lv1 bio beers pdf

Tiny Particles, Tiny Particles, BIG RESULTSBIG RESULTS© Microfluidics

Introducing the new low volume Microfluidizer the LV1

small sample volume (1-20ml), high pressure (30KPSI) and near total sample recovery.

Multiple systems scalable from 1 ml to gallons per minute. Perfect for R&D, discovery, discovery exploratory, small clinical studies, pilot scale right through to production scale systems

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The new low voulme 1–20ml Microfluidizer the LV1

To learn more about the LV1 or the differences between the Microfluidizers, homogenizers and the French Press please go to www.microfluidicscorp.com and go to our Webinars Tab or continue with this presentation

Bill Travers Manufacturer’s Representative Mobile: 410-746-0488 [email protected]

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Perfect for Nanoemulsions | Nanodispersions | Liposomes |

Cell Disruption | Continuous Crystallization(Replace your obsolete French Press)


New from Microfluidics

Don’t Worry – We Lowered the Volume• Samples as small as 1 mL• Same interaction chamber platform• Payback measured in days for high value samples• Near total sample recovery• Ideal for academic and biotechnology research• Fully scalable to larger lab, pilot and production• Shear rate at 30,000 psi: 1.23 X 107 s-1


Discover the Difference:Microfluidizer® Processors vs. Homogenizers, Sonicators and the French Press

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Discussion Topics

• Microfluidizer® Technology– Highest shear– Narrow distribution– Scaleup guaranteed

• Particle Size Reduction vs. Homogenizers– Particle size– Uniformity– Repeatability– Process efficiency– Customer Case Study: Corixa (now GSK)

• Cell Disruption vs. Sonicators and French Press– Rupture rate– Protein yield– Temperature control– Ease of use

• Questions and Answers


Tiny Particles, Big Results

Who We Are• Founded in 1983 outside Boston, MA• 3,000 processors in 50 countries• Technology Center (virtual tour online)

Microfluidizer Processors• Uniform particle size reduction• Cell disruption with high protein recovery• Continuous crystallization of nanoparticles

M-110P “plug n’ play” benchtop lab model

M-110EH-30 pilot/production scale

M-700 series for commercial production

LV1 Low Volume (1 mL capable) processor

Nanoemulsions | Dispersions | Liposomes | Cell Disruption | Deagglomeration | Continuous Crystallization


Heart of the Technology

Fixed-Geometry Interaction Chamber- Microfluidics exclusive- Generates incredibly high shear and impact forces- Precisely engineered microchannels- Repeatable and scalable results- Diamond or ceramic construction

More than Machines- Proof of Concept- Process Development- Preventive Maintenance- Safeguard Spares™- Scaleup consulting- MRT Development


Applications and Benefits

Select Applications Proven Benefits

Vaccines & adjuvants Improved bioavailability

Oncology (injectables) Controlled drug delivery

Asthma (inhalables) Sterile filtration

Antibiotics Extended shelf life

Steroids Continuous processing


E. coli High rupture rates

Yeast Maximized protein yield

Algae Temperature control

Mammalian Lower pressures/fewer passes

Plant, insect, etc. No contamination

Pharmaceutical Biotechnology


Carbon nanotubes Uniform dispersions

Inkjet inks Less energy required

Coatings & sealants Consistent product quality

Fine chemicals Easy to use and clean

Polymers & waxes

Agrochemicals

Chemical EnergyMore efficient catalysts and scaleable processes for fuel cells, batteries, photovoltaics and biofuels

CosmeticsBrighter colors and controlled actives delivery for premium and natural lipsticks, hair sprays, clays, etc.

Food/NutraceuticalsNew ingredient flavors, time-release vitamins and encapsulated odors of nutrients such as Omega-3


Microfluidizer TechnologyMicrofluidizer Technology

How It Works


Microfluidizer Processor Schematic

Inlet Reservoir

Intensifier Pump

Pressures up to 40,000 psi

(276 MPa)

Cooling Jacket Outlet

Interaction Chamber

Product


Z Chamber – Lab Z Chamber – Production

Interaction Chambers

High Shear Zones

High Impact Zone

Low Pressure Outlet P2

High Pressure Inlet P1

High Pressure Inlet P1

High Shear Zone

Low Pressure Outlet P2

High Impact Zone

Specifications• No moving parts• Diamond or ceramic material• Shear rates up to 107 s-1 • Velocities over 400 m/s • Diameters 50-300 microns• “Y” and “Z” shapes available • Controllable mixing to 25-50 nm• Demonstrated scalability

On the Other Hand…Valve homogenizer systems use variable processing geometry, leading to inconsistent pressure, shear and results


Shear Rates for Various Technologies

Agitator

Sawtooth Blade

Closed Rotor

Rotor-Stator

Colloid Mill

Homogenizer

Microfluidizer Processor


Particle Size ReductionParticle Size Reduction

Vs. High Pressure Homogenizers


Under Pressure

Pressure Profile Comparison• Homogenizers operate at peak pressure for mere moments (~7%)• Microfluidizers process effectively the entire product stream to the desired pressure (and consistent shear) during each processing stroke


Oil-Water Emulsion Results

Particle Size• At each pass, Microfluidizer results are twice as small• Even after 5 passes, the homogenizer can’t meet results of a single Microfluidizer pass

Uniformity• Due to consistent shear, Microfluidizer results are exceptionally narrow• Homogenizer displays larger particles and higher variance – leading to a less stable emulsion


• Median particle size (D50) AFTER: 45 nm

0

2

4

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1 10 100 1000

Freq

uenc

y (v

olum

e %

)Particle Size (nanometers)

BEFORE

AFTER

AFTER

O/W - Drug Nanoemulsion (Cancer Drug)


Before After 15 passes

0 p 1p 2p 3p 4p 5p 7p 10p 15p

Process pressure: 18,000 psi (1241 bar) Chamber: F20Y (75 microns)

Final product is translucent

O/W - Drug Nanoemulsion (Cancer Drug)


Median = 0.075 m

Median = 14.53 m

Before

After

Emulsion: Oxygen Carrier (Perfluorocarbon) in a Cosmetic Application


Liposome Results

Particle SizeConsistent with emulsions, average particle sizes with Microfluidizer are approximately half the size

UniformityVariability advantages are even more pronounced here – the homogenizer sample contains multiple peaks

Microfluidizer Processor

Leading Homogenizer

Pass 1 113 nm 268 nm

Pass 2 95 nm 228 nm

Pass 3 72 nm 183 nm

Did You Know? Microfluidics was originally founded as a liposome producing company


• 0.1% DNA plasmid solution was encapsulated inside a Palmitoyl oleoyl phosphatidyl choline liposome

• DNA intact after processing

DNA Encapsulation in a Liposome


Example – De-agglomeration

Malvern NanoS (no sonication)

Mean (nm) Mean (nm) d10 (nm) d50 (nm) d95 (nm)NA NA Unprocessed 348.8 226 137 209 384

1 136.1 92 52 84 1662 127.1 87 52 81 1511 256.7 119 55 102 2532 154.7 111 53 91 247

30% by wt.

CeO2 in water

Horiba LA 910 (1 min sonication)

G10Z30,000

20,000

Formulation Chamber Pressure (psi) Pass

024

68

1012

14161820

0.01 0.1 1 10Diameter (um)

Fre

qu

ency

(%

)

2 passes 1 pass

unprocessed

Unprocessed 2 passes G10Z 30k


• SWNTs* in high viscosity mineral oil

• Application: Golf clubs

*From CarbonNanotechnologies Inc.

Magnification 20,000x

Unprocessed 1 Pass H30Z-G10Z @ 158 MPa

10 Passes H30Z-G10Z @ 158 MPa 20 Passes H30Z-G10Z @ 158 MPa

Carbon Nanotubes Dispersed in a Liquid Media


Critical Quality Criteria Leading Homogenizer Microfluidizer

Average Particle Size 185 nm after 15 passes 141 nm after 3 passes

Goal: <150 nm

Polydispersity 43% above 200 nm 0.5% above 200 nm

Goal: <10% above 200 nm

Active Concentration 15% loss of actives 1% loss of actives

Goal: <2% loss of actives

640 cm2 17 cm2

Microfluidics reduced filter area required by over 97%

Customer Success Story: Corixa (now GSK)

Vaccine Adjuvant Nanoemulsion

Based on these data, Corixa switched from their leading homogenization equipment to a Microfluidics-powered production environment


Cell DisruptionCell Disruption

Vs. Sonicators and the French Press


Why is Cell Disruption Important?

• The generation of important enzymes, proteins and other products form microbes has been developed and used for the last 40 years

• Cell rupture is required any time that products from cell sources must be removed from inside the cell

– Recombinant proteins are often grown in E. Coli or S. Cerevisiae which do not excrete proteins

– Algae cells currently being used for biofuel generation must be lysed to access bio diesel precursor


Shear Impact

Microfluidizer French Press Sonicator

• Precise shear control

• Disrupt resilient cells (e.g. yeast, algae)

• More effective lysis of shear-sensitive cells (e.g. >99% of E. coli with a single pass)

• Lower levels of shear generated even at higher pressures

• Has difficulty rupturing tough cells

• Requires more passes for bacterial cells

• Uses cavitation to generate shear – typically much lower than high pressure methods

• Increasing shear results in unwelcome higher processing temperatures, as well

The Microfluidizer has the highest commercially available shear rates and can control pressure precisely for shear-sensitive applications


Keeping Your Cool


• Cooling coils ensure product comes out at a low temperature

• Low residence time (~0.5s) within chamber minimizes excessive heating at any point

• Cooling tray at output minimizes denaturation

• Heats samples during processing – product comes out hot

• No native cooling options

• Collection vessel must be manually iced to limit high-temp residence time

See thermal image, above• Cells near the tip experience extreme temperatures

• Cells further away remain cool and potentially unlysed

• Must be actively adjusted by turning the system on and off

• Keeping sample on ice does not prevent localize overheating

One of the most important factors in maximizing protein yield and minimizing denaturing is product temperature


Repeatability


• Fixed-geometry interaction chamber and constant pressure pumping system ensure uniformity

• Enables use of lowest pressures and passes possible to rupture more cells

• Depends on a manually operated valve

• Speed of human user’s valve turn determines actual applied shear

• Not a trustworthy or repeatable method

• Relies on cavitation to generate shear

• Probe oscillation produces widely variable

Controlling shear and pressure rates allows researchers to reduce energy and heat applied while achieving target rupture rates


Commercial Viability


• Microchannels align in parallel applies consistent shear to samples ranging from 1 mL to 100 liters/minute

• Process and scaleup consulting enhances efficiencies and reduce passes for customers

• Demonstrating viability helps universities with grant applications

• Volume is limited to lab-scale

• That said, cannot process small samples, leading to waste of materials

• Batch processing restricts volumes

• Lack of repeatability

• scaleup is impossible

Success in the lab is great – but doesn’t mean much to a business unless results are repeatable at production volumes


Speed Limits


• Typically completes a processing cycle in 90 sec

• Includes cleaning

• Requires fewer passes

• Very slow drip process regardless of sample size – good for coffee, bad for labs

• An average cycle lasts 30 minutes

• Dangerous to clean – head alone can weigh 30 lbs (14 kg)

• Ranges from seconds to several minutes per cycle

• Cycle time is less predictable due to the constant monitoring and on/off adjustment necessary to prevent sample overheating

Microfluidizers are designed to be easy to use and clean, with fewer passes required, to support busy lab research teams


Additional Considerations

• Aeration – Difficult to remove oxygen from French press syringe. Product often creates froth or foam during loading

• Sonic Boom – Sonicators are loud and are often operating in isolation. Even in a sound-enclosure, OHSA recommends ear protection, which may not guard against long-term ear damage

• Ongoing Support – The French press is no longer manufactured or supported except by third-parties and is quickly becoming obsolete

Tiny Particles, Tiny Particles, BIG RESULTSBIG RESULTS© Microfluidics 34

Comparison to Other Mechanical Methods

Microfluidizer Homogenizer Bead Mill

Continuous Yes Yes No

Scalable Yes Limited Yes

Optimal Temp Control

Yes Yes No

Contamination Free

Yes Uncertain No

Minimum Volume

1ml* 10 ml 1 ml

Constant Shear Rate

Yes No No

Shear Rate Potential

Highest High Medium

* Now available with the LV1 Microfluidizer processor


BEFORE AFTER

Process pressure: 18,000 psi (1241 bar)Chamber: H10Z (100 microns)

Shear rate: 5.58 X 106 s-1

E. Coli

Microfluidics users typically rupture >99% of E. coli cells in a single pass


Yeast Lysis (S. Pombe)

Unprocessed 1 pass ~60% lysis

5 passes ~95% lysis 10 passes ~99% lysis

Process conditions: 30,000 psi (2070 bar)

Chamber: G10Z (87 microns)

Shear rate per pass: 6.94 X 106 s-1

Microfluidizers are tough on cells (even yeast) and gentle on proteins


Lysis of Haploid S. Pombe on 110EH – 30,000 psi

02040

6080

100120

140160180

0 1 2 3 4 5 6 7

Pass #

mg

so

l pro

tein

/g c

ell

• The maximum recovery of soluble protein is achieved at 5 passes

• Further passes appear to cause more protein to denature than are liberated by the additional lysis


Mammalian, Plant and Insect Cells

Example: MammalianPerformed for NC State University Gene Therapy Center to release viral vectorsfrom human embryonic kidney cells

– Cells processed with the Microfluidizer processor for 1 pass yielded expected amount of protein

Before - Unprocessed After - 1 Pass

Process conditions: 1 pass at 5000 psi (535 bar)

Chamber: H30Z (200 microns)


• Used to produce more complicated proteins (do not require post translational modifications)• Require much lower shear rates


Algae Cells

• As the supply of fossil fuels diminishes, the need for renewable fuel sources will increase

• Biofuels from algae cells are appealing because they grow quickly and can directly convert CO2 to longer chain oils which can be easily converted to biodiesel

• Cells must be ruptured in order to gain access to oil• There is a wide variety of algal cells which all require different shear rates to

rupture


Algae Cell Disruption Example #1

Process conditions: 1 passes 10,000 psi (690 bar)Chamber: H10Z (100 microns)



BEFORE AFTER

Process conditions: 3 passes 20,000 psi (1380 bar)Chamber: G10Z (87 microns)


Algae Cell Disruption Example #2


Microfluidizer Processors – Summary of Benefits

Proven Results• Highest possible shear• Fixed-geometry interaction chamber• Less energy & fewer passes• Easy to use and clean• Scaleup guaranteed

Particle Size Reduction• Smallest particle sizes• Most narrow distribution• Targeted actives delivery• Simplified downstream processing & less filter area

Cell Disruption• Single processor capable of E. Coli, Yeast and Algae• High protein recovery

• Temperature control• Low residence time• Controlled shear (lower pressures required)

• No contamination• 1 mL sample volumes available with the new LV1


Thank You for Your Time

Two Ways to Ask Questions• New! Raise your virtual hand & we’ll un-mute you so you can talk live• Type your question into the chat field and we’ll ask it for you

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Current Promotions- 5% discount on LV1 in 2010 (Promotion Code: LV1-2010)- Up to 15% off interaction chambers in 2010 (Promotion Code: CHAMBERS10)-Customers! Visit the Customer Center or www.microfluidicscorp.com for referrals, news and more

Plus, Over 50 Questions in the Queue… Nanoparticles Cell Lysis Applications Technology LV1

•Emulsion success criteria•Maximizing stability•Shear alters compound?•Smallest possible size?•Water-insoluble APIs•Extending shelf life

•Large-scale capabilities•Pretreatment •Insect cells•System running dry•Cellulosic materials

•IV formulations•CNT’s•Coatings•Pigment dispersion•Polysaccharide size•Diatom processing

•Range of viscosity limits•Reliability•Expected chamber life•Heat Microfluidizer?•5-10mL samples in 1 hr?•Flammable liquids

•Product recovery•Cooling efficiency•Throughput