Novel 3D Nano-template Approach to
Protein Crystallization
Daryl R Williams
Surfaces and Particle Engineering Laboratory (SPEL)
Department of Chemical Engineering
Imperial College London
South Kensington Campus, London SW7 2AZ
Email: d.r.williams@imperial.ac.uk
Web: www.imperial.ac.uk/spel
3D Nanotemplates –
A Step Forward in Developing Crystallisation as
a Tool for Purification of Biopharmaceuticals
Today’s Outline
Why is protein crystallisation
relevant in bio-processing?
What do we know about it so far?
Where is the challenge and what
is the novelty in our approach?
What did we find so far and what
are the conclusions made?
How do our findings affect the
wider academic audience and
industry?
Approval Year
2010
2009
2008
2007
2006
2005
2004
2003
2002
2001
2000
1999
1998
1997
1996
1995
1994
1993
1992
1991
1990
1989
1988
1987
Recombinant
1986
1985
1984
400
1983
1982
Cumulative Approval of Bio-Pharmaceuticals
BioPharmaceuticals in Development
450
Non-Recombinant
350
300
250
200
150
100
50
0
BioPharmaceuticals - Current Challenges
Scope: > 6000 biopharmaceutical products in pipeline ($XXX billion)
Challenges :
o Developing understanding of solution aggregation/ denaturation mechanisms
o Enhanced product stability and bioavailability
o Reliable method for purification and bioseparation
o High end-product cost- partially manufacturing costs
o Alternate processes to chromatographic processes
o Different product options for product delivery
Current Status of Protein Crystallisation
Scope:
180,000 proteins prepared awaiting structure determination
Success So-far:
Crystal structure determined for only 2% of cloned proteins
Challenge:
Understanding and Controlling Protein Nucleation
Obtain Diffraction quality Crystal
Develop biomanufacture process
Manufacture:
Only one biopharmaceutical currently crystallized - insulin
What are the Benefits of a Protein Crystallisation Process in
Biopharmaceutical Manufacture?
Isolation and Purification
Sustained Release (Stability)
Handling/Processing/Delivery (Formulation)
Engineering to Suit Purpose
Background –Effect of Different Surfaces in Controlling Nucleation
Effect of Different Surfaces
Crystallised
Minerals
(McPherson et
al., 1988)
Natural Proteins – Hair
(Abrahams et al., 2007)
Surface Roughness
(Curcio et al., 2010)
Effect of Surface Porosity
Bioactive Glass with
disordered porosity
(Saridakis et al., 2006)
CNT based material
Layered Silicates
(Asanithi
et al., 2009)
(Kengo et al., 2008)
Effect of Surface Chemistry
Preferred orientation of
crystal (Tsekova et al.,
1999) (Matzger et al.,
2011)
Porous Surface
Flat (Non-porous)
Surface
Simulation based studies explaining
effects of surface porosity on
crystallisation
(van Meel et al., 2010)
Polystyrene beads as
nucleant
(Kallio et al., 2009)
Effect on crystal density
(Tosi et al., 2008)
Surface selective
crystallisation of protein
crystal polymorphs (Matzger
et al., 2011)
The Challenge/ Drawbacks
Insufficient
knowledge of
protein crystal
nucleation.
Poor scientific
understanding on
the effect of
heterogeneous
nucleants.
The
Challenge/
Drawbacks
No Correlation between nucleant
surface physicochemical properties and
protein surface which allows systematic
design of nucleants for targeted protein
crystallisation.
3D Nanotemplates – Controlled Topography and Surface Chemistry
Surfaces with Controlled Surface Porosity and Surface Chemistry
Inter-Pore Distance
Inter-Pore
Distance
Pore Diameter
Pore
Height
Pore Diameter
3D Nanotemplate
Design and Synthesis of Novel 3D Nanotemplates
Surfaces with Controlled Surface Porosity and Surface Chemistry
16.0 3.0nm
6
F127 w TMB
F127 w Xylene
22.0 5.0nm
5.5 1.5nm
5
3.5 1.0nm
P123 w/o swelling agent
11.0 3.0nm
F127 w/o swelling agent
nano-porous glass w/o
sacrif icial template
Pore Volume (cm³/g Å)
4
3
2
1
0
10
100
1000
Pore Diameter (Å)
Crystallisation of Proteins on 3D Nanotemplates
Surface Preferential Crystallisation of Proteins
450kDa
(a)
(b)
232kDa
24 Sub-Unit
Complex
Trypsin (22kDa) (3.5±1.0nm)
106kDa
Ferritin
(450kDa)
Ferritin
(450kDa)
(17.0±5.0nm)
(22.0±5.0nm)
Con A (106kDa) (11.0±3.0nm)
4(b)Sub-Unit
(Tetramer)
(a)
(c)
67kDa
Trypsin (22kDa) (3.5±1.0nm)
Con A
A (106kDa)
(106kDa) (11.0±3.0nm)
(11.0±3.0nm)
Con
Ferriti
Single Sub-Unit
14 – 24kDa
Protein Molecular Weight
(c)
Trypsin (22kDa)
(22kDa) (3.5.0±1.0nm)
(3.5±1.0nm) Con A (106
Trypsin
(scale bar 200m except thaumatin)
Nucleant Pore Diameter Resulted in Preferential Crystallisation
3.5±1.0nm
5.5±1.5nm
11.0±3.0nm
22.0±5.0nm
Shah, U.V., Williams, D.R., Heng,
J.Y.Y., Crystal Growth & Design (2012)
12(3) 1362-1369
Surface Preferential Crystallisation – What co-relates to Pore
Diameter?:
RH
o Hydrodynamic Radius is a radius of the
molecules considering the effect of
solvent (Hydro) and shape of molecule
(Dynamics).
RR
Rg
Comparison of hydrodynamic radius (RH) to other
radii for proteins
Rg : Radius of Gyration
RM: Hypothetical radius for a hard sphere with the
same mass and density as protein molecule
RR: Radius established by rotating the protein
about the geometric centre.
% Volume
30
25
Lysozyme
20
Concanavalin A
15
10
5
0
0
5
10
15
20
Hdrodynamic Radius (nm)
25
Representative results of hydrodynamic size
measurements of proteins under crystallisation conditions
Experimental Validation of the Relationship Proposed
Crystallisation of Proteins – Effect of Specific Surface Porosity
A – Lysozyme + No PEG 4k
B
60
Lysozyme (w\o PEG-4000)
A
Lysozyme (1% PEG-4000)
C
50
(a)
(b)
First crystal observed
only on 3.5±1.0nm
Lysozyme (10% PEG-4000)
40
% Volume
(c)
(d)
B – Lysozyme + 1% (w/v) PEG 4k
30
(a)
(b)
First crystals observed
only on 5.5±1.5nm
20
10
B – Lysozyme + 10% (w/v) PEG 4k
0
0
1
2
3
4
5
6
Hdrodynamic Radius (nm)
7
8(a)
9
Hydrodynamic radius measurements for lysozyme + (1 or 10% (w/v) PEG)
in lysozyme crystallisation conditions
10
(b)
First crystals observed
only on 11.0±3.0nm
Relationship Between Protein Hydrodynamic Diameter and
Nucleant Pore Diameter resulting Preferential Crystallisation
Crystallisation of Proteins – Effect of Specific Surface Porosity
Ferritin (450kDa)
Protein (Molecular Weight)
Catalase (232kDa)
Concanavalin A
(106kDa)
Albumin (67kDa)
Typsin (24kDa)
3D Nanotemplate Pore Diameter
Thaumatin (22kDa)
Protein hydrodynamic diameter
Lysozyme (14kDa)
0
5
10
15
20
25
Pore Diameter/ Protein Hydrodynamic Diameter (nm)
30
Crystal at Protein Concentration Obtained from Bio-Reactors
13-15nm
Crystallisation at Hereto -Lowest Supersaturation (50 – 92%)
Any other heterogeneous nucleants
3D Nanotemplate
Catalase (232kDa)
Protein Systems
54% –NH2
70% –CH3
Concanavalin A
(106kDa)
Albumin (67kDa)
92% –OH
50% –OH
Lysozyme (14kDa)
1
10
100
Protein Concentration (mg/ml)
1000
Crystallisation from Protein Mixture on 3D Nanotemplates
Preferential Crystallisation of target molecule prepared from the same
source and under same crystallisation conditions.
(a)
40
RNAse
35
Lipase
Volume (%)
30
25
20
15
10
Lipase crystals on 3D
Nanotemplate with pore
diameter 5.5±1.5nm
5
0
0
2
4
6
Hydrodynamic Radius (nm)
8
10
dh-b
dh-a
ϕ
Hydrodynamic Radius of RNAse and
Lipase measured using DLS
Selective
Crystallization,
when ϕ ≈ dh
RNAse crystals on 3D
Nanotemplate with pore diameter
3.5±1.0nm
(scale bar 150m)
Shah, U.V., Jahn, N.H., Williams, D.R., Heng, J.Y.Y.,
Journal of Crystal Growth (2013) Manuscript in
Preparation
Surface Preferential Crystallisation of Different Crystal Habits on surfaces
of 3D Nanotemplates:
(a)
(c)
Crystallisation of different crystal habits for
proteins
(b)
(d)
Crystallisation of Concanavalin A on surfaces functionalised with (a) phenyl (b) dodecyl (c) chloro
(d) control surface (scale: 200m)
Inset images represents schematic 3D representation of crystal habits – Concanavalin-A (b) Round
Shape Rhombic Tetrahedrons (c) Smooth Faced Crystals(d) habit type-I
Shah, U.V., Williams, D.R., Heng, J.Y.Y., Crystal Growth
& Design (2013) Manuscript in preparation
(a)
(c)
(b)
(d)
Crystallisation of Catalase on surfaces functionalised with (a) phenyl (b) dodecyl (c) chloro (d)
control surface (scale: 200m)
Proteins Crystallisation – A 3D Nanotemplate Approach
Preferential
Crystallisation
3D- Nanotemplates
Trypsin (22kDa) (3.5±1.0nm)
Con A (106kDa) (11.0±3.0nm)
Ferritin (450kDa) (17.0±5.0nm) Trypsin (22kDa) (3.5±1.0nm)
Lowest
Concentration
Protein Purification
dh-b
dh-a
ϕ
Selective
Crystallization,
when ϕ ≈ dh
Co
Key Messages
o Correlation between Protein Hydrodynamic Diameter and
Nucleant Surface Pore Diameter
o Rational design of pore for crystallizing specific protein of
interest
o Crystallisation at Protein Concentration Achievable from
Bio-Reactors
o Target Protein Molecule Crystallisation from Protein
Mixture
Industrial Impacts of Findings:
o Protein molecules crystallised at lowest ever achieved concentrations –
High Efficiency.
o Systematic rational strategy for crystallisation – applicable to protein
purification and protein structural determination.
o Solid state dosage forms which are crystalline, alternate delivery options
and improved product stability.
o Potential for habit
nanonucleants.
and polymorph
discovery
/
control using
o Have crystallised >12 different proteins varying in MW from ~10kDa to
~500kDa
o Currently working on crystallisation of MABs using these same methods
Acknowledgements
Imperial College – Jerry Heng, PF Luckham, MR Roberts, D Tsekova
University of Surrey - JF Watts and S Hinder
King’s College London – J Lawrence, L Kudsiova
Team SPEL
Especially – Umang Shah, M Allenby, C Hayles-Hahn, S Huang,
N Jahn, T Lapidot, GD Wang, Y Wang – Colleagues
THANK YOU!
Thank You
Additional Slides
SPEL’s Templates – 3D Nanotemplates – Controlled Surface Chemistry
Functionality
-OH group
- NH2 group
- Cl group
- C6H5 group
- CF3 group
- CH2CH3 group
θ
11.30 2.80
33.20 4.10
82.60 3.50
109.10 4.90
118.40 4.60
128.60 3.20
Our Approach
Preparation of nucleant
with controlled surface
properties to isolate its
influence of nucleation
and crystallisation
Crystallisation of
biological macromolecule
varying in MW, sub-units,
size on surfaces prepared
Utilise understanding
developed to engineer
surfaces with required
surface properties to
crystallise target
proteins
Understanding physicochemical co-relation
between tuned surface
properties and biological
macromolecule
Preparation of Nano-Porous Glass and Surface Functionalisation:
Preparation of nanoporous surface with
tuned surface porosity and chemistry
Synthesis of Anodised
Alumina Oxide (AAO)
with method reported by
Muller et al.
Preparation of
Nanoporous glass with
Sol-gel method
Tuning porosity
of nanoporous
glass by altering
catalyst molar
ratio
Control porosity
using sacrificial
templates –
surfactant, cosurfactant,
swelling agent
etc.
Controlling the
porosity by
changing
different
electrolyte
Silanisation of Bioactive
glass with different
chemistries
Synthesis of AAO
with controlled
aspect ratio
Coating the AAO
with silica layer
Silanisation of AAO
templates with different
chemistries
Crystallisation of different
complex proteins
Engineering
surfaces for
crystallisation of
target proteins, on
basis of
understanding
developed
Tuning interpore distance by
altering
anodisation
voltage
Understand the
effect of surface
morphology and
surface chemistry
on crystallisatio of
proteins
Schematic describing nano-porous
glass synthesis protocol adopted
Functional
Group
CH3
(Dodecyl)
C6H6
(Phenyl)
Cl
(Chloro)
I
(Iodo)
Silane Reagent
NH2
(Amino)
(3-Aminoproply)
triethoxysilane
Dodecyltriethoxy silane
Triethoxyphenylsilane
Dichlorodimethylsilane
(3-Iodopropyl)
trimethoxysilane
Schematic describing
silanisation protocol adopted
Pore formation Mechanism:
Two different strategies for formation of
mesoporous (A) cooperative self assembly
(B) Liquid Crystal templating methods
Micropore volume (spheres) and mean micropore
diameter (triangle) determined by nitrogen
adsorption for xerogel samples as a function of
sol synthesis pH (Meixner et al., 1999)
Silanisation Mechanism:
Silane Reagent
Dodecyltriethoxy silane (Sigma Aldrich, UK) (Cat No.
44237)
Triethoxyphenylsilane (Fluka Analytical, Germany)
(Cat No. 79223)
Dichlorodimethylsilane (Sigma Aldrich, UK) (Cat No.
440272)
(3-Iodopropyl) trimethoxysilane (Sigma Aldrich, UK)
(Cat No. 58035)
Silanisation of Glass with (3- mercapto propyl) tri-methoxy silane
(3-MPTS) in
i.The reactive groups of physisorbed silane are hydrolysed by the
surface water on a hydrated silanol (glass) surface,
ii.followed by condensation which leaves the silane covalently bound
to the oxide surface,
iii.Thermal curing of the film, which, in cross-linking the free silanol
groups, reduces the effect of hydrolysis of one or more of the siloxane
linkages to the surface
(3-Aminoproply) trimethoxysilane (Sigma Aldrich,
UK) (Cat No. 281778)
Crystallisation of Proteins – A Method:
Preparation of Precipitant Solution
Flow chart of method for setting up
experiments in typical hanging drop vapour
diffusion method
Schematic of Protein
Crystallisation Protocol
Name of
Protein
Molar
Weight
(kDa)
Thaumatin
22
Trypsin
24
Concanavalin
A
106
Catalase
232
Buffer
Solution
(Solvent
water)
50 mM PIPES
pH 6.8
100 mM Tris
pH 8.4
10 mM Tris pH
8.5;
20 mM CaCl2;
20 mM MnCl2
100 mM Tris
pH 8.4
Precipitant
Solution
340 mM
Na- K Tartrate
30-32% (w/v)
(NH4)2SO4
1M (NH4)2SO4
in 20 mm Tris
pH 8.0
5% (w/v) PEG
4K,;
5% (v/v) MPD
Final
Protein
C
(mg/ml)
2.0 - 11.5
12.0
1.5 – 17.5
Preparation of Protein Solution
Trypsin
32% (w/w) Solution of Ammonium
Sulphate in 100 mM Tris Buffer
(pH 8.4)
Trypsin
20.0 mg/ ml of Trypsin from
porcine pancreas in 100 nM Tris
Buffer
(pH 8.4)
Thaumatin
0.34M Solution of Sodium
Potassium Tartrate in 50 mM Pipes
Buffer (pH 6.8)
Thaumatin
2.0 – 12.5 mg/ml Thaumatin from
Thaumatococcus daniellii in 50mM
Pipes Buffer (pH 6.8)
Catalase
5% (w/v) PEG 6K, 5% (v/v)
Solution of MPD in 100 mM Tris
Buffer (pH 7.5)
Catalase
16 mg/ml Catalase from bovine
lever in 100 mM Tris Buffer
(pH 7.5)
Concanavalin A
1.0 M Solution of Ammonium
Sulphate in 20mM Tris Buffer
(pH 8.0)
Concanavalin A
15 mg/ml Concanavalin A from
Jack bean in 20 mM CaCl2;
20 mM MnCl2 prepared in 10mM
Tris Buffer (pH 8.5)
Filteration
The precipitant solution and protein solution
are filtered using 0.22µm filter (Ultrafree-MC,
Millipore,USA) before any further
experiments.
Protein Crstallisation
Crystallisation trials are carried out at 18 0C
except for Concanavalin A (4 0C) by sitting
drop and hanging drop vapour diffusion
technique using well tissue culture tray
Hanging Drop
The well is filled with 750 µl
precipitant solution. The 2µl
mixture (1:1(v/v)) of the protein
solution and the precipitant
solution is applied on the surface of
the control and surface modified
samples and using this samples
the well is sealed and maintained
at 40C.
Sitting Drop
Control and surface modified
samples are placed in the well. The
well is applied with 400µl (1:1 (v/v))
mixture of the protein and
precipitant solution. The well is
sealed with the wax maintained at
4 0C.
11.5
Characterisation
The process of crystallisation is
monitored using the Olympus
Microscope Camera System
Crystallisation Mechanism Slides
Hydrodynamic Radii and Mechanism of Nucleation :
o Thermodynamics:
Free energy barrier of nucleation as a function of spherical radii
4/3𝜋𝑟 3
∆𝐺 = −
∆𝜇 + 4𝜋𝑟 2 𝛼
𝑣
Differentiating the equation above with respect to r and set it to zero
𝑟 ∗ = 2𝑣𝛼/∆𝜇
Here, ∆𝜇 = 𝑘𝑇𝑙𝑛(𝑆) where, S is the ratio of actual solution concentration to
the concentration of solution at equilibrium. It can be observed from this
equation that critical nucleus radii is an inverse function of supersaturation
Replacing the value for kT in the equation above from the Stoke Einstein
equation
𝑟 ∗ = 2𝑣𝛼/𝑘𝑇𝑙𝑛(𝑆)
𝑟 ∗ = 2𝑣𝛼/𝑟𝐻 6𝜋𝜂𝐷𝑙𝑛(𝑆)
where, rH is hydrodynamic radii, η is viscosity and D is diffusion coefficient.
Hydrodynamic Radii and Nucleation – Two Step Perspective:
•
•
o
o
o
The size of a high-density
cluster (be it solid or liquid like)
can be defined as the number of
connected particles, Np
The number of solid like
particles belonging to a given
crystal nucleus by Ncrys
For proteins or colloidal solutions concentration difference
can be parameter to distinguish between dilute solution and
dense liquid, where as structure is the deciding factor
between the dense liquid and crystal.
Crystallisation of proteins from solution can transit along
two parameters, i.e. concentration and structure.
Formation of crystals from the dilute solution follows path
in which first formation of dense liquid phase from the
dilute liquid takes place (difference in concentration) and
from the dense liquid crystalline nucleus formed within the
droplet (Difference in Structure).
Hydrodynamic Radii and Nucleation – Role of Porous Nucleant:
o Considering two step nucleation mechanism, crystal formation is from high dense
protein/ colloidal solution.
o The nucleation barrier for formation of such high dense phase is lowered by the
interaction with the surface functional groups.
o For such droplets protein concentration is considerably higher than rest of droplet
hence the supersaturation (S) is also higher.
o As supersaturation is considerably higher, the critical nucleus size required for stable
nucleus formation can be as low as single molecule. (r* ∞ 1/ln(S)).
o As the protein crystals are hydrates and solvates, the hydrodynamic radii of the
solution is important
Role of Porous Substrate:
o If pores are tuned of the same size of hydrodynamic
diameter of crystallisation solution (solution in droplet), it
can not only form nuclei within also stabilise it as well due
to local immobilisation of protein molecule with in the pores.
o Diffusion of protein solution within the pores follows the
capillary rise, which can be illustrated by Figure.
Controlling Crystal Density and Habits – Effect of surface chemistry –
Crystal Density
o Effect of surface chemistry of the templates on controlling crystal size and crystal
density.
Ferritin Crystal
Thaumatin Crystal
Tsekova, Daniela S., Heng, Jerry Y. Y., Williams, Daryl R. Chemical Engineering Science (2012) Manuscript Accepted,
doi: 10.1016/j.ces.2012.01.049