principle Mobile Phase Feed column Continuous Countercurrent Chromatography

24. The feed and solvent inlets are now placed between the segments and are moved each time a segment is moved from one end to the other Basic Principle Mobile Phase Feed column Continuous Countercurrent Chromatography

25. Products are removed by bleeding off a carefully calculated flow at suitable exit points. This changes the velocity of the bands in the column and forces the products to move toward the ports This ensures that the column segments are clean before they are moved and that the solvent can be recycled directly back through the system Mobile Phase Basic Principle Mobile Phase Feed column Continuous Countercurrent Chromatography

26. True Moving Bed

27. Binary Separation in a True Moving Bed Desorbent Raffinate Extract Feed Extract Raffinate Time : t Time : t +  t Reference: Linda Wang, Perdue University

28. Binary Separation in a True Moving Bed Desorbent Raffinate Extract Feed ExtractRaffinate Time : t + 3  t Feed Time : t + 2  t Desorbent Reference: Linda Wang, Perdue University

29. Binary Separation in a True Moving Bed Desorbent Raffinate Extract Feed Extract Raffinate Time : t + 4  t Time : t + 5  t Reference: Linda Wang, Perdue University

30. TMB to SMB Since it’s very difficult to move solids, true countercurrent chromatography does not exist. Instead, the bed is broken into many fractions and their movement is simulated by changing the inlet and outlet ports

31. Simplified SMB - 1 Feed Solvent ExtractRaffinate Feed Solvent ExtractRaffinate The system is started..... A frontal elution separation occurs in Section 3. 1234

32. Simplified SMB - 2 Feed Solvent ExtractRaffinate Feed Solvent ExtractRaffinate The separation continues..... Eventually the front of pure product 1 reaches the outlet. It is distributed between the final Section and the product port

33. Feed Solvent ExtractRaffinate Simplified SMB - 3 Feed Solvent ExtractRaffinate Finally, the mixed product reaches the outlet. To avoid collecting impure material, it is necessary to move the columns 1 position upstream.

34. Feed Solvent ExtractRaffinate Simplified SMB - 4 Feed Solvent ExtractRaffinate The frontal separation continues; at the same time, the slow moving product starts to separate from the tail of the mixed product band in Section 2 Eventually the fast moving product again reaches the outlet and more pure product is collected.

35. Feed Solvent Extract Raffinate Simplified SMB - 5 When the mixed band reaches the end of Section 3 its tail has left Section 2 (if the separation has been correctly designed) and only pure product 2 remains in Section 2. Feed Solvent Extract Raffinate To avoid collecting impure raffinate, the columns are moved once more. Now, the pure component 2 is in Section 1.

36. Feed Solvent Extract Raffinate Simplified SMB - 6 Feed Solvent Extract Raffinate The second component is now collected at the Extract port while the separation continues in Sections 2 and 3. The faster component reaches the Raffinate port and is again collected; note that the outlet concentrations are neither constant nor concurrent.

37. Feed Solvent Extract Raffinate Simplified SMB - 7 Feed Solvent Extract Raffinate Eventually, the mixed zone reaches the raffinate port and the columns are again switched. This simplified system is now in a steady state mode and will continue to cycle. Switch

38. The moving of the bed is simulated by moving the points of feed and mobile phase addition, as well as the points of raffinate and extract removal while keeping the column positions fixed. Time = 0 Extract Feed Raffinate Mobile Phase Feed Raffinate Time = 1 Mobile Phase Extract Packed Column

39. The zones are made up of one or more columns Six-column SMB System Eight-column SMB system IIIIIIIV IIIIIIIV IIIIIIIVIIIIIIIV SMB Configurations

40. SMB Operation

42. Theory – Governing Equations For another day… Maybe

43. Theory – Working Equations / Definitions k’ 1 = capacity factor = (t r -t 0 ) / t 0 α = k’ 2 / k’ 1 R s = 2* (t r1 -t r2 ) / (w 1 -w 2 )

44. SMB – Method Development 1.Start with linear batch experiments 2.Increase either mass or volume of load to overload the column 3.Calculate isotherm 4.Determine resistance to mass transfer (if important) 5.Calculate necessary flow rates 6.Optimize (either on-the-fly or with a proven model)

45. Linear Chromatography

46. Batch Chromatography Experiments Feed concentration –As concentrated as possible to minimize disruption to Zone III velocity –Need to run batch experiments at appropriate concentrations and solvents Desorbent composition –Solubility of products –Strength Trade-off between time and mobile phase utilization Sorbent –Capacity, selectivity, resolving power

47. Feed Concentration Feed concentration: Consider two systems –A: Concentrated feed –B: Dilute feed Run batch experiments to examine effect of concentration

48. Desorbent composition Multiple trade-offs: Solubility of products and effectiveness of the solvent –Not always complimentary –Often solubility dictates solvent composition Speed –Low k’ = high throughput More wear and tear on equipment Larger system needed –Large k’ = low throughput Less wear and tear Smaller system acceptable

49. Choice of Sorbent Capacity: higher = better? Selectivity: higher α = better? Resolving power: higher R s = better?

50. Linear Chromatography

51. Volume Overloading

52. Batch Chromatography to SMB Initial Operating Conditions Determine optimal feed concentration, stationary phase and mobile phase composition (highest α with lowest capacity factors) Calculate isotherm and mass transfer resistances Either use software package or rules of thumb to generate initial SMB flow rates

53. Zone velocities v I = v Recycle + v D v II = v I - v X v III = v II + v F v Recycle = v III - v Raff Solvent Mass Balances – Flow Rates IIIIIIIV vIvI v II v III v Recycle vXvX v Raff vFvF vDvD Overall Mass Balance v D + v F = v X + v Raff

54. Flow rates Commercial SMB design models available –Given batch results from 5-10 column experiments Flow rate, feed concentrations, retention times Solubility data –Predict zone velocities, productivities –Problems: Usually assumes simple adsorption model and lumped mass transfer coefficients Often difficult to interpret overloaded chromatograms Rules of Thumb –Educated guesses based upon batch results from linear and overloaded experiments V II and V III ratio (based upon retention times) V I to flush back-side of slowest component from zone I Feed concentration and flow rate based upon solubility data and solvent mass balance

55. Period The period is the time a column stays in one zone also called switching time. Changing the period has the effect of changing all 4 zones simultaneously, thus either speeding up or slowing down the solutes

56. Example of switching time

57. SMB Optimization Independent variables: –Flow rates Recycle, Desorbent, Raffinate, Extract, Feed –Period (switching time) –That’s it. Procedure: –Get the system bound, manipulate the flow rates to maximize throughput at required purity

58. SMB Optimization IIIIIIIV vIvI v II v III v Recycle vXvX v Raff vFvF vDvD Questions: What is the effect of increasing the Zone I flow rate? –How would one accomplish this? Zone II? Zone III? What if the system is underutilized (i.e., more feed can be added to the system) – how would one do this without affecting the other zone flow rates?

59. Two component SMB System Feed Desorbent Extract Raffinate Conc. I II III IV Bed Position

60. SMB Optimization IIIIIIIV vIvI v II v III v Recycle vXvX v Raff vFvF vDvD Questions: Extract contains too much of the weakly adsorbed species – what do you do? If situation was reversed?

61. Two component SMB System Feed Desorbent Extract Raffinate Conc. I II III IV Bed Position

62. SMB Optimization IIIIIIIV vIvI v II v III v Recycle vXvX v Raff vFvF vDvD Questions: Extract contains too much of the weakly adsorbed species – what do you do? If situation was reversed?

63. Two component SMB System Feed Desorbent Extract Raffinate Conc. I II III IV Bed Position

64. Examples of SMB

65. Two component SMB System

66. Multi-component System Single-component pulse data Reference: Linda Wang, Perdue University

67. Multi-Component SMB System Desorbent Extract (2, 3) Feed (1, 2, 3) Raffinate (1) I II IIIIV 1 Fast Solute 2 Intermediate Solute 3 Slow Solute Concentration Bed Position Reference: Linda Wang, Perdue University

68. Complete Separation in Tandem SMB Column Number 05101520 0 0.5 1 C i /C F,i Des.Ext.FeedRaf. Sulfuric Acid Glucose Acetic Acid 05101520 0 0.5 1 C i /C F,i Des.Ext.FeedRaf. Reference: Linda Wang, Perdue University

69. Profiles of a Parallel SMB Glucose yield: 94% Glucose purity: 99% Reference: Linda Wang, Perdue University

70. Other Questions?