Development of novel membrane structures for enhanced ...
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Engineering Conferences InternationalECI Digital Archives
Advanced Membrane Technology VII Proceedings
9-13-2016
Development of novel membrane structures forenhanced purification of plasmid DNA using smallpore size ultrafiltration membranesAndrew L. ZydneyThe Pennsylvania State University, USA, [email protected]
Ying LiThe Pennsylvania State University, USA
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Recommended CitationAndrew L. Zydney and Ying Li, "Development of novel membrane structures for enhanced purification of plasmid DNA using smallpore size ultrafiltration membranes" in "Advanced Membrane Technology VII", Isabel C. Escobar, Professor, University of Kentucky,USA Jamie Hestekin, Associate Professor, University of Arkansas, USA Eds, ECI Symposium Series, (2016).http://dc.engconfintl.org/membrane_technology_vii/11
Ultrafiltration for Purification of Nucleic Acids (DNA and RNA)
Andrew L. Zydney Distinguished Professor of Chemical Engineering
The Pennsylvania State University
ECI Advanced Membrane Technology Cork, Ireland September 13, 2016
Nucleic Acid Therapeutics
• DNA Therapeutics and Vaccines • Replace malfunctioning / missing gene (e.g., Hemophilia)
• In situ production of antigen to generate provide immunization against viral infections (e.g., Ebola, Zika, Flu)
• RNA Therapeutics• Small-interfering RNA, microRNA, antisense RNA • Inhibit / silence specific genes • Applications in cancer, diabetes, cardiovascular disease
• Thousands of clinical trials completed or currently underway
Glybera – 1st approved product
Lipoprotein Lipase Deficiency (LPLD) • Very rare disorder – one per
million • Glybera à dramatic improvement
in lipoprotein metabolism
• 1st gene therapy product
• Approved in Europe (not in U.S.) in 2013
Glybera – 1st approved product
• Cost is for treating a single patient!
Glybera – 1st approved product
• Cost is for treating a single patient!
• Total mass of DNA ≈ 0.1 µg à $10 trillion / g
• Significant challenges in purification, with exciting potential opportunities for membrane technology
DNA Transmission vs Flux
3.0 kbp plasmid DNA RDNA ≈ 73 nm Rpore ≈ 9 nm (300 kD MWCO) 10 mM Tris-EDTA buffer
§ DNA transmission negligible at low filtrate flux
§ DNA transmission increases to >60% at high flux, even though DNA is nearly 10x the size of the membrane pores
DNA Transmission vs Flux
3.0 kbp plasmid DNA RDNA ≈ 73 nm Rpore ≈ 9 nm (300 kD MWCO) 10 mM Tris-EDTA buffer
§ DNA transmission negligible at low filtrate flux
§ DNA transmission increases to >60% at high flux, even though DNA is nearly 10x the size of the membrane pores
DNA Transmission – Stirring
3.0 kbp plasmid DNA RDNA ≈ 73 nm Rpore ≈ 9 nm 10 mM Tris-EDTA buffer
§ DNA transmission independent of stirring speed at any given flux (even in absence of stirring)
§ Increase in transmission at high flux not due to concentration polarization
Effect of DNA Size on Transmission
1000 kDa Ultracel membrane
Transmission independent of DNA size
3.0 kbp9.8 kbp
17.0 kbp
Flow-Induced Elongation • Elongational flow into pore stretches plasmid
Low Flux No Elongation
Minimal Transmission
Flow-Induced Elongation • Elongational flow into pore stretches DNA
• Longer DNA elongate more easily
Low Flux No Elongation
Minimal Transmission
High Flux Elongation of DNA
Significant Transmission
DNA Elongation
Video provided by Ron Larson, University of Michigan
Membrane Fouling § Significant fouling
seen during DNA UF with transmission dropping to zero at long filtration times
§ Rate of fouling increases significantly with increasing DNA concentration
§ Impractical for large scale DNA purification
DNA “Trapping”
• Large DNA molecules become trapped at entrance to narrow pores due to incomplete elongation leading to membrane fouling
• Need to find way to more effectively elongate the DNA
30 µm
Filtration
Pre-stretching in UF
• Approach – use membranes with flow from substructure to skin
• Passage through membrane substructure should “pre-stretch” the DNA, minimizing DNA trapping in pores in UF skin layer
Filtration
Pre-stretching - Fouling
• Use of asymmetric UF membrane in reverse orientation (skin down) completely eliminates fouling
• Pre-stretched DNA doesn’t become trapped at pore entrance
Skin-downSkin-up
Pre-stretching – Optimization
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Pore Diameter in Open Layer, dp (µm)
Sie
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, So
Supercoiled DNA, Jv = 60 µm/sBiomax 300 kDa membrane
UF Layer
Open Layer
• “Composite” membrane formed by placing MF on top of UF membrane
• Optimal pore size for pre-stretching appears to be around 0.1 µm
• New opportunity for membrane design
DNA Isoform Purification
• DNA exists in different topological isoforms
• Supercoiled isoform is desired for therapeutic applications à open-circular and linear isoforms are considered process impurities (removed to <5% of total DNA)
Supercoiled Open-Circular ‘Single-nick’
Linear
DNA Isoform Purification
• DNA exists in different topological isoforms
• Hypothesis – Differences in elongational flexibility of different isoforms may provide opportunities for purification of desired supercoiled DNA
Supercoiled Open-Circular ‘Single-nick’
Linear
Sieving of DNA Isoforms Linear & Open-circular forms generated by
enzymatic digestion of Supercoiled
300 kD membrane 10 mM Tris-EDTA
LinearSupercoiledOpen-circular
• Much higher transmission of linear DNA -> most easily elongated
• Transmission of supercoiled DNA intermediate
• Nearly complete retention of open-circular DNA due to low flexibility
DNA Isoform Purification by UF 36 LMH 97 LMH
104 L/m2/h 17 kbp DNA Permeate
17 kbp DNA, 300 kD membrane, 10 mM Tris-EDTA buffer
Supercoiled DNA Purification 36 LMH 97 LMH
Permeate Permeate Feed Feed
Open-Circular
Linear
Supercoiled
3.0 kbp DNA, 300 kD membrane, 10 mM Tris-EDTA buffer
Reverse orientation
Normal membrane
• “Pre-stretching” dramatically increases selectivity
Pre-stretching – Selectivity
SupercoiledLinear
RNA Ultrafiltration
• RNA transmission increases with increasing filtrate flux, similar to behavior seen with DNA
• No effect of stirring, suggesting that transmission is again due to flow-induced elongation
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Filtrate Flux, Jv (µm/s)
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RNA Ultrafiltration
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23 bp 70 bp 120 bp
TE Buffer + 10mM NaCl
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23 bp 70 bp 120 bp
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• Transmission of single stranded RNA is significantly different for the different RNA species
• Maximum transmission seen with 70 bp (≈23 kDa) RNA
Flux = 110 µm/s
RNA Structures
23 bp
120 bp
70 bp
Summary • Nucleic acid transmission through UF
membranes due to flow-induced elongation à different isoform flexibility leads to separation
• Pre-stretching DNA in large pore region significantly reduces fouling while increasing selectivity à opportunity for developing membranes with novel pore morphology
• RNA ultrafiltration depends on RNA structure • Exciting opportunities for development of
membrane processes for nucleic acid purification
Acknowledgements • Dave Latulippe (now at McMaster University)• Ying Li (current PhD student)• National Science Foundation (funding)• Millipore (membranes)