Presentation of our technology and applications in pharma/biotech and nanotech.

2. Field-flow fractionation
Sample
External field
th
ad
re
B
Thickness
Flow
External field
Parabolic flow
Thickness
Detector
v1
v3
v2

3. AF4
Flow Flow
In Out
Depletion wall
(polymer, glass)
Spacer
UF membrane
Frit (ceramic, metal)
Cross flow Accumulation
Out wall (polymer,
metal)

4. AF4: working range
molecules particles
Atom Molecule Solids
1E0 1E2 1E4 1E6 1E8 1E10 1E12
Molar Mass
1 nm 10 nm 100 nm 1 µm
Radius
-Macromolecules: proteins, protein complexes,
nucleic acids.
-Nanopartcles: viruses, virus-like particles
liposomes, lipoproteins,
protein aggregates, subcellular components.
-Microparticles: large protein aggregates, whole cells.

5. AF4: normal mode
kT
D=
6πη rh
x
Axial flow
⎛ xU x ⎞
C( x) = C0 exp⎜ −
⎜ D ⎟
⎟
⎝ ⎠
l
z
C
Cross flow F = fU
V ∝ 1/D ∝ rh
r

6. Hyphenation
AF4 retention depends on D and, then, on Mr
AF4-based Mr measurements do not account for
non-ideality effects
D Mr relationship depends on analyte conformation
Hyphenation with uncorrelated methods multiplies
the amount of analytical information
Hyphenation of AF4 with:
Multi-Angle Laser Scattering Detection (MALS)
Fluorescence Detection (FD)
Mass spectrometry (MS)

7. Size/shape characterization by AF4-MALS
MALS rg or RMS – mass average (root mean
square) distance of each point in a molecule/NP
from the molecule/NP center of gravity.
10nm – 1 P m
AF4 rh or Hydrodynamic radius – radius of a
sphere with the same diffusion coefficient of the
analyte.
1nm – 100 P m

8. Light scattering properties
1. The amount of light scattered is directly proportional to the
product of the molar mass and the sample concentration.
[The amount of light scattered (divided by the incident light
intensity) by a solution into a particular direction per unit
solid angle in excess of the amount scattered by the pure
solvent is directly proportional to the product of the
weight-average molar mass and the concentration. R(θ), in
the limit as θ→0, ∝Mc]
2. The variation of scattered light with scattering angle is
proportional to the average size of the scattering molecules.
[The variation of light scattered with respect to sin2θ/2, in the
limit as θ→0, is directly proportional to the average
molecular mean square radius. dR(θ)/dsin2θ/2 ∝ <rg2>]

9. What does rg tell about our analyte?
=∑
ri 2 mi
rg2
M
hollow sphere: rg2 = a 2
solid sphere: rg2 = 5 a 2
3
L2
Random coil polymer with average end-to-end length L: r
g
2
=
6

10. What’s the hydrodynamic radius?
Rh Rh Rh
+
_ H2O H2O
+
H2O
+ H2O
H2O

11. rg / rh: a conformational index
3-arm star polymer
solid sphere
Same rh
rg rg
ρ= = 0.77 ρ= ≈ 1.4
rh rh

12. QDs: size-dependent fluorescence
nm
S. Kim et al. Nat. Biotechnol. 2004, 22, 93

13. Biocompatible, water-soluble QDs
0.0 3.0 6.1 9.2 12.7 17.0 23.0 34.8
1000
1,0
15 nm Vis @ 533 nm
Refractive Index
Rayleigh Ratio 100
0,8
Relative Scale
rms radius (nm)
0,6 3-5 nm 10
0,4 1
3-10nm
?
0,2
0,1
0,0
0,01
0 2 4 6 8 10 12 14
Retention time (min)
(ca. 3 nm)

14. AF4-MALS-FD of water-soluble QDs

15. AF4-MALS-FD of fluorescent silica NPs
FLUORESCENCE FRACTOGRAM (λex: 325 nm; λem: 500 nm)
3,5 100
90
3,0
80
2,5
70
rms radius (nm)
2,0 60
100 nm
Fl (LU)
50
1,5
40
1,0
30
0,5 20
10
0,0
0
0 5 10 15 20 25 30
Time (min)

16. AF4-MALS-FD of fluorescent silica NPs
Unbound
BLUE + GREEN BLUE + GREEN NPs
NPs
NPs (497 nm)
Unbound (463 nm)
Elution Time (min)
λex 325 nm
BLUE > GREEN FRET
AF4-FD gives direct evidence of FRET between the tandem dyes (BLUE > GREEN)
This can be used for fine tuning of NP optical properties by changing the dyes ratio

17. Viruses
Denser inside
courtesy from MedImmune Inc.

18. Virus-like NPs
courtesy of G. Winter et al. (2007)

19. Liposomes
Liposomes or phospholipid vesicles emerged
during the past 25 years as versatile and
potent carriers for drugs and diagnostics,
both, low-molecular weight compounds and
peptide-/protein-drugs and genetic material.
The size distribution of liposomal drug
carriers is of key interest because size not
only affects the vesicle’s in-vitro
characteristics such as the amount of drug
that can be accommodated, but also its in
vivo behaviour such as circulation time in the
blood-stream upon i.v.-injection, and
consequently also biodistribution.
Schematic drawing of small unilamellar
l i p o s o m e - d r u g c a r r i e r Liposomes smaller than 70 nm can escape
with hydrophilic drug in the aqueous through the fenestrae of the liver sinus.For
compartments and lipophilic drug liposomes bigger than 200 nm, the steric
incorporated into the phospholipid bilayer shield appears to be less efficient.

20. Liposome PSD: SEC-PCS vs. AF4-MALS
Egg phosphatidylcholine (egg PC, E-80)
liposomes by Lipoid GmbH, Germany
The AF4-MALS approach has slight advantages being less time consuming, having
lower preparative effort and thus shows less sources of error than SEC-PCS. However,
AFMALS has limitations for very small liposomes. An additional online-coupling of PCS
to the MALS detector might improve the detectability of very small liposomes.
S. Hupfeld et al. J. Nanosci. Nanotechnol. 6, 1–7, 2006

21. Filled vs. unfilled liposomes
Radius versus elution time for a filled and unfilled liposome sample
160
unfilled liposome
filled liposome
RMS- Radius (nm)
120
80
40
0
0 40 80 120 160
Elution time (min)
courtesy from Wyatt Technology Europe GmbH

22. Phospholipid nanovesicles for ophtalmic use
Effect of cholesterol uptake
1.2
2
0.5 mL LipimixTM
detector voltage (V)
1.0 1 20 ng cholesterol
0.8
Effect of change in osmolarity
0.6
0.4
350
2.7 mOsm
10.0 20.0 30.0 40.0 50.0 60.0
27 mOsm time (min)
300
Root mean square radius (RMS, nm)
270 mOsm 1: native LipimixTM nanobeads
250 300 mOsm
2: higher-order structures
200
0.7 2 0.5 mL LipimixTM
150 200 ng cholesterol
detector voltage (V)
0.6
100
1
50
0.5 2
0 0.4
0 5 10 15 20 25 30 35 40 45 50
Retention time (min)
10.0 20.0 30.0 40.0 50.0 60.0
time (min)

23. Layer-by-layer coated gold NPs for blood-blain barrier drug delivery
d=15 nm
G Schneider, G Decher Nano Letters (2006), 6, 530-536
Poly Allylamine Sodium Polystyrene
Hydrochloride (PAH) Sulfonate (PSS)
MW = 15 kDa
MW = 4.3 kDa

24. AF4-MALS of multilayered gold NPs
Au-PAH/PSS (Au core: 7.5 ±1.5 nm)
50
PSS NPs
45
40
35
[
Free polymer separated
rms radius (nm)
Signal Intensity
30
from the NPs 25
20
15
10
5
Au-PAH/PSS/PAH 0
80 0 2 4 6 8 10 12 14
Retention time (min)
70
UV signal @ 230 nm ( ), MALS signal @ 90° ( )
PAH 60
rms radius (nm)
Signal Intensity
50
NPs 40
Higher state aggregation
for triple-layer NPs
30
20
10
0
0 5 10 15 20
Retention time (min)

25. F4 for analysis of protein products
F4 advantages
Wide Range of Applicability
Gentle Separation Mechanism
Broad Mobile Phase Options
F4 can be used to study high-MW
protein products under native
conditions and in formulation buffers

26. Eclipse-DAWN HELEOS of BSA
molar mass vs. time/volume
BSA 1mgmL 60uL 490um 3zu1 04[5Runs].vaf
4mer
3mer
molar mass (g/mol)
2mer
1.0x10
5
1mer
20.0 25.0 30.0 35.0
time or volume
FlFFF-UV fractogram and molar masses measured by on-line MALS

27. What makes protein drugs different?
Protein drugs differ from low molecular weight drugs in terms of
structure, source, analysis, formulation, and administration
Protein drugs can undergo a variety of degradation reactions
at the level of primary structure and higher-order structures
The stability of a protein drug very much
depends on how it is formulated
Protein aggregates are an important class of degradation products
that is difficult to tackle analytically and formulation-wise
Aggregation can lead to the formation of soluble or insoluble
aggregates, reversible or non-reversible aggregates,
covalent or non-covalent aggregates
Aggregates can vary in size from small dimers to large fibrils and be
composed of native or misfolded protein molecules

28. Protein aggregation: what’s the problem?
Despite enormous technological advances made in the production and
formulation of protein drugs, the understanding, detection, and prevention
of aggregate formation remain major pharmaceutical challenges
Aggregates not only can have a reduced potency or show different
pharmacokinetics, but also – even at extremely low aggregate levels –
can cause serious safety problems
Clinical implications of protein aggregates in a formulation are currently
largely unpredictable and likely to depend on the aggregate species

29. Protein aggregation: analytical challenges
In spite of the enormous progress made in analytical
technologies to examine the chemical and physical integrity
of protein aggregates, their full characterisation is not as yet possible
A major complicating factor in the analysis of protein aggregates
is that several aggregate types, in minute amounts, can
coexist in one formulation, yielding a heterogeneous product
The analytical challenges of studying
early-stage, low levels of aggregated protein are huge:
1. it is difficult to pick up minor fractions of aggregated species
in the presence of excess native protein
2. no single technique can detect all possible aggregates,
so complementary techniques are necessary
F4 or SEC combined with MALS are increasingly used to measure
the physical properties of protein aggregates

30. Protein aggregation is a method-dependent issue
We need the right toy…..

31. AF4-MALS of prion aggregates

32. β-Amyloid protein (Aβ) aggregation in AD
The Aβ derives by secretase
cleavage from the
transmembran, amyloid
precursor protein (APP)
Three forms of Aβ: the Aβ1-42
peptide is most hydrophobic,
most aggregating, and then
most neurotoxic form of Aβ
• Aβ1-42 is most lipophilic, and exists in two
conformations: relaxed or a-strain bundled
• Its size does not protect the inner
lipophilic part from conformational changes
• This originates self-assembling into
oligomers, protofibrils, and insoluble fibrils

33. Aggregation of an amyloid peptide: Aβ1-42
UF Aβ 1-42 CE
FILTRATED trimers-undecamers
RETAINED dodecamers
50 kDa
H y d r o d y n a m ic r a diu s
(nm)
1 4 7 10 13 17 21
0,08 500
Rh ~ 5 nm L ~ 1.5 µm
(AU)
0,06
MW ~ 60 kDa
rms radius (nm)
400
Rayleigh Ratio 0,06
2 2 0 n m
300
0,04
t0 0,04
days 200
0,02
@
0,02
100
A b s
0,00 0
0,00 0 5 10 15 20 25 30
0 2 4 6 8 10 Retention time (min)
Retention time (min)
AF4-UV AF4- MALS

34. Aβ 1-42 aggregation follow-up by AF4 and MALS/1

35. AE 1-42 aggregation follow-up by AF4 and MALS/2

36. AF4 of IgG
IgG pharmaceutical formulations in PBS
m AU
50
m AU ZOOM
3.5
3
40 2.5
2
1.5
1
30
Ab1 0.5
Ab2
0
-0.5
Ab3 -1
8 10 12 14 16 mi
20
Ab6
10 monomer dimer
0
8 10 12 14 16 m in
MONOMER DIMER

37. AF4-MALS of Abs
High molar mass
aggregates

38. Abs: IgG self-association /1
Two different IgG samples show
same molar mass but different retention

39. Antibodies: IgG self-association /2
Zoom-in: dimer retention identical in both the IgG samples
Only the monomer self-associates

40. IgG aggregation: „invisible“ and „visible“ particles
Fluorescence photomicrographs of Antibody A (from Novartis Pharma AG) dissolved in
0.1% acetic acid containing 50 mM magnesium chloride (A) and in 10 mM phosphate buffer
pH 7.1 (B). No aggregates were visible in 0.1% acetic acid containing 50 mM magnesium
chloride, even though protein concentration was high (94 mg/ml). Antibody A solution in
phosphate buffer (0.8 mg/ml) showed many spherical aggregates, with a mean
diameter of 3.18 μm
B. Demeule et al. (2007) BBA 1774:146-153

41. AF4-MALS of the IgG „invisible“ particles
Antibody A solution in 0.1% acetic acid containing 50 mM magnesium
chloride. The monomer peak at 12.5 min shows a molecular weight of 170
kDa, whereas the aggregates peak at 18.5 min exhibits molecular weight
ranging from 1 to 2 million Da. A magnified view reveals a smaller peak
that can be a dimer.

42. Pre-MS method for protein analysis
ProsCons
2D PAGE
↑Resolution, visualization, cost
↓Time, recovery, manual, off-line
FFE
↑Unlimited throughput
↓pI-based selectivity only, ampholines
CZE
↑Resolution, “nano-scale”
↓Saline buffers, on-line coupling issues
RP LC
↑ Automated, desalting, easy on-line coupling
↓Sample denaturation, adsorption, recovery
SEC
↑Size/shape-based selectivity
↓Sample entanglement
Immunoaffinity
↑ Specific protein depletion
↓Co-depletion, desalting, cost
Beads
↑Sample “equalization”, simple
↓Co-depletion, desalting

43. F4 as pre-MS step for protein analysis
Broad application range
High molecular weight proteins
Protein complexes, aggregates, organelles
Soft fractionation mechanism
Biocompatible mobile phases
Preservation of native conditions
Evaluation of D
Independent Mr determination
MS-compatible mobile phases
No ionization suppression

44. AF4 with nanoLC-ESI/MSMS for proteomics
1.0
Relative Rayleigh ratio
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80
Retention time (min)
Enzymatic hydrolysis
(trypsin)
nanoLC – ESI/MSMS

45. Fraction collection from AF4
LDL VLDL
1.0
7 8
UV (280 nm)
Relative Intensity
0.8 Rayleigh Ratio
0.6
0.4
0.2
0.0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Retention time (min)
1.0 113000
80000
Relative Intensity
0.8 HDL +
51800
0.6
HAP 34700
30000
0.4
22000
0.2
0.0
0 1 2 3 4 5 6 7 8
Retention time (min)
SDS-PAGE

46. Molar mass distribution in the fractions
8
Increasing retention time
7
Fraction number
6 Lower-Mr
5 components
found in the
4 fractions of
3 higher-Mr
components
2
1
0
10 100 1000 10000
Molar mass (KDa)

47. Protein identification in VLDL fraction
1.0
Relative Intensity
8
8 UV (280 nm)
0.8 Rayleigh Ratio
0.6
0.4
0.2
0.0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Retention time (min)
NAME ACC NUM SCORE MASS
Serum albumin precursor P02768 1178 71317
Keratin, type II cytoskeletal 1 P04264 800 66018
Complement C3 precursor P01024 281 188585
Keratin, type I cytoskeletal 10 P13645 268 59711
Keratin, type II cytoskeletal 2 epidermal P35908 265 66111
Ig kappa chain C region P01834 255 11773
Dermcidin precursor P81605 221 11391
Alpha-2-macroglobulin precursor P01023 194 164600
Transthyretin precursor P02766 191 15991
Ig gamma-1 chain C region P01857 180 36596
Prothrombin precursor P00734 170 71475
Alpha-1-antitrypsin precursor P01009 153 46878
Apolipoprotein C-II precursor P02655 148 11277
AMBP protein precursor P02760 137 39886
Apolipoprotein A-II precursor P02652 136 11282
Keratin, type II cytoskeletal 6A P02538 123 60162
Ig lambda chain C regions P01842 119 11401
Alpha-1-acid glycoprotein 1 precursor P02763 108 23725
Haptoglobin precursor P00738 100 45861
Apolipoprotein A-I precursor P02647 98 30759
Inter-alpha-trypsin inhibitor heavy chain H4 precursorQ14624 96 103489
Ig kappa chain V-II region Cum P01614 93 12782
Protein S100-A7 P31151 91 11433
Beta-2-glycoprotein 1 precursor (Apolipoprotein H) P02749 89 39584
Apolipoprotein C-III precursor P02656 85 10846
Ceruloplasmin precursor P00450 85 122983
Alpha-1-antichymotrypsin precursor P01011 64 47792
Apolipoprotein E precursor P02649 63 36246
Nuclear mitotic apparatus protein 1 Q14980 41 239214
Myeloid/lymphoid or mixed-lineage leukemia protein O14686 40 570046
Ig alpha-1 chain C region P01876 38 38486
Abnormal spindle-like microcephaly-associated prote Q8IZT6 37 413192
Immunoglobulin J chain P01591 36 16041
Development and differentiation-enhancing factor 2 O43150 35 112835
Sodium/potassium-transporting ATPase alpha-2 cha P50993 32 113505
Apolipoprotein C-I precursor P02654 32 9326
Calmodulin-like protein 5 Q9NZT1 31 15911

48. Interactomic networks in VLDL fraction
Fraction 8 8
Fraction
Dermcidin precursor
Dermcidin precursor Prothrombin precursor
Prothrombin precursor
Apolipoprotein A-II precursor
Apolipoprotein A-II precursor Keratin, ,type IIII cytoskeletal6A
Keratin, type cytoskeletal 6A
Transthyretin precursor
Transthyretin precursor
Apolipoprotein C-I precursor
C-I precursor
Apolipoprotein
Keratin, type II cytoskeletal 1
Keratin, type II cytoskeletal 1
Apolipoprotein A-I precursor
Apolipoproteinprecursor
A-I
HSA precursor
Serum albumin precursor
Apolipoprotein C-III
Apolipoprotein precursor
C-III precursor
Igalpha chainC region
-1
Haptoglobin precursor
Haptoglobin precursor
Keratin, type II cytoskeletal 10
Keratin, type cytoskeletal 10 IgIg gamma-1 chain C region
gamma-1 chain C region
Ig kappa chain C region
Ig kappa chain C region
Ig lambda chain C regions
Apolipoprotein C-IIprecursor
C-IIprecursor
Apolipoprotein

49. Hollow-fiber FlFFF (HF5)
The HF5 cross-flow is generated by the elution flow, which splits
into a longitudinal and a radial direction: no depletion wall, only
accumulation wall
Cross-flow
outlet Hollow Fiber
r
rf
Channel Vin z Vout
sleeve
Cross-flow
Inlet connection Tee connection Outlet connection
(from injector) (to detector)

50. HF5: prototype channel
1/8”
PE fitting
1/8”
1/8”
1/8” PEEK Tee
Teflon tube
cPVC / PSf 1/8”
1/8” HF membrane PEEK ferrule
PEEK ferrule 24x0.08 ID cm
PEEK Nut (1/8”)
Hollow Fiber
Ferrule (1/8”)
Teflon sleeve Union Tee
(1/8”)

51. HF5: advantages
– Potentially disposable
No risks of run-to-run sample carry-over
– No memory effects when coupled with other techniques
Reduced sterility issues
– Easier work with biological samples
– Low channel volume
Low sample dilution
– High detection sensitivity
Short analysis time
– Highly suitable to hyphenation

52. F4MS for protein analysis
HF5 of BSA:
1
fractionation of
oligomers
1: monomer
2: dimer
3: trimer
4: tetramer
2
3 4
+50
+45
100
100
M = 66397.8
+55
+40 (monomer mass)
% %
+36
+60
0 mass
mass
56000 58000 60000 62000 64000 66000 68000 70000 72000 74000 76000 78000 80000
0 m/z
1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850
ESI MS multicharge spectrum ESI MS mass spectrum