Transcript 2004-2008 Plan Meeting

Illkirch, June 2011
Current Progress on
Advanced Systems for
Glycomics Sample
Preparation
Split, 10-2-2013.
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HighGlycan
Presentation 2013
Outline of MP Bio Presentation
 Introduction
 Scientific Portion: Surface chemistry of enzymes
important for glycan sample preparation
 Kits for glycoproteins extractions
 New FastPrep-5G instrument
 Other possible forms of collaborations
HighGlycan
Presentation 2013
MP Bio New contact people
 Dr. Daniel Weissbart; Dr. Véronique Karsten
 Dr. Kumar Bala, Dr. Jhony Orbulescu, Jeffrey Whyte
 Financial/Legal: Mr. Barthel / Mr. Stankovitch.
 Lear: Mr. Fracois Barthel
Consultants:
 Prof. Miodrag Micic, Cerritos College & UCI
 Prof. Roger M. Leblanc, University of Miami.
HighGlycan
Presentation 2013
Surface chemistry of enzymes for glycan sample prep
 For the first time studied behavior of enzymes
important for the glycan preparation in the
Langmuir monolayer.
 Future work: Langmuir blodgett film enzyme
immobilization for the purpose of creating an
efficient heterogenous support for columns and
possibly sensors
 Enzyme currently studied: Beta Galactosidase,
future work on l-fucosidase and fucose
dehydrogenase and pngase
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Presentation 2013
Langmuir monolayer studies of beta galactosidases
 What is the Langmuir monolayer technique
 Pressure area isotherm slide
 Pressure surface potential isotherm
 Spectroscopic properties at monolayer
 Future work on the enzyme immobilization in the
form of the Langmuir Blodgett film
 Practical application: heterogenous bio catalyst for
glycans cleavage/sample preparation based on
immobilized Lagmuir Blodgett films on substrate.
HighGlycan
Presentation 2013
What is the Langmuir Monolayer ?
1.
A one-molecule thick layer of an insoluble or
amphiphilic material spread onto water/aqueous
subphase
2.
Works for most proteins and enzymes.
3.
Can be used in a form of the mixed colayers
4.
Can be transferred to a solid substrate surface as
a film,a.k.a Langmuir Blodgett film
5.
In vitro model of biological membrane
6.
Controllable experimental variables
7.
Efficient heterogenous catalysts and sensors
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Presentation 2013
Langmuir Monolayer Technique
50
Air
Water
40
Surface Pressure (mN/m)
Air
Water
KSV Isotherm of beta-Galactosidase
for UV-Vis at Air-Water Interface
Sample Date: 091113
Conc: 0.424 mg/mL
Injection: 300uL
LOT: M5982
Subphase: 0.01M NaCl
30
20
10
0
4000
6000
8000
10000
12000
14000
16000
18000
2
Mean Molecular Area (A /molecule)
Air
Water
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Presentation 2013
Langmuir-Blodgett technique - transfeering highly
packed enzyme monolayer to the solid substrate
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Presentation 2013
Current Lanmguir Monolayer results on beta
galactosidase
 As a model system we have performed a study of
the beta galactosidase from e coli (MP 0215003901)
 Obtained stable and reproducible surface-pressurearea isotherms
 Studied spectroscopic properties in the lanmguir
monolyer
 Current work: Formation of stable isotherms with
Mg2+ subphase
 Monolayer transfer to hydrophilic and hydrophobic
substrates.
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HighGlycan
Presentation 2013
Beta Galactosidase from e coli (MP0214003901)
 β-Galactosidase cleaves terminal galactose
residues, which are β -1-->14 linked to a
monosaccharide, glycopeptide, or oligosaccharide.
The enzyme requires Mg2+ for optimal activity.
 β-galactosidase cleaves lactose into its
monosaccharide components, glucose and
galactose. It also catalyses the transglycosylation
of glucose into allolactose, the inducer of βgalactosidase.
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Presentation 2013
Spectroscopic properties of beta galactosidase, bulk
Separate but
original samples immediately
after going into instrument
2
Intensity
8000000
Emission Spectra of beta-Galactosidase
Sample Date: 090213
Concentration: 0.1722 mg/mL
LOT: M5982
Excitation: 284 nm
Slit Width: 3 nm, 3 nm
No color change, no precipitate
1
6000000
70 nm
4000000
Same sample but degraded
after being in instrument for
10 minutes
1
2000000
Absorbance of beta-Galactosidase
Sample Date: 090213
Concentration: 0.1722 mg/mL
LOT: M5982
0.45
275
Absorbance
354 nm
10000000
281, 0.360
291
0.30
0.15
2
0
0.00
200
300
400
500
600
700
800
250
300
350
Wavelength (nm)
Wavelength (nm)
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HighGlycan
Presentation 2013
Surface pressure area isotherms of Beta
Galactosidease with estimated average mean
molecular surface areas at the 2D solid state film.
35
50
30
Surface Pressure (mN/m)
Surface Pressure (mN/m)
40
Kirbon Isotherm of beta-Galactosidase
Sample Date: 091113
Conc: 0.424 mg/mL
LOT: M5982
Injection: 25uL
Subphase: 0.1M NaCl
30
KSV Isotherm of beta-Galactosidase
for UV-Vis at Air-Water Interface
Sample Date: 091113
Conc: 0.424 mg/mL
Injection: 300uL
LOT: M5982
Subphase: 0.01M NaCl
20
25
20
15
10
5
10
2900
2600
2400
0
0
1000
4000
6000
8000
10000
12000
14000
2
16000
Mean Molecular Area (A /molecule)
18000
1500
2000
2500
3000
3500
4000
2
4500
Mean Molecular Area (A /molecule)
5000
5500
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Presentation 2013
Both spectroscopic and surface pressure area data
indicates stable 2D monolayer solid film
0.040
0.035
50
Absorbance
0.025
0.020
Absorbance of beta-Galactosidase
from UV-Vis at Air-Water Interface
Sample Date: 091113
Conc: 0.424 mg/mL
LOT: M5982
Subphase: 0.01M NaCl
0.015
0.010
0.005
Surface Pressure (mN/m)
0.030
40
091813
Fluorescence at the
Air-Water Interface
beta-Galactosidase
Excitation: 284nm
Emission: 354 nm
Slit Width: 3nm, 3nm
Injection: 50uL
Subphase: 0.1M NaCl
Conc: 0.6081 mg/mL
30
20
10
0.000
0
-0.005
0
10
20
30
Surface Pressure (mN/m)
40
50
7000
8000
9000
10000 11000 12000 13000 14000 15000 16000
Intensity
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Presentation 2013
Beta Galactosidase current work:
 Finalizing first manuscript/communications on
surface properties of monolayer of beta
galactosidase. Intended submission: Colloids &
Surfaces B:Biointerfaces or Langmuir.
 Make reproducible surface pressure area istoherm
with Mg2+ active subphase.
 Transfeer Langmuir Blodgett film to mica and glass
substrates
 Test stability and activity of the solid state LB films
 Second manuscript on LB film transfer and
properties.
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Presentation 2013
Fucose Dehydrogenase, Fucosidase and PNGase
surface chemistry work

Currently working on souricing/ recombinant
expression of Fucose Dehydrogenase, L-Fucosidase
and PNGase

Lanngmuir, Langmuir Blodgett studies

individual enzymes

Enzyme mixtures

Practical applications:

Enzyme mixture for glycan release in the form of

Heterogenous catalysis / immobilized sample prep
“labware”

Secondary outcome: MP Bio will be able to provide low
cost supply of high
 purity enzymes and enzyme mixtures once the
expression is established
HighGlycan
Presentation 2013
Glycoproteins sample prep kit works
 Kits for glycoprotein extractions
Lectin ConA based columns for purification of N-linked
glycoprotein fractions using the
 FastPrep Lysis
 Simplified workflow, bind-wash-elute
 WGA and ConA column
 Procedure :
FastPrep Lysis
Bind;
Wash
Elute
 First samples available for testing Nov-Dec 2013.
FastPrep-24-5G : Next Generaton
FastPrep
Presentation 2013
Key Features
 Speed Range: 4.0-10.0 M/S (from
6.5)
 Time: 1-120 Seconds (from 60)
 Cycles: 1-9 (New feature)
 Pause between cycles: 1-300
seconds (New feature)
 Noise: <60 dB
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 Touchscreen User Interface (all
new)
 >60 Predefined Settings,
MP Recommended
 Will include settings for
FastGlycoProtein kits
 Manual Settings
 Store up to 12 User-defined
Settings (from 5)
 Export Data
 Re-order and tech serve
info for 19 global regions
 Bluetooth® Compatable
 USB Port, App Updates
 Voice Interface that will give run
statistics
 Strobe Light to aid visual lysis
inspection
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Presentation 2013
Other collaboration with Consoritum Participants
 MP bio is an affordable high quallity reagents
supply provider
 90% of Sigma Aldrich offerings
 High quallity reagents, kits and systems:
Biochemicals
Radiochemicals
Immunologicals
Molecular Biology
RIA assays
 Fine chemical and biologicals sourcing and custom
manufacturing group for custom products.
HighGlycan
Presentation 2013
Who we are
MP Biomedicals is a global company committed in the
development, manufacturing and marketing of life sciences
and diagnostic products.
The company is dedicated to provide scientists and
researchers innovative and high performance tools
combined with superior service to help them in their quest
for discovery and thus make the dream of a better life, a
reality.
HighGlycan
Presentation 2013
History
Formerly ICN Pharmaceuticals, MP Biomedicals was formed in 2004 after the
acquisition of various companies in various fields of application and
complementary.
1959, ICN is founded by Milan Panic.
1970, ICN discovers the first broad-spectrum antiviral drug in the world.
Sales reach $ 1.5 billion and several million people were treated.
1986, ICN was the second largest shareholder of Roche.
2002, ICN listed as 49th largest pharmaceutical company in the world with 17 000
employees.
2003, ICN is sold.
2004, Milan Panic founded MP Biomedicals through the acquisition of thirty
companies, including Qbiogen.
HighGlycan
Presentation
2013
MP Biomedicals
world
Santa Ana, CA
in the
Strasbourg, France
European Headquarter
Eschwege Germany
Facilities and Logistics
American Headquarter
Singapore
Asia Headquarter
Solon, OH
Facilities and logistic
Sydney, Autralia
Facilities and logistics
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Presentation 2013
Our Applications
Life Science
Fine Chemicals
Diagnostic
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HighGlycan
Presentation 2013
LifeScience
From molecular biology, cell biology through
apoptosis and immunology products MP
BIOMEDICALS accompanies you at every
stages of your research in life sciences.
Sample preparation
Cell Biology
Many researchers have chosen our products
because they guarantee returns and meet your
requirements.
Molecular Biology
Proteomics
Drug Discovery
We now supply both large universities, as well
as major industries that quests reliable and
innovative products.
Solid Phase Peptide
Synthesis
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Presentation 2013
MP Biomedicals provides more than 12,000
compounds of the K & K library. More than 1,000 of
them are exclusive to MP Biomedicals and can be
found in our product catalog.
Chemicals
Specials Synthetic Reagents
Building Blocks
Protecting &Deprotecting Reagents
These compounds demonstrate our commitment
to provide researchers with the largest selection of
rare and fine chemicals, many of which are
available nowhere else.
Reduction &Oxidation Reagents
Our wide range of compounds includes a variety of
functional groups, both aromatic and nonaromatic.
In this collection, you will find chiral compounds,
inorganic rare earth compounds, amino acids,
organometallic compounds and more.
Rare Earths
We also have a wide range of silica and alumina for
chromatography.
Precious Metal compounds and
catalysts
Amino Acids
Silica & Alumina / Chromatography
Albumins
Absorbents
And more…
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Presentation 2013
Diagnostic
Whether you work with assays for animal testing
or products geared toward human specimens,
we are committed to provide you with kits and
reagents of superior quality.
Auto-immunes Kits
Biogenic Amines
Cardiac Markers
Endocrinology
Easy to use for determining in vitro
concentration of various hormones in human
and mouse samples.
Food Quality
Infectious Diseases
Isocalibrators
Each kit contains all necessary reagents,
including controls, and produces accurate
results within 24 hours
Newborn Screening
Cancer Markers
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Presentation 2013
Examples of Europeans Events
TradeShows
25
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Presentation 2013
Q&A?
THANK YOU !
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