genvault’s ambient-temperature technologies for...

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GenVault’s Ambient-Temperature Technologies for Biospecimen Management

P3G Meeting

Luxembourg

September 30, 2009

1© 2009 GenVault – Risk Free Sample Management

Michael Hogan

Chief Scientific Officer

GenVault Corporation

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The Core Physical Chemistry of GenVault Technologies


H20, Catalysts & Heat

ROS

&

Heat

Non-aromatic

Proteins

DNA RNA

Aromatic

Proteins

Lipids

Oxidation

Rate

Dimension

Hydrolysis

Rate

Dimension

The Chemical Stability of Biomarkers

During Storage

Can be Approximated as a Plane

In “Oxidation, Hydrolysis” Space

(0,0)

Means

No oxidation

Or hydrolysis

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Physical Chemistry of Cryogenics vs Ambient-Temp Preservation

3

• Traditional Cryogenics Solution:

“Collapse the plane” to the origin by cooling the specimen, to slow oxidation & hydrolysis, based on the temp dependence of the chemistry.

Use mechanical energy to “quiet” the sample by cooling,

keeping the underlying chemistry the same.

• The GenVault Ambient Temperature Solution:

“Collapse the plane” to the origin by removing water,

adding ROS scavengers, and eliminating O2 exposure.

Use chemistry to “quiet” the sample: -H2O, -ROS

keeping the temperature the same.

© 2009 GenVault – Risk Free Sample Management

Slide -

The Core Physical Chemistry of GenVault’s Technologies


H20, Catalysts & Heat

ROS

&

Heat

Non-aromatic

Proteins

DNA RNA

Aromatic

Proteins

Lipids

Oxidation

Rate

Dimension

Hydrolysis

Rate

Dimension

The Chemical Stability of Biomarkers

During Storage

Approximated as a Plane

In “Oxidation, Hydrolysis” Space

(0,0)

The

Specimen

Is at rest

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Cryogenics vs Ambient Temp Preservation

5

Practical advantages result from ambient temp stabilization:

Upon removing water and adding ROS scavengers:

The Specimen is Poised at Equilibrium,

in the absence of applied mechanical energy

-No need for energy to maintain -20C, -80C, -190C during storage

-No need for an energy-source backup to mitigate risk

(no generators, no LN reservoirs)

-No need for high-cost to maintain +4C or -20C during shipping

(no cryopacks)

-No need for high-cost, just-in-time, shipping protocols

(international book-rate instead)

Obviating cryogenics: in biobanking & sample sharing © 2009 GenVault – Risk Free Sample Management

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The GenVault Technologies: Span the Biobanking Spectrum


PRESERVE TRANSPORT BANK RECOVER ANALYZE

Dynamic ArchivePersonal Archive

GenConnect GenCode ID

GenTegraGenSolve Automation

GenPlates

Slide - 7

Preserve = GenPlates -> Paper “Pop-Out” Hemispheres

• Treated macroporous Whatman filter paper technology: exclusive license to GenVault

• H2O removed, ROS quenched by protein & additives

• Proven stability, 20 years and counting

• High quality DNA recovery: with GenSolve Enhancements

• Designed for a wide variety of ‘raw’ sample types

blood, buffy coat, homogenized tissue

• Contact inactivation of bacteria and viruses

• Proprietary biological barcode at aliquot level GenCode ID = internal chemical bar code

GenPlate


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“GenSolve “ Technology Enables DNA Recovery from Dried Blood Spots on Very-Old Filter Paper Disks

8

• Use 11-15 year old Blood Spot Cards

• From California Birth Defects Cohort

• Collaborators at March of Dimes & State of California

• Two 3mm punches pooled per donor

• From 1993-1998

• Recover DNA with GenSolve, then Qiagen

• >250ng of DNA from each

• Illumina GWAS on each:

BMC Genetics 2009 Jul 22;10:38.

Whole genome microarray analysis, from neonatal blood cards.

Hardin J, Finnell RH, Wong D, Hogan ME, Horovitz J, Shu J, Shaw GM.


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DNA Recovered from 11-15 year old Filter paper is >50kb


Slide - 10© 2009 GenVault – Risk Free Sample Management

Specimen Name No Calls Calls Total

Call Rate (%)

Primary DNA Isolate

MOD 881 1484 591008 592492 99.75

MOD 892 1581 590911 592492 99.73

MOD 919 1502 590990 592492 99.75

MOD 937 1607 590885 592492 99.73

MOD 920 2524 589968 592492 99.57

MOD 975 2840 589652 592492 99.52

MOD 986 2432 590060 592492 99.59

MOD 001 3240 589252 592492 99.45

DNA from 11-15 Year Old Dried Blood -> high quality GWAS

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Track Samples = GenConnect

• Manages complex ambient-state and cryogenic storage media

• And associated sample data, if needed, in a robust database

• Enables Federated “virtual” biobanking among many sites

• Enables a secured, multiple-user multiple-role collaborator group

GenVault - Ambient Temperature Storage Solutions

Sample and Storage Data

GenConnectLN Tanks Downstream Data

Clinical/ Patient Data

Freezers


Slide - 12

Bank & Transport Samples = GenTegra (DNA & RNA)


Slide - 13

GenTegra-DNA

For purified DNA transport and banking

• Mineral Matrix to dehydrate DNA

• Antioxidants to scavenge ROS

• Water soluble and inert to downstream analysis

• For large (50ug) and small (50ng) DNA alloquots

• No post-recovery cleanup

• Just mix and air dry.

• Microfuge Tube & 96 well plate formats

GenTegra DNA


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GenTegra DNA Performance at the Extremes of Temperature

ctrl

RT 37°C 56°C 76°CAffy 6.0

Infinium 1M

40 kb

40 kb

Frozen Control

GenTegra RT

GenTegra 56°C

GenTegra 76°C

Call RateAffy 6.0 99.50% 99.40% 98.70% 99.20%

Infinium 1M 99.80% 99.70% 99.80% 99.70%

ConcordanceAffy 6.0 99.80% 97.70% 99.70%

Infinium 1M 99.90% 99.90% 99.90%


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GenTegra-RNA: How to make RNA a Stable Biomarker

15

• Inert, mineral matrix, plus antioxidants and Small-MW RNase inhibitors• Stabilizes purified RNA upon contact in the fluid phase (days)• Then long-term stabilization upon air-drying (years)

• Two-fold stabilization @ ambient temperature• In the fluid-phase: during routine lab preparation and incubation,

without bench-top cryogenics• In dry-state: during long term storage

without storage cryogenics• Ideal for global RNA transport among biobanks

• Preserves RNA integrity during exposure to harsh shipping temperature fluctuation (up to +60C)

• $(€) savings in resources and budget; eliminates high cost of shipping wet/refrigerated samples

GenTegra™ RNA

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GenTegra-RNA Development. One Month Dry, at 56C

RT 56 C( ~264 day at RT)

FrozenCtrl

No Matrix

+ Matrix

15ug rat liver RNA(Qiagen)

MatrixDry ON at

RT33days at RT / 56 C

Rehydrate

Bioanalyzer

Electropherogram showing RNA Integrity & Breakdown28S:18S Ratio = ribosomal species 28S and 18S ratio; in part linked to instability of the 28S rRNA structure relative to the 18S RNA. Decrease in the area of the 28S rRNA peak reflects breakdown.

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GenTegra-RNA Development. 20 hrs in Solution, 37C with RNAse

+ Matrix+ RNase A

Fro

zen

ctr

l

+ R

Nas

e A

No

mat

rix

No

RN

Ase

1ug Hela RNA (20ul)

Matrix

RNAse A/120hrs at 37 C gel

+ Matrix+ RNase 1

+ R

Nas

e 1

Slide - 18

GenTegra-RNA Development . One Month Dry, at 56C, with RNAse

Frzn

ctrl

Frzn

ctrl

Frzn

ctrl

+ Matrix

+ RNAse A/1

Dry state storage : RT RT 37 37 RT RT 37 37 RT 37

1ug Hela RNA (20ul)

Matrix

RNAse A/1

Dry ON at RT

4 weeks at RT / 37 C

Rehydrate

10min

90min 37 C

Gel

+ Matrix

+ RNAse A/1

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Representative GenTegra RNA Field TestingDiagnoCure, Quebec City

Diagnostic Testing of the presence of Guanyl Cyclase C (GCC) as

a marker of occult metastases in FFPE pericolonic lymph nodes

from colon cancer patients

DiagnoCure Collaborators: Michel Houde, Guillaume Beaudry, Genevieve Garon, Melanie Robitaille, Josianne Veer

19

A Sample of Third PartyTesting: At a CLIA-Certified Lab

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Testers Validate GenTegra at the Extremes of Temp Exposure

20Risk Free Sample Management Technologies

• FedEx Shipping: can expose samples to up to +50C• US Army Shipping: can expose samples to up to +70C• Laboratory Medicine: fluid sample processing at 25C, 24 hrs

The Standard Test Plan:• Fluid phase RNA in GenTegra at 25C or 37C for 24 hrs• Dry State RNA in GenTegra at 25C, 37C, 56C for 2 weeks• Cells & high-value biopsies: fresh and FFPE• Use LoC Capillary Electrophoresis (Bioanalyser) = physical integrity• Use Q-rtPCR = biochemical integrity

GenTegra™ RNA

20

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Liquid Samples – 24h

• RNA tested (5ug):– Colon (FFPE)

– Lymph Nodes (FFPE)

– T84 cells extracted with GCC method

– T84 cells extracted with Trizol

– HelA cells extracted with Trizol

• Temperature tested:– Room Temperature

– 37 C

Dry Samples – 2 weeks

• RNA tested (5ug):– Colon (FFPE)

– Lymh Nodes (FFPE)

– T84 cells extracted with GCC method

– T84 cells extracted with Trizol

– Liver (Fresh Frozen)

– Hela

• Temperature tested:– Room Temperature

– 37 C

– 56 C

GenTegra RNA Field Testing – FFPE and Frozen Tissues

21

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2 weeks,56°C

2 weeks,FrozenAt -70°C

Dry RNA Samples –T84 cells, Extracted with Trizol

RNA degrades Over two weeks, without GenTegra protection

22

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Liquid RNA Samples –T84 cells, Extracted with Trizol

23

24 hours in fluid solution at 25C or 37C

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Sample ID

Actin Ct Actin CopiesCopies ratio

Mean Ct StdDev CtCopies (10+6) StdDev

RT

m1 14.69 0.78 3.91 2.2 63.4%

m2 14.55 0.24 3.95 0.62 63.9%

m0 13.98 0.26 5.84 0.99 94.4%

37°C

m1 13.36 0.43 9.10 2.57 147.2%

m2 13.87 0.07 6.22 2.82 100.7%

m0 14.27 0.20 4.79 0.65 77.4%

56°C

m1 13.82 0.32 6.54 1.44 105.8%

m2 13.95 0.33 5.98 1.25 96.8%

m0 14.00 0.40 5.84 1.49 94.6%

-70°C 13.98 0.66 6.18 2.45 100.0%

T84 cell RNA: Q-rtPCR, after Two Weeks Dry, 56C

24

Two weeks dry at 56C

Slide -

Sample Name

Actin Ct GCC Ct GCC Copies

Copies ratioMean Ct StdDev Ct Mean StdDev Copies StdDev

RT

m1 24.64 0.10 23.25 0.30 18,305 3,560 82.2%

m2 24.28 0.09 23.01 0.19 21,495 2,807 96.5%

m0 23.54 0.07 23.05 0.23 20,946 3,439 94.0%

37°C

m1 24.91 0.04 23.13 0.31 19,900 4,343 89.3%

m2 24.61 0.13 23.10 0.25 20,243 3,610 90.9%

m0 23.38 0.08 23.06 0.26 20,806 3,806 93.4%

56°C

m1 25.51 0.09 23.27 0.10 17,844 1,178 80.1%

m2 24.88 0.03 23.09 0.19 20,260 2,549 91.0%

m0 23.74 0.12 23.12 0.21 19,950 2,900 89.6%

-70°C 22.98 0.07 22.95 0.13 22,274 2,044 100.0%

Lymph Node RNA: Q-rtPCR, after Two Weeks Dry, 56C

25

Two weeks dry at 56C

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Technology in Development – Elastomer-Chaperone

26

The Elastomer-Chaperone Concept

Store blood on a 150uL Elastomer,

inside a protective tube with internal drying capacity &

2D bar code or RFID tag.

For ambient-temp acquisition, shipping and biobanking.

The Assembled Elastomer-Chaperone

The Cap can be touched to the skin or to a pipette.

The Cap is then screwed into the tube body.

Drying occurs over several hours, internally

Elastomer-Chaperone

Self-contained biospecimen preservation & carrier

2D Bar code or RFID tag = “Header”

• Self-contained sample acquisition, shipping & storage– neonates, adult epidemiology, personalized medicine,

– DNA, most proteins, most small molecules

• 150uL elastomer matrix inside a Chaperone carrier

• Direct contact transfer from a pipet or heal, finger or ear-prick


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Transport - Chaperone Performance - DNA

27

At the DNA level, we have found that 100uL of dried blood can be re-hydrated after up to 34 days of storage at RT or 56C (133F) by addition of the same standard protease solutions used to process fresh blood, followed by standard protease treatment at 56C and then fluid release by a one minute of centrifugation in a spin basket followed by a standard Qiagen Mini prep column.

DNA Source:Storage:

Condition:Amt. on gel:

Elast.34days56oC125ng

Roche

50ng

Elast.34daysRT125ng


Elast.19daysRT125ng


Elast.15daysRT125ng

Blood0daysFresh125ng

Roche

250ng

Blood0daysFresh50ng

% Recovery

DNA Yield:

123.4%

3.0 g

NA111.2%

2.7ug

90.5%

2.2 g

102.7%

2.5 g

99.4%

2.4 g

77.2%

1.9 g

100%

2.4 g

NA100%

2.4 g

DNA Recovered from the Elastomer:Whole blood, stored dry at RT or 56C for 34 DaysNative electrophoresis after Qiagen purification & picogreen quantitation

Bottom Line:Picogreen quantitation shows ~100% recovery based on an expected DNA yield of 2.5+/-0.5µg from the same fresh

blood samples extracted prior to drying.

Gel band intensity shows that DNA remains >>40kb long over storage time


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Transport - Chaperone Performance - Protein


Cancer markers, Acute Phase Reactants and other serum proteins - 1

Analytes were tested at Rules Based Medicine (Austin TX) in a multiplexed fashion via the Luminex-RBM bead immunoassay platform, on 5 different specimen types: (1) serum (SST), (2) EDTA-plasma (EDTA Pls), (3) Heparin-plasma (Hep Pls), (4) Citrate-plasma (Cit Pls) and (5) whole blood (WB) from 4 different healthy volunteers each. 100uL of each sample was applied to 100uL Elastomer elements, each with a different set of chemical stabilizer treatment dried into the Elastomer, and then air dried at RT for a day. Upon drying, these specimens were sealed and then stored at 25C for 28 days. The specimens were rehydrated by adding 130uL of water, incubation at RT for 30 minutes then ejected from the Elastomer by spinning for 5min at 1000g in a microfuge spin basket

Data is presented as percent recovery for a fraction of those 114 non-zero protein analytes, comprising the multiplex panel of the most abundant protein species

Slide - 29

GenVault: P3G Summary

• Numerous ways to Introduce Ambient Temp Technology into Workflow

Replace Cryogenics for Nucleic Acid Transport Among Sites

Replace Cryogenics for Long-Term Nucleic Acid Storage

“The GenVault + Cryogenics Approach”

GenVault for the Nucleic Acids(chemical stabilization of simple linear polymers like DNA & RNA)

Cryogenics for the rest(cell viability & enzymatic activity is more than just a chemical problem)

For Example: at the April 2010 P3G Meeting, 2 hrs away in Chicoutimi

The CARTaGENE-Chicoutimi Biobank

-Automated GenVault “Dynamic Archive” for Blood Biobanking DNA for Genomics

-Liquid Nitrogen & CryoStraws for Serum Biobanking For Proteomics

-80C Freezers For Flash Frozen Tissue

-All Storage Media Linked by GenConnect (Dry-Ambient, -190C, -80C) © 2009 GenVault – Risk Free Sample Management

Slide - 30

CARTaGENE- Chicoutimi Biobank: GenVault + Cryogenics

GenVault’s Ambient-Temp Technology Coexists Nicely with Cryogenics.

GenConnect can Link it all Together


100,000

Plate

GenVault,

Fully

Automated

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