Membrane Extractions & Adsorptions in Separation of Biomolecules: Case Studies & Future Prospects

1. Membrane Extractions and Adsorptions in Separation of Biomolecules from Multicomponent MixturesMd M Hossain*Department of Chemical & Petroleum Engineering, United Arab University, P.O. Box 15551, Al Ain, UAE *

2. Membrane extraction and adsorption from aqueous media Two examples of Biomolecule separation (A) Extraction of an antibiotic (Shengimycin) In a bench-scale Membrane Contactor And using an eco-friendly solvent system (B) Separation of a protein from its mixture With other proteins using a commercially Available Membrane Module

3. Overview • Brief introduction of the “target” compounds • Aims of the projects (A) and (B) • (A)Extraction & Reactive extraction • Results of the antibiotic system in the Module • (B) Results of the protein system in a commercially available adsorber • Concluding remarks from (A) and (B) • Future work

4. (A) Shengjimycin (an antibiotic) • It is a multicomponent antibiotic • Better pharmacokinetic properties (improved absorption and distribution of the antibiotic) • Intended applications for the treatment of - respiratory infections andurinary tract infections

5. Shengjimycin (SJM) • Produced by fermentation and can be separated by any of the available methods • Chromatography –based methods - High performance chromatography - Counter current chromatography - Works well in separating SJM - Some disadvantages

6. Disadvantages of the Method • Very expensive to prepare the samples and process large quantities of product • High consumption of toxic solvent • In situ product recovery difficult • Try a new method such as Reactive extraction

7. Advantages of Reactive Extraction • Extraction at natural pH ? • Higher productivity (in situ removal possible) • Less contamination (Non-dispersive contacting of phases in separation) • Smaller secondary waste production • Large scale modules (commercially available) • Efficient and selective separation

8. (A) Extraction of SJM • Determine a solvent system (less toxic solvent) for possible extraction at natural pH • Examine the solvent alone • Examine the reactive extraction • Evaluate the performance in a module: - high pH (close to fermentation pH) - at higher flow rate

9. Organic phase SJM Aqueous phase SJM Extraction (solvent only) • -Extraction of Shengjimycin (SJM) from the organic and aqueous phase (SJM) • Solubility must be high in the organic phase compared to the aqueous phase (SJM) • Organic phase? New? • By itself (organic alone)? Or • Need a “carrier” for better result Distribution Coefficient (DE) = CSJMorg/CSJMaq

10. Reactive Extraction (need a “carrier” C) • SJM-OH SJM+ + OH- • The mechanism with C (carrier): • C(org)+ SJM(aq)+ OH(aq) CSJM(org) • (CSJM) is transported and more soluble • In the organic phase • Select the carrier which forms a complex CSJM C Organic Phase with “carrier” C OH- + SJM Aqueous Phase (SJM and others

11. Experimental Procedure • First equilibrium experiment with solvent alone: (no carrier) • 2 solvents (Tributyl phosphate and sunflower oil) was tried DE is low (0.1-0.2) with solvent only • Extraction with carrier (Amberlite LA2) DE (Distribution coefficient) = CSJM, Org/CSJM, Aq CSJM = Concentration of SJM Or and Aq the organic and aqueous phases (Org -5ml) C CSJM Organic Phase OH- + SJM+ Aqueous Phase (Aq- 5ml)

12. A Organic phase ASJM Aqueous phase SJM Reactive extraction of SJM • Amberlite LA-2 (A) is a commercially available carrier • Forms an ionic pair with the Shengjimycin molecule A(org) + SJM(aq) ASJM(org) High DE Only requires small amounts of carrier

13. How does the HFMM work? Aqueous Organic Aqueous Phase (outlet) Organic Phase (outlet) CSJM SJM C Shellside Tube-side Organic Phase (inlet) Aqueous Phase (inlet) Hydrophobic Porous membrane

14. Carrier+Sunflower oil as a Solvent phase Sunflower oil worked successfully as a solvent With D E of value 3 - 4 In presence of the carrier, Amberlite-LA2 Compared to other solvents (kerosene) Being tried in the literature Sunflower oil system is considered nontoxic, less expensive and sustainable Upgraded to a bench-scale membrane module

15. Contacting in hollow fibres • Hollow Fibre Module C CSJM Reaction Occurs Aqueous CSJM Complex OH- +SJM + C (org) Organic Pore of Fibre

16. Experimental Procedure Hollow Fibre Module Set-Up • HFM experiments E (%) = (CSJM,Aq,initial – CSJM,Aq,final) CSJM,Aq,initial

17. Advantages of HFMM • Easy to install and operate • Require pressure on the aqueous side to stabilize the interfaces • Membranes are hydrophobic, hence allows only the organic to wet the pores and • The CSJM complex is transported to permeate through the membrane

18. HFMM Results

19. HFMM Results (continued)

20. HFMM Results (continued) Tributyl phosphate Sunflower oil

21. Conclusions • With sunflower oil alone, the percentage extraction of SJM is low (less than 10%-physical extraction) • The % extraction can be greatly improved by using less than 3-5% of Amberlite LA-2 as a carrier • An extraction of 70% was achieved in a small pilot scale module (hollow fibre membrane contactor) • This was achieved at a flowrate of 12-13 L/h within 3-4 hours at the natural pH of antibiotic

22. Recommendations and Future Work • Back-extraction of Shengjimycin from organic phase into another aqueous phase • Carry out HPLC analysis on the extracted Shengjimycin components • Determine the stability of the system e.g. how long the system can perform the extraction

23. (B) Adsorption of Proteins onan Ion- Exchange Membrane • Milk contains two major fractions: Caseins and whey • Milk whey • 20% milk proteins, the majors are: • a-lactalbumin (a-lac),b-lactoglobulin (b-lg), bovine serum albumin (BSA)

24. Membrane-based Particle-based Feed flow & pressure Feed flow & pressure Filtrate Filtrate Separation of Proteins

25. BACKGROUND • Membrane (commercially • Available) in the ion exchange • Proteins of interest:

26. Membrane Selection • Various types of • Membrane used • A commercial Anion • Exchange Membrane • Volume: 3.8 cm 3 • Membrane area: 75 cm 2 • Pore Size: 3 mm

27. Stand Clamps Reservoir of Feed Titration Stand Glass Blown Nipple Pump Inlet Leur lock fitting Peristaltic Pump Pump Outlet to Membrane EXPERIMENTAL LAYOUT

28. EXPERIMENTAL – General • Adsorption from aqueous solution • Desorption with salt solution • Concentration and volume varied • Feed volume = 100 ml solution • Flow rate = 15 ml/min • Natural pH (no adjustments)

29. EXPERIMENTAL – General • Protein adsorption from aqueous solution • At protein concentrations similar to whey • a-lac concentration: 1.28 mg/L • BSA concentration: 0.32 mg/L • b-lg concentration: 3.22 mg/L • Natural pH (no adjustments)

30. RESULTS – Single Component

31. RESULTS – Single Component Initial Reservoir Volume: 100ml, Flow Rate: 15ml/min

32. RESULTS – Robustness Initial Reservoir Volume: 100ml, Flow Rate: 15ml/min

33. Desorption of proteins at 30 min

34. A solution of BSA and a-lactalbumin

35. RESULTS – A binary mixture

36. FUTURE WORK • Studies with “real” whey sample • Effect of minor components • Purification from eluted solution • HPLC analysis • Continuous process • Industrial conditions

37. Acknowledgements (Antibiotic work) - Kam Rothman Student from the department of Chemical & Materials Engineering, Univewrsity of Auckland, New Zealand - Professor Chenghang Sun of the • Institute of Medicinal Biotechnology, Chinese Academy of Medical sciences, Beijing, PRC, for supplying the samples and references on Shengjimycin work

38. Acknowledgements(protein adsorpton work) • Collin Hendersen (Undergraduate student) • Ameera Fares (postgraduate student, UAE University) • Emirates Foundation (Abu Dhabi, UAE) Grant No. #2010/088, in the Science and Engineering Program. The authors also acknowledge the support from the UAE University. • supported by the Emirates

39. Thank you !!