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