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HyperGRO™: Plasmid DNA Manufacturing Platform

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HyperGRO™Plasmid Fermentation Reduced metabolic burden Toxic/unstable plasmid production Up to 3.5 g/L volumetric yield Sublicensed to CMOs for cGMP

e.g. Aldevron, Eurogentec, VGXI, Leidos

Antibiotic-free Expression Vector kits

NTC Technologies & Services Synthetic Gene Design/Contract Cloning Antibiotic-free vector backbone retrofit

NTC8 Series

Transgene

RNA-OUT

pUC origin

Plasmid Manufacture

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HyperGRO™: Industry Leading Scalability of Plasmid Vectors Proprietary fed-batch bacterial

fermentation process

Produces up to 10x the yield oftypical processes

Dramatically improves thescalability of plasmid productioncompared with traditionaltechniques

Results in the production of moreusable, high quality plasmid forgene therapy and DNA vaccinepurposes, without increased cost

Up to 3.5 g/L plasmid yield

Licensed to 5 cGMP CMOs

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High Yield Manufacture Three cGMP facilities have successfully utilized NTC’s HyperGRO™

fermentation process for high yield manufacture of NTC’s RNA-OUT Vectors

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Three Pronged Business Strategy Creating valuable IP enabling gene-based drugs

• 12 Granted US Patents• 8 Pending US Patent Applications

Providing design, development and manufacturingof gene-based medicines to industry partners

Technology transfer and licensing• HyperGRO™ plasmid fermentation process - 5 licensed

CMOs• NTC RNA-OUT Marker vectors - 7 licensed vector users• Nanoplasmid™ vectors - >20 Biotech/Pharma companies

evaluating

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Plasmid Production Platform: Intellectual Property

Granted US Patent 6

High specific yield

High biomass yield

Generic platform: compatible with many different plasmids and antigen gene inserts

Reduce cost

Plasmid Manufacturing Goals

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Medium composition C:N ratio Trace minerals

Metabolic burden & cell stress High copy number plasmid Plasmid encoded protein over-expression (e.g.

kanR gene product)

Process Factors

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Initial temperature 30°C for minimum plasmid copy number during biomass accumulation Reduces copy number and metabolic burden

Temperature shift 30°C 42°C during nutrient limited growth to induce high copy number plasmid production Slow growth, low protein expression reduces metabolic burden

HyperGRO™ Inducible Fed-Batch ProcessDesigned for temperature sensitive pUC origin-containing plasmids

Carnes and Williams. US Patent 7,943,377; EP1781800 (multiple international equivalents) Carnes et al. (2011) Biotechnol Bioeng 108: 354 Williams et al. (2009) Biopharm Int 22: 46 Williams et al. (2009) Biotechnol Bioeng 103:1129 Carnes et al. (2006) Biotechnol Appl. Biochem 45:155

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Time

X(t), T(t)

μ = μmax

X(t) V(t)

F(t)

μ = 0.12 h-1

Batch phase Fed-batch phase Sf

F(t)=F0eµt

30°C

42°C Step shift Cell density

Slow ramp

HyperGRO™ Inducible Fed-Batch Process

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Overall Plasmid Yield (mg/L)

Specific Plasmid Yield(mg/L/OD600)

E. coli DH5α/gWizGFP Fed-batch 30 42°C shift at 35 hours

HyperGRO™ pDNA Fermentation Plasmid:1100 mg/L - Biomass: OD600 111

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E. coli DH5α/NTC7485 Fed-batch 30 42°C 2130 mg pDNA/L

HyperGRO™ pDNA Fermentation

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Analysis indicated 98.7% supercoiled plasmid in final sample

Plasmid Quality Analysis GE Healthcare Plasmid Select Xtra analysis

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Inducible Fed-batch process enables productionof plasmids with direct or inverted repeats (e.g.Retroviral vectors) and unstable, dimerization-prone sequences (e.g. Multiple shRNA vectors)

Unstable plasmids are stabilized by maintaininglower copy number at 30°C during biomassgrowth

Yield increased by temperature shift prior toharvest

Production of unstable plasmids

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M) 1 kb DNA Ladder1) Seed culture 30-42°C

2) 30°C, 24 OD600, 28 mg/L3) 30°C, 33 OD600, 41 mg/L4) 42°C, 81 OD600, 680 mg/L5) 25°C, 70 OD600, 695 mg/L

37ºC Process

M 1 2 3 4 5 6 7 8 9

VLTrap stabilization using 30-42°C inducible process:

6) 37°C, 10 OD600, 64 mg/L7) 37°C, 13 OD600, 90 mg/L8) 37°C, 15 OD600, 83 mg/L9) 37°C, 90 OD600, 214 mg/L

(Arrow denotes deletion product arising from LTR recombination in 37°C process)

Production of unstable plasmids

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Process

Yield Quality

Viability (seed stock toxicity)

Plasmid Intrinsic FactorsOrientation/composition of vector elements

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Process must also consistently produce acceptable amounts of suboptimal plasmids, or plasmids with suboptimal inserts (e.g. inserts designed de novo from emerging pathogen sequences) Toxic proteins or peptides from cryptic promoters

Unusual DNA structures (Z-DNA, triplex)

Plasmid Factors: Intrinsic Poor Yield

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Reduce cell stress during creation of seed banks and during the fermentation process

Example:

Influenza hemagglutinin (HA) genes from H1, H3, and H5serotypes evaluated

Genes from H1 and H3 serotypes are known to lower plasmidyields

Seed banks were created at 30°C and 37°C and evaluated infermentation

Overcoming Plasmid-Intrinsic Poor Yield

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† H1 and H5 are in pDNAVACCUltra backbone. H3 is in gWiz-D backbone. * Fermentation yield from 30°C glycerol stock with H3 antigen in pDNAVACCUltra backbone was 1005 mg/L.

Plasmid antigen†

Glycerol stock preparation temp.

Glycerol stock viable

Fermentation yield (mg/L)

H1 influenza 30°C Yes 252

37°C No Inviable cell line

H3 influenza 30°C Yes 570 * 37°C No Inviable cell line

H5 influenza 30°C Yes 1290

37°C Yes 1260

Effect of cell banking temperature:

Overcoming Plasmid-Intrinsic Poor Yield

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Time

X(t)

μ = μmax

X(t) V(t)

F(t)

μ = 0.12h-1

Batch phase

Fed-batch phase

Sf

S≈0

T = 30-42°CpH = 7.0DO = 30%

Reduced temp Temp induction

Scale up: 300L GE Healthcare Uppsala, Sweden

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Scale up: 10L to 300L Plasmid Yield Time Course

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Scale 10 L 300 L Volumetric

plasmid yield 1560 mg/L 1184 mg/L

Specific plasmid yield 16.6 mg/L/OD600 16.9 mg/L/OD600

Cell density 93 OD600 71 OD600

Scale up: Yield Summary

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Used for GMP production of DNA vaccines by NIH’s0Vaccine Research Center (VRC)Yields > 1 g/L at 50 L & 100 L scale

VRC DNA Vaccine Production

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Specific plasmid (mg/g DCW) Plasmid (mg/L)

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HyperGRO™ Production of VRC pDNA

A. Growth and volumetric and specific plasmid yield from an NTC 10L DH5α/VRC2439fermentation starting from 30°C transformed cell bank Temperature was shifted from 3042°C at 30 h EFT.

B. Analysis of Harvest plasmid sample by 1% agarose electrophoresis; left lane: 1kb PlusDNA Ladder (Invitrogen), right lane: 1µg VRC2439 plasmid from harvest sample.

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Nelson et al. (2013), Hum Vaccin Immunother 9:2211

Antibiotic-free HyperGRO™ fermentation: cGMP plasmid

VGXI, Inc., utilized cGMP fermentation with NTC’s HyperGRO™ fermentation platform to manufacture Admedus’s Herpes Simplex Virus 2 DNA vaccine for their Phase I clinical trial.

VGXI achieved reproducible plasmid yields of up to 1.8 g/L at the 320L scale without the use of antibiotic selection

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NTC9385R Nanoplasmid™Scalable high yield Manufacturin g using HyperGRO™ Fermentation

NTC9385R Nanoplasmid™ vector growth and plasmid yield profile of a 10L HyperGRO™ fed-batch fermentation using slow ramp temperature induction. Peak Nanoplasmidyield of 2420 mg/L.

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3042°C

High plasmid yields up to 3.5 g pDNA/L

High specific plasmid yields up to 25 mg/L/OD600, 50 mg pDNA/g DCW (5% of total)

High productivity: up to 50 mg/L/hour

Compatible with NTC’s antibiotic-free sucrose selection

Low metabolic burden processimproved production with unstable & toxic plasmids

Scaled to 100 L & 320 L GMP production at >1 g/L

HyperGRO™ Key Advantages

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Currently licensed for use at GMP facilities in the USA, includingthe VRC and four CMOs, and GMP facilities in Europe and India

Research grade pDNA manufacturing at NTC and GMP productionof clinical grade by HyperGRO licensee provides customer withprocess continuity

HyperGRO™ Licensing and Tech Transfer

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NTC Autolytic Plasmid DNA Manufacturing Platform

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Alkaline lysis Widely used in laboratories Scale-up issues (specialized mixing) Large volumes

Lysis technology for plasmid purification

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Lysozyme/heat lysis Lower volume

Efficient plasmid recovery requires addition ofrecombinant lysozyme

Availability of recombinant lysozyme would be anissue for production of a DNA vaccine in apandemic situation*

Hoare M, et al. Biotechnol Prog 2005; 21:1577-1592.

Lysis technology for plasmid purification

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Compatible with NTC’s inducible fed-batch plasmid fermentationprocess

High yielding plasmid production strains express chromosomallyencoded lysozyme

o Phage λ endolysin

o Heat inducible promoter(no inducer required when used withNTC’s inducible fed-batch plasmid fermentation process

o Cytoplasmic production(no lysis during fermentation)

Autolysis of harvested cells resuspended in membranepermeabilizing buffers

NTC plasmid production in autolytic E. coli host strains

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RF 546: NTC4862 (DH5α SacB host) RF 547: NTC3016 (autolytic DH5α SacB host)

137 OD6002.2 g pDNA/L 6.3 mg pDNA/g WCW

125 OD6001.9 g pDNA/L 6.6 mg pDNA/g WCW

High yield in HyperGRO™ NTC8 series AF vector is produced at similarly high

yields in non-autolytic and autolytic hosts.

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117 OD6003.5 g pDNA/L 9.9 mg pDNA/g WCW

RF 561: NTC8 series AF vector in NTC3016 (autolytic DH5α SacB host)

High yield in HyperGRO™ NTC8 series AF vector is produced at record high yield of 3.5 g/L

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Autolysis of harvested cells

DH5α

DH5α with 500 U/ml Epicentre

Ready-Lyse™Lysozyme

NTC autolytic

plasmid host

30 OD600 resuspensions and 30 minutes at room temperature: STET buffer 8% glucose, 50 mM TrisHCl, 50 mM EDTA, 2% Triton X-100

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Autolytic cells are resuspended under low salt, acidic conditions to release the cytoplasmic endolysin and permeabilize the cell wall to plasmid DNA without complete cell lysis.

PeptidoglycanDisrupted by endolysin(e.g. λ R) or lysozyme

Outer membraneDisrupted bypermeabilizer(e.g. EDTA)

Inner membraneDisrupted by detergent(e.g. Triton X-100)

Bacterial chromosome

Plasmids1. Resuspend cell paste with 20

volumes Autolytic AcidicExtraction Buffer (30mMsodium acetate, 10 mM EDTA,8% sucrose, pH 5.2).

2. Add Triton X-100 to 0.1% andstir for 30 minutes at roomtemperature.

3. Remove cell debris/gDNAbysolid/liquid separation andrecover the pDNA-containingsupernatant.

Carnes AE, Hodgson CP, Luke JM, Vincent JM, Williams JA. Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification. Biotechnol Bioeng. 2009 Oct 15;104(3):505-15

Acidic Autolytic Plasmid Extraction

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• 2 grams RF 547cell paste = 13.2mg pDNA

• 40 mL AutolyticAcidic Extraction,30 minutes

• Centrifuged 30min at 4000 x g.

• Recoveredsupernatant

• Adjusted to 0.6MNaCl and 1% TritonX-100

• Purified overGiga-scale weakanion exchangecolumn

• Final yield:8.4 mg pDNAA260/A280 = 1.8 64%purification yield

1 µg of the purified pDNA on agarose gel

From fermentation RF 547:NTC8 series AF vector in NTC3016 (autolytic DH5α SacB host)

Small Scale Test Purification

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Example Large Scale Process

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Step Volume (mL)

DNA conc. (mg/mL)

Total DNA (mg)

pDNA (%)

gDNA (%)

pDNA recovery (%)

Autolytic acidic extraction (30 min), 30 min 12,000g centrifugation.

1000 0.228 228 98.61% 1.39% 69%

KDS protein removal, 30 min 12,000g centrifugation.

1176 0.233 274 99.25% 0.75% 82%

Mustang Q (10 mL capsule, 2 cycles) anion exchange

544 0.498 271 99.66% 0.34% 82%

LRA impurity adsorption & buffer exchange to TE

100 2.2 220 99.7% 0.30% 66% overall downstream yield

Large scale autolytic purification From fermentation RF 547:NTC8 series AF vector in NTC3016 (autolytic DH5 α SacB host) 50 g fermentation cell paste = 331 mg pDNA

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Large scale autolytic purification

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Agarose gel electrophoresis of process samples

1. 1 kb DNA ladder2. Extract after cell debris removal3. Extract after KDS precipitation4. Mustang Q lysate flow through (cycle 1)5. Mustang Q wash (cycle 1)6. Mustang Q lysate flow through (cycle 2)7. Mustang Q wash (cycle 2)8. 1 kb DNA ladder9. 1 µg of miniprep purified pDNA from initial

extract shown in lane 210. 1 µg of final large scale purified pDNA

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Acidic Autolytic Extraction vs. Alkaline Lysis

Standard Alkaline

Lysis

Modified Alkaline

Lysis

Autolytic Acidic

Extraction

Total reagent mass (kg) 4.32 13.03 0.17

Total lysate volume (L) 30 100 20

Total lysis reagent cost $93.79 $173.73 $8.95

Dramatic reduction in raw materials and lysis volumes

Reduce cost by 90%, reagent mass by 96%, lysate volume by 33%versusstandard alkaline lysis Reduce cost by 95%, reagent mass by 99%, lysate volume by 80%versusmodified alkaline lysis (used for Mustang Q processing) Potentially 2x final yield vs. alkaline lysis processes (NTC historical data)

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