Analysis of Polymers and

Protein Nanoparticles
using Asymmetrical Flow
Field-Flow Fraotionation (AF4)
Stephan Schuttes, Kathrin Mathis, Jan Zillies, Klaus Zwiorek, Conrad Coester and Gerhard Winter,
Ludwig Maximilians University, Department of Pharmacy, Pharmaceutical Technoîogy and Biopharmaceutics, Munich, Germany.

Gelatin nanoparticles are used as a novel, biodegradable and well-tolerated drug carrier in tumour
therapy and numerous other diseases. For a valid formulation approach in clinical studies an in-depth
understanding of the molecular weight characteristics of this biopolymer is essential. Asymmetrical
Flow Field Flow Fractionation (AF4) analysis was shown to be a fast and reliable analysis method for
the pre-formulation screening of standard hydrophilic and hydrophobic prototype gelatins and also for
mucoadhesive thiomers.

Polymer and protein-based drugs are becoming more
common in the pharmaceutical industry and asymmetrical
fiow field-flow fractionation (AF4) offers a number of
advantages over existing techniques, particularly for
formulations containing polymers and nanoparticles. The
interest in AF4 has grown since Giddings' break-through work
in the nineties' and the subsequent research by Fraunhofer^
has helped the technique gain wider acceptance and use.
By combining this technique with muitiangle light-scattering
detectors (MALS), polymeric active ingredients can now
be separated from polymeric excipients and particulate
structures such as nancparticles or virus-like particies
Expert knowiedge and practical expertise in this technique
is, however, somewhat iimited, but the advantages of AF4 are
becoming more widely accepted. This article describes novel
work in polymer and nanoparticle research and highlights the
importance and potential of AF4 in this emerging field.
In practice established methods for protein and polymer
analysis, such as reversed-phase high performance liquid
chromatography (RP HPLC). size exclusion chromatography
(SEC) and fluorimetry are often limited (e.g., shear force
destruction of protein aggregates in stability studies due
to the presence of the stationary phase or the need for
fluorometric moieties in the sample). A typical packed column
for these techniques requires high pressure — but this is not
the case for AF4.
The separation principle of AF4, however, is based on a
differentiai flow of a solvent in a channel and a respective
perpendicular cross-flow to separate the analytes only by
hydrodynamic properties in a fast, non-destructive,
one-phase separation mechanism. One phase, non-destructive
separation ensures very gentle separation conditions for
iarge biomolecute analysis and aggregate detection, a rapid
cost-effective analysis time and less unwanted interactions
because of the small surface area of the channel compared
with packed columns. Additionally, the smallest molecules
can be analysed simultaneously with iarge proteins,
aggregates or nanoparticles, which rneans AF4 offers a wider
dynamic range than other Chromatographie methods.

390 LC'GC Europa September 2009

 Schuttes et al.
Figure 1 shows a schematic diagram of this separation
channel. In theory macromolecules between 1 kDa and
several GDa or particles between 2 nm and 100 pm can be
separated by AF4.^
Nanoparticles have the potentiai to offer numerous
advantages for targeted, safe and effective drug delivery,^''
especially when made of biocompatible and biodegradable
polymers. Such nanoparticles could be useful for the targeted
delivery of drugs to improve bioavailability, sustain drug
effects at the target site, solubilize drugs for intravascular
delivery arid improve the stability of therapeutic agents against
enzymatic degradation.^ Additionally, by modulating the
polymer characteristics, the release of a therapeutic agent
from nanoparticles can be controiled to achieve the desired
therapeutic efficacy.^
Gelatin nanoparticles, for example, are a promising
drug carrier system for future applications in various new
approaches in gene therapy.''° Innovative gelatin prototypes
with modifications of the backbone offer a further improvement
regarding enhanced drug loading and modified targeting
properties in vivo.
The aim of this investigation was to characterize the moiar
mass profile of two newly modified gelatin prototypes by AF4
and compare them with standard geiatin and fractionated
geiatin, where low molecular weight (Imw) gelatin was
Figure 1 : Schematic drawing of an AF4 channel.
Spacer
Filtration
Membrane
Frit
Crossflow
Figure 2: Molecular weight distribulion of standard Sigma gelatin. UV (curve) and MALS (dotted line) signal.
1.0E+10 -1
ro 1.0E + 08 -
.^ 1.OE+06 -
i -0.6 's
-| 1.OE + 04 -
o -0.4 S
i 1.OE+02 -
1. Ô do
Time (min)
392
removed from high molecular weight (hmw) gelatin. A thorough
knowledge of the molar mass profiles ¡n these highly
Imw-reduced samples and in the prototype samples is
mandatory for all nanoparticle formulation steps from gelatin
as weil as from other polymers such as chitosan. Hence, we
also examined the molecular weight fractions of highly different
chitosan-thiomer batches modified in two ways with sulphydryl
groups and prompting different viscosities and chain lengths.
In the course of method development we determined the
optimum settings in terms of optical detector parameters as
well as the correct type of membrane and elution profile for the
separation of hydrophobic large proteins.
With this data, an improved formulation process for
nanoparticles was developed based on the traditional
desolvation method developed by Coester."
KEY POINTS
• New developments in the fields of liposomes, cells,
bacteria and especially antibodies will increase the
demand for a fast and reliable analysis method such as
AF4 particularly when the standard separation methods
for polymeric and colloidal analytes reach their limits.
• Hydrophobically modified large proteins can be sized
by AF4.
• The analysis of gelatin bulk material by the combination
of asymmetrical flow field-flow fractionation and
muitiangle light scattering was accomplished in
continuation of earlier studies from Fraunhofer.
• Prototype hydrophobic gelatin nanoparticles were
prepared with altered physicochemical surface
properties for targeted drug delivery to the brain.
• Modern automatic microviscosimetry was used to
determine molecular weights of polymers and compare
¡=i the results with AF4.
p1.2
-10 §
-0.8 f
03.
-0.2 I
-0,0
40
LC«GC Europe Septer^ber 2005
 Schultes et al.

The physicochemical characterization of all nanoparticies A (Bloom -175) from porcine skin (Sigma Aldrich, Munich,
was done by static tight scattering {SLS) and ccmpared with Germany) was analysed in this study. As control experiments
established gelatin nanoparticle formulations in terms of size, in turn, measurements with two customized Gelita batches
zeta potential and polydispersity index (PI). (VP306/VP413-2) that possessed less than 20% (w/w)
peptides < 65 kDa were conducted. The AF4 analysis of
Experimental these gelatins was performed on an AFIOOO-FOCUS system
AF4 measurements of gelatins: As well as two (Postnova, Landsberg am Lech, Germany) coupled with a UV
hydrophobicatly modified gelatin prototypes (Gelita. detector (UV100 Thermo Scientific, Egelsbach, Germany),
Eberbach, Germany} called MS and MA, with succinate an Rl detector (n-1000 WGE Dr. Bures, Germany) for
and dodecenylsuccinate residues, standard gelatin type concentration detection and a static light-scattering detector
(miniDAWN Wyatt Technology Corporation, Dernbach,
Germany) for molecular weight determination. The laser
Table 1; Induence of the different membrane types on recovery wavelength accounted for 690 nm, while slice collection was
rale and repeatability (defined as the intra day repeatability of set to 1200. For molar mass determination the refractive index
a 100% value in per cent of six replicates). (Rl) increment was set to 0.174 ml/g and the second virial
Membrane and cut-off Recovery Repeatability coefficient was set tc 0. The separation was achieved using
Regenerated cellulose (5 kDa) 92.3% 97.4% a PBS buffer pH 6.0 as the mobile phase, a channel with
350 \}m height and an ultrafiltration membrane consisting of
Regenerated cellulose (10 kDa) 95.7% 98.5%
regenerated cellulose with 5 kDa cut-off (Postnova), All proteins
Nitrocellulose (5 kDa) 86,5% 91.9% were dissolved in analysis buffer at a concentration of 2.5%,

Figure 3: UV signal (continuous line) and molecular weight (dots) calculated from respective UV and MALS data resulting from AF4
analysis of gelatin bulk material VP413-2 developed and provided from Geflta; the circle marks !he low molecular-weight fraction.

1.0E4-10 -.

g 1,0E+08 -1.0

-0.8
.ra I.OE-t-06
-0.6
4 1,0E + 04 -
-0,4
1.0E+02 -
-0.2 w

1.0E + 00 -0.0
10 40
Time (min)

Figure 4: Mean molecular weight fractions calculated from respective UVand MALS data resulting from AF4 analysis of (1) gelatin
bulk material purchased from Sigma-Aldrich, (2) gelatin bulk material VP306. (3) VP413-2 developed and provided from Gelita. and
of (4) gelatin sediment obtained after the first desolvation step from the manufacturing process of the gelatin nanoparticles.

5,0 ^
trad

4.0 -

£
3,0 -
lal 1

2.0 -
c
0
ftei

1.0 -
Q

0.0
1.00E + 04 1.00E + 05 1,00E+06 1.00E + 07 1 OOE + 08 1.00E + 09
Molecular weight (Da)

394 LC*GC Europe September 20Q9

 Schultes et al.

The channel flow-rate accounted for 1 mL/min, while the
cross flow was adjusted to 0,05 mL/min over 10 min and then
reduced to 0 mL/min, which resulted in a totai measurement
period of 20 mm.
AF4 measurements of chitosans: The modification of
the chitosan materials (Sigma) was conducted according
to protocols described elsewhere.'''^ Chitosan low viscosity
modified with W-acetylcystelne (Sigma) (lyophilized);
chitosan low viscosity modified with thiobutylamidine (Sigma)
(lyophilized); chitosan low molecular weight modified with
A/-acetylcysteine (lyophilized) and chitosan low molecular-
weight modified with thiobutylamidine (lyophilized) were
investigated with the same AF4 hardware set-up as for
gelatin. The solvent and the running buffer for the chitosan
samples were made of 0.3 M acetic acid, 0.2 M sodium
acetate and sodium hydroxide/hydrochloric acid q.s. The
pH was adjusted to 4 at a chitosan concentration of 0.1%. A
membrane consisting of regenerated cellulose with a
cut-off of 10 kDa was used in a 350 |jm separation channel.
The detector's dn/dc was set to 0.163 mL/g and the second
virial coefficient to 0. For chitosan the cross flow was set to
1,0 mL/min at a channel flow of 1,0 mL/min while the focus
time amounted to 350 s. The complete measurement period
was 25 min.
Automatic microviscosimetry: Microviscosimetry
experiments were performed using an AMVn
microviscosimeter (Anton-Paar, Ostfitdern, Germany).
The viscosity of all investigated polymer solutions was
determined by examining the rolling time of a steel sphere
under the influence of gravity in an inclined cylindrical
tube filled with the sampie liquid. To ensure a constant
temperature of 40 °C ± 0.01 "C, a built-in peltier was used.
The viscosity was then calculated using the laws of Stoke:
F,= 3'TT\*d'v [1]
with F being the frictional force, t] the fluid viscosity and d
the diameter of the spherical object. The molecular weight
was approximated with the Mark-Houwink equation.^"^
Nanoparticle formulation: Nanoparticles from Sigma
gelatin were prepared using the established two-step
desolvation technique.'^ In brief, a first desolvation step was
performed with acetone (Sigma) as an antisolvent to remove
the low molecular weight fractions of Sigma gelatin. After
resolvation of the sediment with 40 °C warm highly purified
water, nanoparticles were formed in a second desolvation
step under constant stirring by addition of acetone at
pH 2,5. The in situ nanoparticles were then stabilized by
crosslinking with glutaraldehyde (Sigma). Before further

Table 2: Molecular weight of Sigma gelatin and modified gelatin prototypes determined by automatic microviscosimetry compared
with AF4 (tnean value).
Batch Molecular weight (g/mol) AMVn Molecul Correlation coefficient
GNP 231 kDa 158 kDa 0.9469
MS-Geiatin 203 kDa 218 kDa 0.9312
MA-Gelatin 500 kDa 395 kDa 0.8946

Figure 5: AF4 signals of the examined gelatin batches MALS (dots) and UV signals (curves)- Key represents MALS signals.

1,00E+09 6,00

l.OOE+08 A
. . .. ,,- 5,00

o
1,00E+07

1.00E+06
in
Molar mass (g,

UX signa[| (volt)

1.00E+05

1.00E+04

1,00E+03
nA •

•
Sigma gelatin

MS gelatin
3.00

2,00

1.00E+02 1 ^ MA gelatin

1W 1,00
1.00E + 01

1.00E+00 ^
1 xs * ^
•^-^^^S^^^^^^^M^»^^^^^^ 0.00
O.OC 5.00 10.00 15.00 20,00 25.00
Elution time (min)

396 LC'GC Europe September ?0n9

 Schultes et al.

analysis the nanoparticles were centrifuged and washed
twice with highly purified water.
The MA and MS prctotype gelatin nanoparticies were
prepared frcm a 1% solution of geiatin by a novel desolvaticn
method including severai modifications to the previously
mentioned two-step desolvation method; for succinylated
gelatin the pH was set to 10, while for dodecenylsuccinylated
gelatin the pH could be varied in a range from 2.4-10 to
reach particles in the lower nanometer range. For a higher
precision in the desolvation and to ensure a good size
control, a peristaltic pump (Miniplus 3, Abimed Gilson,
Langenfeld, Germany) was combined with an immersed
needle for the first time where acetone is added via a 26 G
steel needle (Sterican, Braun. Emmenbrueoke, Germany)
into the stirred solution, Crosslinking was achieved by
the addition of glutaraldehyde, however, at a different
concentration and pH.^^
The chitosan nanopartioles were prepared by ionic gelation
in 0.05% aoetio acid solution al a polymer concentration
of 2.5% (w/v) and a pH of 5.5. After complete dissolution
of the polymer at 40 "C, a 0,2% (w/v) solution of sodium
triphosphate pentabasic (Sigma) as the counter polyanion
was added dropwisely (5 mL/min) under constant stirring
until nanoparticles were formed. The final crosslinking was
done by adding 10 pL of a 1 mM iodine (Sigma) solution.
The anions and iodine were removed by dialysis against
0.1 MHCIover 12 h.
Nanoparticle characterization: The size distribution of
all nanoparticles was analysed in aqueous dispersion by
SLS (LA-950 laser difractometer, Retsch Technologies,
Haan, Germany). Each size vaiue and corresponding
polydispersity index was the mean of 10 subruns. Static
iight scattering was used to screen larger agglomerates.
All experiments were conducted with a refractive index
of 1.59 (iabs = 0.01) and highly purified water as the
dispersion medium. The zeta potential of the nanoparticles
was measured with the Zetasizer Nano (Malvern) and flow
through cells in highly purified water under controlled ionic
strength conditions. All measurements were oonducted in
triplicate.

Table 3: Size and distribution of the nanopariicles as measured by SLS and ¿eta potential {n=3).
Batch Zeta (mV) Size (nm) PDI Span
GNP f 27 - 4 213 ± 22 0.005 0.422
MS-NP -36 ± 8 362 i 45 0.053 0.523
MA-NP -13 ± 2 193 ± 18 0,107 0.721
Chitosan low viscosity +21 ± 2 272 ± 19 0.103 0.881
Chitosan low moiecuiar weight +27 ± 9 290 ± 24 0.140 0.923
Chitosan-TBA low viscosity + 12 ± 4 412 ± 37 0,231 0.786
Chitosan-NAC low viscosity +32 ± 12 304 ± 5 0.432 0.699

Figure 6: Cumulative weight fractions of chitosan samples.

Cumulative molar mass

1.0 -1

0.8 -

r 0.6 -
I Chitosan low viscosity
ay
I 0.4 H Chitosan low molecular weight

o Chitosan-TBA low viscosity

0.2 - Chitosan-TBA low molecular weight

Chitosan-NAC low viscosity

0.0
1 I
1.0X10^ 1.0X106 1.0X10^ 1.0X108 1.0X109

Molar mass (g/mol)

398 LC-GC Europe September 2009


Results and Discussion
AF4 measurements of gelatins: At first, gelatin bulk
material from Sigma-Aldrich was analysed to gain a
benchmark for the following investigations of the modified
samples. The molar mass was determined to comprise sizes
ranging from 10 kDa to above 10000 kDa (Figure 2), which
confirmed the data reported by Fraunhofer''' and exceeded
the findings from size exclusion high performance liquid
chromatography-MALS (SE-HPLC-MALS) anaiysis^^ by
more than one order of magnitude. The discrepancy between
sizing data obtained from SE-HPLC and AF4 refiects the
fundamental differences between these two separation
techniques. While SE-HPLC separation takes place in a
packed column, AF4 uses an open channel leading to iower
hydrostatic pressure and therewith tower shear forces on the
samples are encountered during analysis. High molecular
weight specimens particularly suffer from degradation by
increased shear forces and are, therefore, preserved and can
be detected during AF4 analysis.^^
Figure 2 also shows how the high molecular weight
fractions of gelatin almost eiute over the whole experimental
period. As a result of the broad variety of molecules
possessing different molar masses in gelatin buik material, a
baseline separation of particular portions is excluded. Thus it
was decided to only apply a weak separation force to expand
the elution of the blend of molecules over a prolonged
period to demonstrate the broad molecular variety within
gelatin. A thorough understanding of the molecuiar weight
distribution of naturai polymers Is essentially important for the
formulation scientist. As will be discussed later, by delpeting
certain fractions of gelatin, smaller and more homogenously
distributed nanoparticles can be generated.
Figure 7: Cumulative molar mass distribution within the gelatin samples.
Cumulative molar mass distribution
0.90
0.80
uiative weight fraction
0.70
0,60
0,50
0.40
b
• Sigma gelatin
O 0.30
• MS gelatin
0.20
! • " MA gelatin
0.10
1 .OOE+00 1.00E+01 1.00E+02
1.00E+03
Molar mass (g/moi)
www.chromatographyonline.com
Schultes et al.
While very fast analysis is, of course, always possible
with AF4 we demonstrate thai the broad molecular
weight distribution of the analysed gelatins has important
implications in nanopartciie formulation. In this particular
experiment, however, our goal was to highlight the broad
molecular weight distribution of these gelatins, rather than a
superfast analysis (which is, of course, possible).
As only the high molecular weight fraction of Sigma
gelatin can be used for the preparation of homogenous
nanoparticles it generally has to be processed by two-step
desofvation. Manufacturing experiments in turn, conducted
with two customized Gelita batches (VP306/VP413-2)
that possessed less than 20% (w/w) peptides < 65 kDa
resulted in successful one-step desolvation synthesis of
gelatin nanoparticles exhibiting equivalent size and size
distribution.^^ These findings reveal the restriction that has
to be especially made for the presence of low molecular
weight portions in gelatin batches designated to one-step
desolvation. The successful depletion of the low molecular
weight fraction of gelatin is demonstrated in Figure 3,
In addition, gelatin sediment obtained from two-step
desolvation after the first desolvation step — as the
result from fraotionation and used for the preparation of
nanoparticles — also underwent AF4 analysis. Data from
these experiments and from gelatin bulk material are
displayed as function of their mean molecular weight in
Figure 4.
Interestingly the clear shift of the mean molecular weight of
the gelatin sediment (4) by more than one order of magnitude
compared with the bulk material (1) is not a prereq^jisite for a
successful one-step desolvation. A mean molecular weight
between 400 and 500 kDa determined for the Gelita batches
1 /
1/1/
il J
1.00E+04 1.00E+05 1.00E+06 1.00E+07 1,00E+08
399
 Schultes et al.
VP306 and VP413-2 was already sufficient to allow the new
production process. Thus, derived from these findings and
the specification of the applied gelatin batches a mean
molecular weight of -500 kDa and a threshold of a maximum
of 20% (w/w) for the portion of low molecular weight fractions
< 65 kDa could be defined as prerequisite for the successful
manufacturing of gelatin nanoparticies by a one-step
desolvation procedure.
The mean molecular weight of gelatin sediment ranges
clearly above the one of the Gelita batches, which may
not only be attributed to even more reduced amounts of
peptides < 65 kDa far below 20°/ô in the sediment. Thus,
the fractionation of gelatin bulk material during two-step
desolvation supposedly led to a depletion of molecular
weight fractions bigger than 65 kDa,
In the process of method development for the analysis
of MA and MS gelatins, several ultrafiltration membrane
types were tested. After experiments with different materials
and cut-off values regenerated cellulose with a cut-off of
10 kDa proved to be the most adequate. The results in
terms of recovery rate, repeatability and signai quality are
presented in Table 1. Regenerated cellulose Is a very low
protein binder and therefore ideally suited for analyses that
require maximum sample recovery. In addition the membrane
possesses a good solvent resistance with both aqueous and
organic solvents, and is able to work over a wide pH range.
The derivated hydrophobic prototype gelatins showed a
molar mass distribution from 140-10000 kDa and
200-100000 kDa for MS and MA, respectively. While the
average molecular weight of MS was 218 kDa, MA showed
an average molecular weight of 395 kDa. The motar mass
distribution of standard gelatin was found to be between
140-1000 kDa with an average molar mass of 158 kDa.
(Figure 5). This is in accordance with the prior studies, where
even fractions up to 10000 kDa were detected. The average
recovery was about 97.4%.
Figure 8: Comparison of the calculated molecular weights with
disulphide bonds and free suiphhydryl groups in the chitosan
samples. The CS-NAC low-molecular-weight-sampie was too
viscous for analysis.
Molecular weight vs thiol groups and disulpliidB bond
-Thiolgrouos -DiSLiphtae Bonds
400
AF4 Measurements of Chitosans
For the analysis of chitosan, the cumulative mass
distribution was plotted for a better comparison with the
geiatin sampies. As seen in Figure 6 the distribution of
molecular weight fractions in chitosan is much broader
than for gelatin (Figure 7). Interestingly, chitosan Imw
modified with thiobutylamidine as a potentiai nanoparticle
crossiinker showed an increased amount of low molecular
weight fractions compared with the unmodified chitosan
Imw samples. A polymer crosslinking throughout the small
molecular weight range could be the potential reason for this
data. In contrast, the thiobutylamidine modification of iow
viscosity chitosan lead to higher molar mass profiles over the
whole range, which might be based on the natural origin of
chitosan and also on a partial depolymerization during the
suiphhydryl modification process.
Because the chitosans were modified with the potentially
crosslinking suiphhydryl groups we compared the total
amount of free sulphydryl groups and the existing disulphide
bonds to the calculated moiar masses. In Figure 8 these
three parameters were correlated and it was shown, that a
constant amount of disulphide bends throughout the sampies
does not automatically account for higher molar masses
of the polymers. The infiuence of these modifications is
given exemplariiy for the case of TBA-modified chitosan in
Figure 9, For the W-acetylcysteine modification of chitosan
Imw, the viscosity was so high, that a molar mass calculation
could not be made. In all other cases the molar separation
and molecular weight calculation was successful and
reproducible. In conclusion, AF4 analysis was able to give
a good idea of what the molecular weight distribution of the
chitosan samples looks like and where modifications of the
backbone lead to different retention behaviour.
Automstic microviscosimetry: The molecular weight results
from AF4 were in accordance with the calculated values from
automatic microviscosimetry via the Mark-Houwink equation
{Table 2). A correlation factor of 1.0 stands for an optimum
compliance of the calculation with the measured values.
Nanoparticle formulation and characterization: The
prototype gelatin nanoparticles were prepared from a
1% solution of gelatin by a single desoivation method
including several modifications to the above mentioned
two-step desolvation method: the pH for negatively charged
MS gelatin nanoparticles had to be adjusted to 10 for a
homogeneous and small particie size, while MA geiatin with
long hydrophobic side chains lead to nanoparticles in the
whole pH range from 2.5-10. Paying tribute to the changed
physicochemical properties of the protein, inert glass
stirring beads had to be used for the preparation process
to prevent aggregation during desolvation. Furthermore, the
amount of acetone needed for desotvation and the amount of
glutaraldehyde as a crossiinker had to be optimized because
several changes in the functional groups of the protein had
occurred during the prior modification.
Resulting MA nanoparticles were 193 nm ± 16 nm (mean
± SD; n = 3) in size with a polydispersity index (PDl) of 0.107,
while MS nanoparticles were larger, with 362 nm ± 22 nm
(mean ± SD; n = 3) and a PDl of 0.053. Statistical analysis
of the data was performed by a one way analysis of variance
(Table 3),
Chitosan nanoparticles prepared from the modified
samples resulted in small and stable nanoparticles (Table 3).
LC«GC Europe September 2009
 Schultes et al.
However, their polydispersity index was much higher than for
the gelatin nanoparticles.
Conclusion
With the precise determination of the moleoular weight
distribution of modified gelatin and chitosan prototypes
by AE4, an important basis for further studies ccncerntng
the development of small, uniform and stable gelatin and
chitosan nanoparticles for drug delivery purposes was
achieved.
The analysis of gelatin bulk material by the combination
of asymmetrical flow field-flow fractionation and multiangle
light scattering was accomplished as a continuation of earlier
studies frcm Fraunhofer.^ At first, their basic results obtained
for gelatin bulk material applied fcr the manufacturing of
gelatin nancparticles by two-step desolvation cculd be
confirmed, Seccndly, mean molecular weights of new
customized gelatin quality characterized by the depletion of
low molecular weight fractions during production could be
successfully classified in between the mean molecular weight
determined for gelatin bulk material from Sigma-Aldrich and
gelatin sediment obtained from two-step desolvation.
These results demonstrated the impact of the low
molecular weight fraction of gelatin for the manufacturing
of gelatin nanoparticles and contributed to further
understanding and description of gelatin nanoparticle
synthesis by desolvation. This was at least feasible in a cne-
step attempt using particular batches of the customized
Gelita materialJ^ The cne-step desolvation is not onty
straightforward in terms of technological aspects because it
simplifies the manufacturing procedure, but is also especially
interesting for regulatory considerations. A simplified
Figure 9: Molar mass signals of chitosan samplei
1.0x108
1.0X10^
1,0X106 -
Time (min)
402
formulation process will increase the chance for regulatory
approval and make post-approval changes easier and
faster to realize due tc the process being less complex.
The simplicity of the whole gelatin nanoparticle formulation
process makes them attractive for FDA approval.
One of the major drawbacks cf the two-step desolvation
is that gelatin nancparticles are produced with bulk material
obtained from the first desolvation step that is not exactly
defined (i,e., varying iaboratcry equipment may lead to
different fractionation outcomes in terms of molecular weight).
The first desolvation step within the two-step desolvation
method requires a manual discarding of the supernatant,
leaving the whole process with a formulator based variable.
Just recently we standardized this process using a newly
developed instrument that reprcducibly fractionates the
gelatins the same way each time. The successful application
cf the one-step desclvaticn for gelatin nanoparticle synthesis
circumvents this problem.
It was further shown that hydrophobically modified large
proteins can be sized by AF4. As with ali quantitative analysis
tools the application of the correct separation material,
in this case the right membrane type and cut-cff had to
be taken into close consideration during the process of
method deveiopment- We can state that the AF4 method
described provides a fast and reliable tool for the analysis of
prcteins with a broad molecular weight distribution. This was
confirmed not only with prototype gelatin samples, normal
gelatin, but also with modified chitosan batches at different
thfol-bond crosslinking rates. The adapted desolvation
process proved to be a reliable way of producing
mono-modal and small-sized nanoparticles confirmed by
static light scattering. With the negative zetapotential of the
Chitosan-low molecular weight
Chitosan-TBA-low molecular weight
15 20
LC'GC Europe September 2009

prototype nanoparticies, these carriers can be used for
electrostatic attachment of positive charged drug molecules,
such as methotrexate. Additionally, the new prototype
nanoparticles possess different surface properties that hold
promise for a different body distribution pattern. Further in
vivo studies are in progress to analyse the properties of those
new gelatin nanoparticles.
In the future AF4 will definitely be used to a greater extent
for the analysis of polymers, proteins and nancparticles. New
developments in the fields of liposomes, cells, bacteria and
especially antibodies wiil raise the demand for a fast and
reliable analysis method such as AF4, particularly when the
standard separation methods for polymeric and colloidal
analytes come to their limits, AF4 may be the new method of
choice.
Stephan Schuites, Kathrin Mathis, Klaus Zwiorek and
Jan Ziilies are scientists in the group of Dr Coester and
Professor Winter, all involved besides their individual projects
in pharmaceutics in the development of AF4 applications.
Conrad Coester is principal investigator at the LMU.
Department of Pharmacy. He is specializing on
nanoparticulate drug delivery, focusing on numerous
applications of gelatin nanoparticles.
Gerhard Winter is a full professor for Pharmaceutical
Technology and Bicpharmacy. After 12 years in the biotech
industry he now focuses his research at the LMU since 1999
on parenteral dosage forms including depot systems, colloids
and especially protein drugs.
TOSOH
TOSOH BIOSCIENCE
Schultes et al.
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