new materials for protein chromatographyfundacionsabercomo.org.ar/wp-content/uploads/2019/...cryogel...
TRANSCRIPT
Prof. Dr. Mariano GrasselliLaboratorio de Materiales Biotecnológicos
Universidad Nacional de Quilmes-IMBICE (CONICET)ARGENTINA
New materials
for protein chromatography
Proteins
Ionizing
radiationh.n
Xen+
e-
since1999.
R&D in:
LaMaBio: research focus
Laboratorio de Materiales Biotecnológicos
Polymers &
nanomaterials
Functional
materials
DegradationCross-linking
Intramolecular Intermolecular
1st Physic events -> Ionisation
2nd Chemical events
**
*
*
Interaction of ionising radiation with macromolecules in solution
Other chemical reactions
- Grafting polymerisation
- Bulk radiation-induced polymerisation
Other chemical reactions
- Grafting polymerisation
- Bulk radiation-induced polymerisation
Preparation and characterization
of different adsorptive materials
for downstream processing
Radiation processing
Sample irradiation
PISI (CNEA-Ezeiza)
Multipurpose Cobalt-60
Irradiation Source
Upstream
Bioprocess
Downstream
Production of recombinant
proteins in cell cultures
Up to 80 % of production cost
Bioprocess
Production is moving to…
Disposable single-use technology
Simplification of the validation process
In-situ validation
High incidence in the production cost
Validation provided
by the supplier
Cost reduction by mass validation
during manufacturing
Disposable
single-use
technology
Disadvantages:
• Few global suppliers
• Strong technological dependence
Bioprocess simplification and intensification
Single-use bioreactor
systems
…higher challenge to
the downstream process
to intensify and improve
economy.
…improvements of recombinant clons
and cell culture formulations
What is happening with intensification in the Downstream Processing?
Beaded chromatography matrices
allows to prepared customised columns
Pre-packed columns….
www.gelifescience.com
Columns became in a black box…
…therefore it is still necessary matrices
in beaded-shape?
One of the main bottleneck of protein adsorptive materials…
Faster process` Fast
Standard matrices
Slow
Bead diameter
(200-300 mm)
Convective transport
Diffusive transport
Diffusion
mass transport driven by protein diffusion…
POROSTM
Thermofisher
Convection
Matrices non-based on beads
Faster chromatographic process FastSlow
Standard matrices
POROSTM
Thermofisher
Diffusion
CIMmultus™
BIAsepartions
Convection
Independent of molecular
diffusion
Channels
(diam 1-10 mm)
Convective transport
Diffusive transport
Convection
Matrices non-based on beadsConvective transport
Diffusive transport
Convection
…better mass transport properties
Disposable adsorptive columns will be based on monolith porous materials!!
Diffusion
Convection
Adsorptive membranes
Protein adsorptive process
Purification of proteins of industrial interest:
• Lysozyme from white egg
• Lactoferrin from whey
Hollow-fiber membranes
Why grafting polymerization?
L
L
L
L
Standard chemical monolayer modificationMacroporous membrane
Low internal surface ~15m2/g
Grafting
polymerization
20 mg/ml Lysozyme ( )
Monolayer
adsorption
Multilayer grafting
100-120 mg/ml Lysozyme
L L L
LL L
LL L
L L LL
LLL L
Multilayer
adsorption
PES membrane
PES= polyethersulfone
GMA= glycidyl methacrylate
DMAAm= dimethyl acrylamide
1 mm
Highly homogeneous membrane modification
PES-g(DMAAm-GMA) membrane
1 mm
Radiation-induced
grafting modification
Grasselli, M. et al.
Radiation Physics and Chemistry 55.2 (1999): 203-208.
PES membrane
PES-g(DMAAm-GMA) membrane
Productivity
A.M. Ventura et al., Journal of Membrane Science 321 (2008) 350–355
Lysozyme recovery from egg-white
Commercial adsorptive membranes
Mustang®
(PALL)
Sartobind®
(Sartorius)
~12 mm
Negative chromatography
is a win-win situation
(removal of minimum amount of contaminants from large volume of sample)
Adsorptive membranes
• Base material (membrane) is very expensive
• High amount of material is required
• Filtration is a big business
Current status….
Cryogels
(Porous) Monolithic columns
Others matrices non-based on beads
Reticulated foams
Cryogels
Prepared by bulk polymerization of under cryogenic temperatures
Modificado por polimerización por injerto
Capacidad media
F.M. Plieva, et al., J. Chromatogr. B 807 (2004) 129
Patented by Protista Internacional
1
Poros 40 mm diámetro
Cryogels
Cryogels
Flexible structure
Low binding capacity
Posibility to improve the capacity
by grafting polymerization
Material Capacity (mg g-1)
Cryogel SP 3 - 8
Potential applications:
•Recovery of virus and bacteria
•Recovery of eucariotic cells
Advantages
• Low back pressure
• It allows to work with particulate samples
Disadvanges
• Low adsorption capacity
• Complex synthesis (polymerization at low temp.)
• Patented by Protista International
• There are no commercial products
Cryogels
Monoliths
Frantisek Svec introduce polymeric stationary
phases
High-speed and high-efficiency analytical
columns
Continuous porous materials
Stationary phase for analytical chromatography
From microspheres to monoliths: synthesis of porous supports with tailored properties by radiation
polymerization
Grasselli, M., Smolko, E.E., Hargittai, P.and Safrany, A
Nuclear Instruments & Methods in Physics Research Section B 185 (2001) 254-261.
From microspheres to monoliths…
Bulk radiation-induced
polymerisation
Increase
monomer
concentration
Capillary electrophoresis
microextractor
In-situ
bulk radiation-induced
polymerisation
Reference:
Agarose beads have diameters in the range of 200-300 mm
Mass-transport enhacement
www.biaseparations.com
Commercial monolithic columns
Challenge:
preparation of high-volume porous columns
Advantages
• Chromatography in minute scale
• System similar to conventional chromotography
• Competitive in capacity for high MW proteins and
DNA purification
Disadvanges
• Low capacity for medium / low MW proteins
• Limitation on the preparation of large cartridges
• Limited availability of industrial products
Monolithic columns
Matrices non-based on beads
Faster chromatographic process FastSlow
Standard matricesStandard matrices
POROSTM
Thermofisher
POROSTM
Thermofisher
DiffusionDiffusion
Standard matrices
POROSTM
Thermofisher
Diffusion
CIMmultus™
BIAsepartions
Convection
Convective transport
Diffusive transport
?
x y
z
Beads vs Fibres
• Keep mechanical strength with lower diameter
• Industrial and widely available material
(cellulose fibers)
Diffusion in 3D
Bead diameter
(200-300 mm)• Reduction of the diffusion path
Diffusional
path
x y
Diffusion in 2D
Fiber diameter
(20-30 mm)
Radiation-induced
grafting modification
~ 50% poly(methacrylate) is
added into the cellulose
matrix keeping the original
shape
M. Fernandez-Lahore and M. Grasselli. "Composite material."
U.S. Patent Application No. 12/449,956.
Confocal microscopy
Sepharose SP beads Adsorptive SP fibres
IgG-Cy3 dye_
Confocal microscopy analysis
Adsorbent fibre modules
Bed volume: 150 mL
Composite cellulose fibres
Diameter: 40 mm
(hydrate)
Composite Fiber
Assembly (non-woven fabric)
‘Roll’ packing
Packing for biomass tolerance
Adsorption from crude feedstock
Column: Roll-packed column
Sample: Yeast Cells 1.5 % disrupted +
Lysozyme 5 g/L
Adsorption Elution
Artificial mixture as model process
Column: Roll-packed column
Sample: Yeast Cells 1.5 % disrupted + Lysozyme 5 g/L
Flow: 150 cm/h (5ml/min)
Sample volume: 45 ml
Buffer A: PO4 20 mM ClNa 0.1 M pH 7.5
Buffer B: PO4 20 mM ClNa 1 M pH 7.5
Lys Inj A4 A11 B8 B1
10% B
60% B
100% B
SDS-PAGE
Advantages
• Adsorption to larger flow rate to conventional columns
• Biomass tolerance
• Low working pressure
• Reduces the complexity and cost of equipment
• Capability to mass production
Disadvantages
• Intermediate adsorption capacity
• No commercial products
Adsorbent fibre modules
Polyurethane foams
Non-reticulated Reticulated
Reticulated PU foams
Photo magnifying glass
Radiation-induced
grafting modification
Sanchez, M., M. Grasselli, and L.J. Martinez. "Bi-structured matrix for solid reactants purification
and handling and methods for obtaining said matrix." U.S. Patent Application No. 15/534,872.
Hydrodynamic and adsorptive studies
Static binding capacity Dynamic binding capacity
Hydrodynamic behaviour
Exchanging mass
with stagnant zones
No plug-flow condition
is fulfilled in these
materials
Axial dispersion ….adsorptive
capacity is reduced
Advantages
• Low cost industrial material
• Adsorption process at larger flow rates than
conventional columns
• Biomass tolerance
• Very low working pressure
• Reduces the complexity and cost of equipment
Disadvantages
• Complex hydrodynamic properties
• Under development
Reticulated PU foams
Composite fibres showed the significant technical
performance and potential industrial production to
be used as disposable protein chromatography
Remarks
Thank you…
Laboratorio de
Materiales
Biotecnológicos
Acknowledgments:
Prof. M. Fernández Lahore
Current R&D topics on
materials for biotechnology
1- Disposable functional
micropipette tips
Internal Tip coating with hydrogelsInternal Tip with hidrogel onto foams
Internal coating
or foam
SiO2 nanoparticles Adsorptive hydrogel
2- Track-etched membranes
for biosensors
Membrane surface
pH 7pH 4
Nano channels
3- Protein Nanoparticles prepared
by radiation-induced crosslinkingby gamma rays/EB
Albuminsolution
Radiation synthesis of seroalbumin nanoparticles.Espinoza, S. L. S., Sánchez, M. L., Risso, V., Smolko, E. E., & Grasselli, M. Radiat. Phys. Chem., 81 (2012) 1417-1421.
Preparation of protein nanoparticle by dynamic aggregation and ionizing-induced crosslinkingAchilli, E., Casajus, G., Siri, M., Flores, C., Kadłubowski, S., Alonso, S. D. V., & Grasselli, M. (2015).Colloids and Surfaces A 486 (2015) 161-171.
gdegradation
EtOH/Water0º C
Albumin NP
g
Potential applications for veterinary vaccines
Multilayer protein coating
Au-NPs
TEM pictures
Diam ≈ 30 nm
Diam ≈ 70 nm
AFM picture
Radiation-induced preparation of core/shell gold/albumin nanoparticles.Flores, C.Y., Achilli, E. and Grasselli, M., 2018. Radiation Physics and Chemistry, 142, 60-64
Radiosynthesis of Gold/Albumin Core/shell Nanoparticles for Biomedical ApplicationsFlores, C.Y., Achilli, E. and Grasselli, M., 2017. MRS Advances, 2, 2675-2681.
4- Hybrid Nanoparticles prepared
by radiation-induced crosslinkingby gamma rays/EB
Au
Potential applications in imaging diagnosis
10 kGyg
Au/BSA-NPs