Laboratory Dialysis

1. Laboratory Dialysis Cassidy Markee Director of Laboratory Division

2. Spectrum’s Core Technology: Membrane Separation • Spectrum offers products for two kinds of Membrane Separation (membrane, systems, components and accessories): • Membrane Dialysis – passive diffusion through a semi-permeable membrane – 100 to 1,000,000 Daltons • Membrane Filtration – applied pressure across a selective membrane. 3 types based on size range: • Macrofiltration – 5 μm and up • (Woven Mesh Screens) • Microfiltration – 0.05 – 5 μm • (Direct Flow and Tangential Flow) • Ultrafiltration (Molecular filtration) – 5 kD to 0.05 μm • (Tangential Flow only)

3. Why use laboratory dialysis? Many Advantages: • Simple, easy & quick set-up • Inexpensive & disposable • Very gentle separation (no pressure or shear stress) • Doesn’t require monitoring • Dissolved solutes remain in solution • Solutes are unaltered or damaged • Maintains macromolecule concentration • Purify low MW solutes • Total product recovery Only 2 Major disadvantages: • Very slow (1 – 2 days) • Molecules should be 100 X size difference

4. Understanding Dialysis Passive diffusion of solutes across a semi-permeable membrane from an area of high concentration (sample) to an area of low concentration (dialysate/buffer) until equilibrium is reached. Driving Force: CONCENTRATION GRADIENT The higher the concentration gradient the faster the rate of dialysis. Opposing Force: EQUILIBRIUM As you approach equilibrium, the rate of dialysis slows down.

5. Mode of Operation: Gentle diffusion across a membrane (high conc. in sample) (low conc. in buffer) (low conc. in buffer) (equilibrium) (equilibrium) (high conc. in sample) (equilibrium)

6. 4 Fundamental Dialysis Applications • Purification – elimination of low MW solutes Protein purification HPLC sample prep Removal of free amino acids or nucleotides • Modification – changing ionic / chemical environment to affect activity Desalting pH Change Buffer Exchange • Isolation – controlled exposure for reactivity Separation of small target species Electro-elution • Equilibrium Dialysis – Binding Studies Antibody & Ligand Enzyme Activity

7. Typical Dialysis Parameters • Sample & Dialysate (buffer) Concentrations • equal except for retained and passing molecular species • Sample to Dialysate (buffer) Ratio • at least 1 : 100 - 500 by volume • Membrane MWCO (pore rating) • 50-90% of the retained species molecular size • Volume to membrane surface area (SA) ratio • Length (10-15 cm) x FW (24-30 mm) = 2-5 ml/cm • Temperature • Room Temperature (20-25 Celsius) • “Static” Dialysis with stirring • Buffer Changing • Three times at 3-4 hrs, 6-8 hrs & 16-24 hours • Duration • 1 – 2 Days

8. Molecular Weight Cut-Off (MWCO) Indirect measure of MW (solute size) retained by approximately 90-95%. Below the curve retained Above the curve passes

9. Selecting MWCO for Max Yield and Purity • Max Yield: MWCO smaller than product size. • And Max Purity: MWCO 100-1000X bigger than low MW contaminants. Ex: 100 kD to retain 98% IgG AND remove 99% Vit B12

10. Select Lower MWCO: Yield vs. Purity If only 10 X size difference between product (Cyt C) & contaminant (Vit B12): • Lower MWCO with 80-100% product retention for max yield BUT sacrifice purity. (Ex: 20 kD MWCO- 88% Cyt C yield & 12% Vit B12 contaminant)

11. Select Higher MWCO: Yield vs. Purity If only 10 X size difference between product (Cyt C) & contaminant (Vit B12): Lower MWCO with 80-100% product retention for max yield BUT sacrifice purity. (Ex: 20 kD MWCO- 88% Cyt C yield & 12% Vit B12 contaminant) Higher MWCO with 50-80% product retention for max purity BUT sacrifice yield.(Ex: 50 kD MWCO- 76% Cyt C yield & 2% Vit B12 contaminant)

12. Spectrum Dialysis Membranes: 2 Grades • Standard Grade - natural cellulosic polymer: 1 Type • Regenerated Cellulose (Standard-RC) MWCO - DISADVANTAGE: Limited range with loose selectivity PURITY - DISADVANTAGE: Trace levels of heavy metals & sulfides TOLERANCE - ADVANTAGE: Good temp (121 C), pH (2-12) & chemical resistance COST - ADVANTAGE: Most economical APPLICATIONS: General dialysis, eliminating small ions (desalt, pH change, Buffer exchange, etc.) • Biotech Grade – synthetic cellulosic polymer: 2 Types • Cellulose Ester (Biotech-CE) MWCO - ADVANTAGE : Broad range (100 – 1,000,000 D) with best selectivity PURITY - ADVANTAGE: Ultra-pure – no trace heavy metals & sulfides TOLERANCE - DISADVANTAGE: Limited temp (37 C), pH (2-9) & chemical resistance COST - DISADVANTAGE: More costly APPLICATIONS: Critical separations & molecular purifications (protein, oligos, macromolecules, etc.) • Regenerated Cellulose (Biotech-RC) MWCO ADVANTAGE: Small range of MWCO’s with good selectivity PURITY ADVANTAGE: Ultra-pure – no trace heavy metals & sulfides TOLERANCE ADVANTAGE: Good temp (121 C), pH (2-12) & chemical tolerance COST DISADVANTAGE: Most costly APPLICATIONS: Requiring purity and tolerance (sample prep with organics, etc.)

13. Laboratory Dialysis: 4 Membrane Configurations • Dialysis Tubing • Most Economical • Many Flat Widths (FW) • Large Qty – bulk rolls • Evaluation – Trial kits • Requires closures • 3 Types: Standard-RC, • Biotech-RC & Biotech-CE • Disposable • Dialysis Devices • Built-in convenience • Easy-to-use • Saves time • Disposable • Ultra-pure membrane • 1 Type: Biotech-CE • Flat Sheet & • Dialysis Systems • Chamber separations • Large volume samples • Small volume samples • Equilibrium studies • 1 Type: Standard-RC • Hollow Fiber • MicroDialysis • Microvolume dialysis • Encapsulation w/ epoxy • Implant for in vivo studies • Potted bundle or loose HF • 1 Type: Standard-RC

14. Dialysis Tubing: Standard Grade RC • Dry Standard Grade RC • Spectra/Por 1: 6-8 kD MWCO, general application • Spectra/Por 2: 12-14 kD MWCO, high perm / special FW • Spectra/Por 3: 3.5 kD MWCO, general application • Spectra/Por 4: 12-14 kD MWCO, general application • Spectra/Por 5: 12-14 kD MWCO, paper for wet strength • Flat Width (diameters): Many for different sample volumes • Quantity: Bulk 15 or 30m; and Trial Kits 5m (S/P 1, 2 & 3) • Preparation: Soak 30 min in water to remove glycerin • Storage: Dry room temp (avoid humidity) • Pre-treated Standard Grade RC • Spectra/Por 6 – Pre-wetted for convenience; 10 meters • Spectra/Por 7 – Pre-cleaned to remove trace heavy metals & sulfides; 5 meters • 8 MWCO’s: 1, 2, 3.5, 8, 10, 15, 25 & 50 kD; Flat Width (diameter): Many for different sample volumes • Quantity: S/P 6- 10m/roll & S/P7- 5m/roll • Preparation: Flush with water to remove preservative (0.05% sodium azide & 1% NaCl) • Storage: 4°C in azide, benzoate or formaldehyde preservative (avoid drying out or freezing)

15. NEW Spectra/Por 1, 2 & 3 Dialysis Trial Kits Six Standard RC Dialysis Tubings in 5 meter Lengths (dry) • Spectra/Por 1: 32 mm & 50 mm FW • Spectra/Por 2: 25 mm & 45 mm FW • Spectra/Por 3: 18 mm & 54 mm FW • “T” at end of standard PN designates a trial size length, Ex: 132720T • Small Quantity Benefits: • Costs less than 15 and 30 m/roll • Ideal for Small Quantity Experiments • Qualify membrane for application • Compare performance of multiple MWCO’s

16. Dialysis Tubing: Biotech Grade • Biotech CE – Best selectivity & ultra-pure • 9 concise MWCO’s • 0.1-0.5, 0.5-1, 3.5-5, 8-10, 20, 50, 100, 300 & 1000 kD • 4 tubing flat-widths (10, 16, 24 & 31 mm) • Bulk (10 meter) and Trial (1 meter) sizes • Pkg’d wet in 0.05% sodium azide (rinse w/ water) • Store at 4 C in azide, benzoate or formaldehyde • Biotech RC – Best compatibility & ultra-pure • 5 MWCO’s • 3.5, 8, 10, 15 & 25 kD • 2 tubing flat-widths (10 & 16 mm) • Bulk (10 meter) • Pkg’d dry with glycerin (soak in water to remove) • Store dry at room temp (avoid humidity)

17. NEW Biotech CE Dialysis Trial Kits 9 Biotech CE Dialysis Tubings in 1 meter Length (wet) • Trial size available for all 9 MWCO’s • 0.1-0.5 kD, 0.5-1 kD, 3.5-5 kD, 8-10 kD, • 20 kD, 50 kD, 100 kD, 300 kD & 1000 kD • Biotech CE: 16 mm FW only • “T” at end of standard PN designates a trial size length, Ex: 131270T • Small Quantity Benefits: • Costs less than 10m/roll • Ideal for Small Quantity Experiments • Qualify membrane for application • Compare performance of multiple MWCO’s

18. Closures for Dialysis Tubing: 2 Classes Universal Closures: All tubing grades & types • Use with Standard RC, Biotech CE & Biotech RC • 5 sealing width sizes (50, 70, 90, 110 & 150 mm) • Nylon – sinks in water • 6 colors available for sample identification Spectra/Por Closures: Standard RC tubing only • 5 sealing width sizes (12, 23, 35, 55 & 75 mm) • Polypropylene – floats in buffer • 3 functional types available: • Standard Closure – Aids in buoyancy • Weighted Closure – Aids in vertical orientation • Magnetic Closure – Aids in buffer stirring • Paired Standard & Weighted – buoyancy & orientation • 4 colors available for sample identification

19. Why use pre-assembled dialysis device? Disadvantages of Dialysis Tubing: • Requires closures • More likely to leak • Requires headspace for buoyancy and vertical orientation • More difficult to load & retrieve sample Advantages of Dialysis Devices: • Very easy handling & convenient • Self-contained & complete • Self-buoyant and self-orienting • Allows in-process testing • Total sample recovery • Disposable

20. Disposable Dialysis Devices • Float-A-Lyzer G2 (FAL-G2) • For ultimate convenience of small volumes • Micro Float-A-Lyzer (μFAL) • For ultimate convenience of micro volumes • Tube-A-Lyzer (TAL) • For increased efficiency combined with convenience Spectrum offers 3 kinds of pre-assembled dialysis devices:

21. Float-A-Lyzer G2 Video Clip

22. Disposable Dialysis Device: Float-A-Lyzer G2 Purpose: Provide ultimate convenience for dialysis of common volumes Design Features: • Biotech CE preformed tubing (16 mm FW, dry) • Pot-sealed on top & bottom in open configuration • Leak-proof screw cap lid with sealing O-ring • 9 MWCO’s (color-coded cap): 0.1-0.5, 0.5-1, 3.5-5, 8-10, 20, 50, 100, 300 & 1000 kD • 3 volume sizes: 1 ml, 5 ml & 10 ml • Floatation ring for buoyancy (included) • Pipette loading & retrieval for 5 & 10 ml (included) and micropipettor loading & retrieval for 1 ml • 12 units/pkg

23. Disposable Dialysis Device: Float-A-Lyzer G2 Benefits: • Superior handling and ease-of-use • Leak-proof with sample protection • Best MWCO selectivity and range (100 – 1,000,000 Dalton) • Ultrapure Biotech CE membrane • Volume specific dilution control • Easy sample loading and retrieval • Total sample recovery • Complete & ready-to-use

24. Disposable Dialysis Device: Micro Float-A-Lyzer Purpose: Provide ultimate convenience for dialysis of micro volumes Design Features: • Biotech CE preformed tubing (10 mm FW, dry) • Pot-sealed on top and clamp sealed on bottom • Leak-proof Luer-lok™ cap • 7 MWCO’s (color-coded cap): 0.1-0.5, 0.5-1, 3.5-5, 8-10, 20, 50 & 100 kD • 2 volume sizes: 100-200 μl & 400-500 μl • Self-floating (“air bladder”) & self-standing • Syringe (no needle) loading & retrieval (included) • Interlocking housing for multi-sample dialysis • 12 units/pkg New - January 2012

25. Disposable Dialysis Device: Micro Float-A-Lyzer Benefits: • Superior handling and ease-of-use • Leak-proof with sample protection • Best MWCO selectivity and range (100 - 100,000 Dalton) • Ultrapure Biotech CE membrane • Open to clamped configuration to maximize SA and minimize volume hold-up • Easy sample loading and retrieval • 100% sample recovery • Complete & ready-to-use New - January 2012

26. Disposable Dynamic Dialysis Device: Tube-A-Lyzer Purpose:Dialysis in 4-12 hours Provides the convenience of a disposable device and the efficiency of dynamic dialysis for faster dialysis Mechanism of Efficiency: • Two membrane defined chambers: sample & buffer • Solutes diffuse through membrane • Circulating buffer sweeps away solutes • Maintains max concentration gradient • Prevents equilibrium • Achieves higher rate of dialysis

27. Disposable Dynamic Dialysis Device: Tube-A-Lyzer Design Feature: Membrane Sample Chamber • Biotech CE preformed tubing (16 mm FW, dry) • Pot-sealed on top & bottom in open configuration • 2 sample volume sizes: 8-10 ml & 25-30 ml • 6 MWCO’s: 0.1-0.5, 3.5-5, 8-10, 20, 50 & 100 kD • Leak-proof Luer-lok™ sample port & cap • Syringe loading & recovery Design Feature: Flow-through Buffer Chamber • Tubular housing encases the membrane chamber • Upper & lower hose-barb ports (3/8” or 6 mm) • Operates with standard peristaltic pump & flexible tubing

28. Disposable Dynamic Dialysis Device: Tube-A-Lyzer Benefits: • Reduces dialysis time from days to hours! • Ready-to-Use: no preparation required • Easy sample loading and in-process testing • Larger buffer volumes • Maintains maximum concentration gradient • Prevents polarization and membrane fouling • Achieves higher sample purities

29. Flat-sheet Dialysis Membrane Standard RC Discs for Spectrum Dialysis Systems: • 3 diameters: 33 mm, 47 mm & 100 mm (50/pk) • 4 membranes: • Spectra/Por 1 (6-8 kD) • Spectra/Por 2 (12-14 kD high perm) • Spectra/Por 3 (3.5 kD) • Spectra/Por 5 (12-14 kD) Standard RC Sheets for other dialysis systems: • Square sheets for cutting (25/pk) • 5 membranes: • Spectra/Por 1 (6-8 kD) • Spectra/Por 2 (12-14 kD high perm) • Spectra/Por 3 (3.5 kD) • Spectra/Por 5 (12-14 kD) • Spectra/Por 5 (12-14 kD large volume)

30. Flat-sheet Dialysis Systems Design concept: Sample chamber and buffer chamber separated by a flat membrane disc. Assembly clamped together by pressure. (a membrane sandwich) • 3 system types depending on application: • MacroDialyzer • For larger sample volumes • MicroDialyzer • For multiple micro volume samples • Equilibrium Dialyzer • For drug and ligand binding studies

31. Flat-Sheet Dialysis System: MacroDialyzer Purpose: Dialysis of larger volumes (200 ml +) Features & Operation: • Equal size sample & buffer chamber (acrylic) • 4 volume sizes: 1 ml, 5 ml, 10 ml & 50 ml • Spectra/Por Standard RC membrane discs • Sealing chamber O-rings • Clamps assure leak-proof assembly • Flow through ports on sample & buffer chambers for circulation of larger volumes • Should be equal pressure or closed system to avoid filtration mode.

32. Flat-Sheet Dialysis System: MicroDialyzer Purpose: Dialysis of multiple micro volumes Features & Operation: • 10-well dialyzer, 500 μl sample volumes • 96-well dialyzer, 150 μl sample volumes • Common buffer chamber with flow through ports • Spectra/Por Standard RC Disc Membranes • Sealing chamber O-ring or gasket • Clamps or screws for leak-proof assembly • Cover plate to avoid sample contamination • Spectra/Por Standard RC membrane sheets

33. Flat-Sheet Dialysis System: Equilibrium Dialyzer Purpose: Studying pharmacokinetics and ligand binding. Compare up to 20 independent reactions to determine drug or binding efficacy. Features & Operation: • 20 reaction cells loaded with equal sample & buffer volumes (1.4 ml half-cell chambers) • 5 cells stacked & spaced per carrier • 4 carriers mounted to drive unit and submerged in temperature controlled water bath. • Continuous rotation of 20 reaction cells • Spectra/Por Standard RC Membrane Discs

34. Hollow Fiber Micro-dialysis Purpose: • Dialysis of micro-volume samples (5 ul/fiber) • Encapsulation & implantation for in vivo studies Features & Benefits: • Standard RC hollow fiber membrane with glycerin • 2 MWCO’s: 13 kD & 18 kD • Fiber dimensions: ID x OD - 200 x 216 um 20 fibers, 15 cm length • Available as loose fibers or potted bundle for easier loading

35. Membrane Dialysis Accessories Dialysis Reservoirs • Polystyrene cylindrical containers ideal for stirring dialysis tubing or devices in buffer • Magnetic stir bar included • Nylon sealing lid prevents evaporation • Two volume sizes – 600 ml & 1800 ml • Spectra/Gel Absorbent: • Concentration of dialysis samples in tubing or device without product loss or contamination • Inert polyacrylate-polyalcohol complex draws and binds water from the sample • Pack with dry powder & then brush away gel • 10 fold sample concentration in 1 hour

36. How do we overcome complicationswith atypical applications and increase efficiency? • Physically modify aspects or components of dialysis. • Membrane • Volumes • Concentrations • Buffer changes • Manipulating operating parameters to perpetuate the Driving Force (Concentration Gradient) and eliminate the Opposing Force (Equilibrium).

37. Parameters to Increase Rate of Dialysis • Sample & Buffer Concentrations • equal except for retained and transferred species • Sample to Dialysate Ratio • 1 : 1000+ by volume • Membrane MWCO • 90-130% of the retained species molecular size • Volume to membrane surface area (SA) ratio • Length (30 cm) x FW (10-16 mm) = 0.1 – 1ml / cm • Temperature • Elevated (36-60 Celsius) • Dynamic Dialysis • Buffer Changing • 4-5 times at 2, 4, 6-8, 8-10, 14-16 & 20 hours • Duration (Resulting) • 0.5 – 1.5 days

38. Common Complications and Issues Associated with Dialysis and Atypical Applications • Membrane impurities • Product purity • Product yield • Membrane Handling • Sample volume dilution • Concentrating the sample • Gel electro-elution • High salt samples • Large volume samples • Small volume dialysis • in vivo implantation research • Dialysis takes too long • Biotech Grade or S/P 7 Membrane • Membrane with higher MWCO • Membrane with lower MWCO • Ready-to-Use Dialysis Device (RDD) • RDD or fully loaded tubing • Spectra/Gel Absorbent • Float-A-Lyzer G2 • Step-down salt concentration of buffer • S/P 5, MacroDialyzer or consider TFF • Micro Float-A-Lyzer • Microdialysis hollow fiber or PVDF implant • Tube-A-Lyzer, Dynamic Dialysis or modify operating parameters

39. 4 Molecular Separation Technologies in the Lab (reference table for technology comparison)

40. Thank You谢谢