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, zydney@engr.psu.edu

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

0

0.1

0.2

0.3

0.4

0.5

0 0.1 0.2 0.3 0.4 0.5

Pore Diameter in Open Layer, dp (µm)

Sie

ving

Coe

ffic

ient

, 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

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Filtrate Flux, Jv (µm/s)

Sie

vin

g C

oef

fici

ent,

So

RNA Ultrafiltration

0

0.04

0.08

0.12

0.16

0.2

23 bp 70 bp 120 bp

TE Buffer + 10mM NaCl

Siev

ing

Coe

ffici

ent,

S o

23 bp 70 bp 120 bp

0.20

0.16

0.12

0.08

0.04

0

•  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)