analysis of polymers and protein nanoparticles using asymmetrical flow field-flow fractionation

8/8/2019 Analysis of Polymers and Protein Nanoparticles using Asymmetrical Flow Field-Flow Fractionation

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Analysis of Polymers andProtein Nanoparticlesusing Asymmetrical FlowField-Flow Fraotionation (AF4)

Stephan Schuttes, Kathrin Mathis, Jan Zill ies, Klaus Zwiorek, Conrad Coester and G erhard W inter,Ludwig Maximilians University, Department of Pharmacy, Pharmaceutical Technoogy and Biopharmaceutics, Munich, Germany.

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

Polymer and protein-based drugs are becoming morecommon in the pharmaceutical industry and asymmetricalfiow field-flow fractionation (AF4) offers a number ofadvantages over existing technique s, particularly forformulations containing polymers and nanoparticles. Theinterest in AF4 has grown since Giddings ' break-through workin the nineties' and the subsequent research by Fraunhofer^has helped the technique gain wider acceptance and use.By combining this technique with muitiangle light-scatteringdetectors (MALS), polymeric active ingredients can nowbe separated from polymeric excipients and particulatestructures such asnancparticles or virus-like particies

Expert knowiedge and practical expertise in this techniqueis, however, somewhat iimited, but the advantages of AF4 arebecoming more widely accepted. This article describes novelwork inpolymer and nanoparticle research and highlights the

chromatography (RP HPLC). size exclusion chrom atography(SEC) and fluorimetry are often limited (e.g., shear forcedestruction of protein aggregates instability studies dueto the presence of the stationary phase or the need forfluorometric moieties in the sample). A typical packed columfor these techniques requires high pressure but this is nothe case for AF4.

The separation principle of AF4, however, is based on adifferentiai flow of a solvent in a channel and a respectiveperpendicular cross-flow to separate the analytes only byhydrodynamic properties in a fast, no n-destructive,one-phase separation mechanism. One phase, non-destructiseparation ensures very gentle separation con ditions foriarge biomolecute analysis and aggregate detection, a rapicost-effective analysis time and less unwanted interactionsbecause of the small surface area of the channel comparedwith packed columns. Additionally, the smallest m olecules


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Schuttes et al.

Figure 1 shows a schematic diagram of this separationchannel. In theory macromolecules between 1 kDa andseveral GDa or particles between 2 nm and 100 pm can beseparated by AF4.Nanoparticles have the potentiai to offer numerousadvantages for targeted, safe and effective drug delivery,^''especially when made of biocompatible and biodegradablepolymers. Such nanoparticles could be useful for the targeteddelivery of drugs to improve bioavailability, sustain drugeffects at the target site, solubilize drugs for intravasculardelivery arid improve the stability of therapeutic agents againstenzymatic degradation.^ Additionally, by modulating thepolymer characteristics, the release of a therapeutic agentfrom nanoparticles can be controiled to achieve the desiredtherapeutic efficacy.^Gelatin nanoparticles, for example, are a prom isingdrug carrier system for future applications in various newapproaches in gene therapy.'' Innovative gelatin prototypeswith modifications of the backbone offer a further improvementregarding enhanced drug loading and modified targetingproperties in vivo.The aim of this investigation was to characterize the moiarmass profile of two newly modified gelatin prototypes by AF4and compare them with standard geiatin and fractionatedgeiatin, where low m olecular weight (Imw) gelatin was

Figure 1 : Schematic draw ing of an AF4 channel.

SpacerFiltrationMembraneFrit

Crossflow

removed from high molecular weight (hmw) gelatin. A thorouknowledge of the molar mass profiles n these highlyImw-reduced samples and in the prototype sam ples ismandatory for all nanoparticle formulation steps from gelatinas weil as from other polymers such as chitosan. Hence, wealso examined the molecular weight fractions of highly differechitosan-thiomer batches m odified in two ways with sulphydgroups and prompting different viscosities and chain lengthsIn the course of m ethod development we determined theoptimum settings in terms of optical detector parameters aswell as the correct type of membrane and elution profile for tseparation of hydrophobic large proteins.With this data, an im proved formulation process fornanoparticles was developed based on the traditionaldesolvation method developed by Coester."

KEY POINTS New developments in the fields of liposomes, cells,bacteria and especially antibodies will increase thedemand for a fast and reliable analysis method such asAF4 particularly when the standard separation m ethodsfor polymeric an d colloidal analytes reach their limits. Hydrophobically m odified large proteins can be sizedby AF4. The analysis of gelatin bulk m aterial by the combinationof asymm etrical flow field-flow fractionation and

muitiangle light scattering was accomplished incontinuation of earlier studies from Fraunhofer. Prototype hydrophobic gelatin nanoparticles wereprepared with altered physicochemical surfaceproperties for targeted drug delivery to the brain. Modern automatic m icroviscosimetry was used todetermine molecular weights of polymers and compare=i the results with AF4.

Figure 2: Molecular weight d istribulion of standard Sigma gelatin. UV (curve) and MALS (dotted line) signal.1.0E+10 -1

ro 1.0E + 08 -.^ 1.OE+06 -i- | 1.OE + 04 -oi 1.OE+02 -

1.

p1.2-10 -0.8 f

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do -0,040


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Schultes et al.

The physicochem ical characterization of all nanoparticieswas done by static tight scattering {SLS) and ccmp ared withestablished gelatin nanoparticle formulations in terms of size,zeta potential and polydispersity index (PI).ExperimentalAF4 measurements of gelatins: As w ell as twohydrophobicatly modified gelatin prototypes (Gelita.Eberbach, Germany} called MS and M A, with succinateand dodecenylsuccinate residues, standard gelatin type

Table 1; Induence of the different m embrane types on recoveryrale and repeatability (defined as the intra day repeatability ofa 100% value in per cent of six replicates).Membrane and cut-offRegenerated cellulose (5 kDa)Regenerated cellulose (10 kDa)Nitrocellulose (5 kDa)

Recovery92.3%95.7%86,5%

Repeatability97.4%98.5%91.9%

A (Bloom -175) from porcine skin (Sigma Aldrich, Munich,Germany) was analysed in this study. As control experimenin turn, measurements with two customized Gelita batches(VP306/VP413-2) that possessed less than 20% (w/w)peptides < 65 kDa were conducte d. The AF4 analysis ofthese gelatins was performed on an AFIOOO-FOCUS system(Postnova, Landsberg am Lec h, Germany) c oupled with a Udetector (UV100 Thermo Scientific, Ege lsbach, Germany),an Rl detector (n-1000 WGE Dr. Bures, Germany) forconcentration detection and a static light-scattering detecto(miniDAWN Wyatt Technology C orporation, Dernbac h,Germany) for molecular weight determination. The laserwavelength accounted for 690 nm, while slice collection waset to 1200. For molar mass determination the refractive ind(Rl) increment was set to 0.174 m l/ g and the second virialcoefficient was set tc 0. The separation was achieved usinga PBS buffer pH 6.0 as the m obile phase, a channel with350 \}m height an d an ultrafiltration membrane consisting oregenerated cellulose with 5 kDa cut-off (Postnova), All proteinwere dissolved in analysis buffer at a concentration of 2.5%,

Figure 3: UV signal (continuous line) and m olecular weight (dots) calculated from respec tive UV and M ALS data resulting from AF4analysis of gelatin bulk m aterial VP413-2 developed and provided from Geflta; the circle marks !he low molecular-weight fraction.

1.0E4-10 -.

g 1,0E+08

.ra I.OE-t-064 1,0E + 04 -

1.0E+02 -1.0E + 00 10

-1.0-0 .8-0 .6-0,4-0.2 w-0.040

Time (min)

Figure 4: Mean molecular weight fractions calculated from respective UV an d MALS data resulting from AF4 analysis of (1) gelatinbulk m aterial purchase d from S igma-Aldrich, (2) gelatin bulk material VP306. (3) VP413-2 deve loped and p rovided from G elita. andof (4) gelatin sediment obtained after the first desolvation step from the manufacturing process of the gelatin nanoparticles.

5,0 ^

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Q

4.0 -3,0 -2.0 -1.0 -0.01.00E + 04 1.00E + 05 1,00E+06 1.00E + 07 1 OOE + 08 1.00E + 09


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Schultes et al.The channel flow-rate ac counted for 1 mL/m in, while thecross flow was adjusted to 0,05 mL/min over 10 min and thenreduced to 0 mL/min, wh ich resulted in a totai measurementperiod of 20 mm.AF4 measurements of chitosans: The m odification ofthe chitosan materials (Sigma) was condu cted accordingto protocols described elsewhere.'''^ Chitosan low viscositymodified with W-acetylcystelne (Sigma) (lyophilized);chitosan low viscos ity m odified with thiobutylamidine (Sigma)(lyophilized); chitosan low molecular weight modified withA/-acetylcysteine (lyophilized) and chitosan low molecular-weight modified with thiobutylamidine (lyophilized) wereinvestigated with the same AF4 hardware set-up as forgelatin. The solvent and the running buffer for the chitosansamples were made of 0.3 M acetic acid, 0.2 M sodiumacetate and sodium hydroxide/hydrochloric acid q.s. ThepH was adjusted to 4 at a chitosan concentration of 0.1%. Amembrane consisting of regenerated cellulose with acut-off of 10 kDa was used in a 350 |jm separation chann el.The detector's dn/dc was set to 0.163 mL/g and the secondvirial coefficient to 0. For chitosan the cross flow was set to1,0 mL/min at a channel flow of 1,0 mL/min while the focustime amounted to 350 s. The complete m easurement periodwas 25 min.

Automatic microviscosimetry: Microviscosimetryexperiments were performed using an AMVnmicroviscosimeter (Anton-Paar, Ostfitdern, Germany).The viscosity of all investigated polymer solutions wasdetermined by examining the rolling time of a steel sphereunder the influence of gravity in an inclined cylindricaltube filled with the sampie liquid. To ensure a constanttemperature of 40 C 0.01 "C, a built-in peltier was usedThe 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 andthe diameter of the spherical object. The molecular weightwas a pproxima ted with the Mark-Houw ink equation. "^Nanoparticle formulation: Nanoparticles from Sigmagelatin were prepared using the established two-stepdesolvation technique.'^ In brief, a first desolvation step waperformed with acetone (Sigma) as an antisolvent to removthe low molecular weight fractions of Sigma gelatin. Afterresolvation of the sediment with 40 C w arm highly purifiedwater, nanoparticles were formed in a second desolvationstep under constant stirring by addition of acetone atpH 2,5. The in situ nanoparticles were then stabilized bycrosslinking with glutaraldehyde (Sigma). Before further

Table 2: Molecular weight of Sigma gelatin and mo dified gelatin prototypes determ ined by automatic microviscosime try c omp aredwith AF4 (tnean value).

Correlation coefficient0.94690.93120.8946

BatchGN PMS-GeiatinMA-Gelatin

Molecular weight (g/mol) AMVn231 kDa203 kDa500 kDa

Molecul158 kDa218 kDa395 kDa

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

o

mag

Moa

1,00E+09

l.OOE+081,00E+07

1.00E+061.00E+05

1.00E+04

1,00E+03

1.00E+02

1.00E + 011.00E+00 ^O.OC

A

i nnA1W1

5.00

6,00

. . . . ,,- 5,00

3.00 Sigma gelatin MS gelatin 2,00

1 ^ MA gelatin1,00

* ^ - ^ S ^ ^ ^ M ^ ^ 00010.00 15.00 20,00 25.00

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Schultes et al.analysis the nano particles were ce ntrifuged and washedtwice w ith highly pu rified water.

The MA and MS prctotype gelatin nanoparticies wereprepared frcm a 1% solution of geiatin by a novel desolvaticnmethod including severai m odifications to the previouslymentioned two-step desolvation method; for s uccinylatedgelatin the pH w as set to 1 0, while for dod ecenylsuccinylatedgelatin the pH cou ld be varied in a range from 2.4-1 0 toreach particles in the lower nanometer range. For a higherprecision in the desolvation and to ensure a good sizecontrol, a peristaltic pump (Miniplus 3, Abimed Gilson,Langenfeld, Germany) was combined with an immersedneedle for the first time where acetone is added via a 26 Gsteel needle (Sterican, Braun. Emmenbrueoke, Germany)into the stirred s olution, Crosslinking was achieved bythe addition of glutaraldehyde, however, at a differentconcentration and pH.^^

The chitosan nano partioles were prepared by ionic gelationin 0.05% aoetio a cid solution al a polymer con centrationof 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 sodiumtriphosphate pentabasic (Sigma) as the counter polyanionwas added dropwisely (5 mL/min) under constant stirringuntil nanoparticles were formed. The final crosslinking wasdone by adding 10 pL of a 1 mM iodine (Sigma) solution.The anions and iodine were removed by dialysis against0.1 M HC Iover 12 h.Nanoparticle characterization: The size distribution ofall nanoparticles was analysed in aqueous dispersion bySLS (LA-950 laser difractometer, Retsch Technologies,Haan, Germany). Each size vaiue and correspondingpolydispersity index was the mean of 10 subruns. Staticiight scattering was used to screen larger agglomerates.All experiments were conducted with a refractive indexof 1 .59 (iabs = 0.01) and highly purified water as thedispersion medium. The zeta potential of the nanoparticleswas measured with the Zetasizer Nano (Malvern) and flowthrough cells in highly purified water under controlled ionicstrength conditions. All measurements were oonducted intriplicate.

Table 3: Size and distribution of the nanopa riicles as measured by SLS and eta potential {n= 3).BatchGNPMS-NPMA-NPChitosan low viscosityChitosan low moiecuiar weightChitosan-TBA low viscosityChitosan-NAC low viscosity

Zeta (mV)f 27 - 4-36 8- 1 3 2+21 2+27 9+ 12 4+32 12

Size (nm)213 22362 i 45193 18272 19290 24412 3730 4 5

PD I0.0050.0530,1070.1030.1400,2310.432

Span0.4220.5230.7210.8810.9230.786

0.699

Figure 6: Cumulative weight fractions of chitosan samples.

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Cumulative molar mass1.0 -1

0.8 -

r 0.6 -Ia yI 0.4 H

0.2 -

0.01.0X10^ 1.0X106 1.0X10^

Chitosan low viscosityChitosan low molecular w eightChitosan-TBA low viscosityChitosan-TBA low molecular weightChitosan-NAC low viscosity

11.0X108

I1.0X109


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Schultes et al.Results and DiscussionAF4 measurements of gelatins: At first, gelatin bulkmaterial from Sigm a-Aldrich was analysed to gain abenchmark for the following investigations of the m odifiedsamples. The molar mass was determined to comprise sizesranging from 10 kDa to above 10000 kDa (Figure 2), whichconfirmed the data reported by Fraunhofer''' and exceededthe findings from size exclusion high performan ce liquidchromatography-MALS (SE-HPLC-MALS) anaiysis^^ bymore than one order of magnitude. The discrepancy betweensizing data obtained from SE-HPLC and AF4 refiects thefundamental differences between these two separationtechniques. While SE-HPLC separation takes place in apacked column , AF4 uses an open channel leading to iowerhydrostatic pressure and therewith tower shear forces on thesamples are encountered d uring analysis. High molecularweight specimens particularly suffer from degradation byincreased shear forces and are, therefore, preserved and canbe detected during AF4 analysis.^^

Figure 2 also shows how the high molecular weightfractions of gelatin almost eiute over the whole experimentalperiod. As a result of the broad variety of m oleculespossessing different m olar masses in gelatin buik material, abaseline separation of particular portions is exclude d. Thus itwas dec ided to only apply a weak separation force to expandthe elution of the blend of molecules over a prolongedperiod to dem onstrate the broad molecular variety withingelatin. A thorough understanding of the molecuiar weightdistribution of naturai polymers Is essentially important for theformulation scientist. As will be discussed later, by delpetingcertain fractions of gelatin, smaller an d m ore homogenouslydistributed nanoparticles can be generated.

While very fast analysis is, of course, always possiblewith AF4 we demonstrate tha i the broa d m olecularweight distribution of the analysed gelatins has importantimplications in nanopartciie formulation. In this particularexperiment, however, our goal was to highlight the broadmolecular weight distribution of these gelatins, rather than asuperfast analysis (which is, of course , possible).As only the high molecular weight fraction of Sigmagelatin can be used for the preparation of homogenousnanoparticles it generally has to be processed by two-stepdesofvation. Manufacturing experiments in turn, conductedwith two customized Gelita batches (VP306/VP413-2)that possessed less than 20% (w/w) peptides < 65 kDaresulted in successful one-step desolvation synthesis ofgelatin na noparticles exhibiting equivalent size and sizedistribution.^^ These findings reveal the restriction that hasto be espec ially made for the presence of low molecularweight portions in gelatin batches designated to one-stepdesolvation. The successful dep letion of the low molecularweight fraction of gelatin is demonstrated in Figure 3,In addition, gelatin sediment o btained from two-s tepdesolvation after the first desolvation step as theresult from fraotionation and used for the preparation ofnanoparticles also underwent AF4 analysis. Data fromthese experiments and from gelatin bulk material aredisplayed as function of their mean molecular weight inFigure 4.Interestingly the clear shift of the mean molecular weight ofthe gelatin sediment (4) by more than one order of magnitudecompared with the bulk material (1) is not a prereq^jisite for asuccessful one-step desolvation. A mean molecular w eightbetween 400 and 500 kDa determined for the Gelita batches

Figure 7: Cumu lative molar m ass distribution within the gelatin samples.

uiatvewgacton

bO

0.900.800.700,600,500.400.300.200.10

1 .OOE+00

Sigma gelatin MS gelatin! " MA gelatin

1.00E+01 1.00E+02

Cumulative molar mass distribution

1 /1 /1 /l J1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1,00E+08


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Schultes et al.VP306 and VP413-2 was already sufficient to allow the newproduction process. Thus, derived from these findings andthe specification of the ap plied gelatin batches a meanmolecular weight of -500 kDa and a threshold of a maximumof 20% (w/w) for the portion of low molecular weight fractions< 65 kDa could be defined as prerequisite for the successfulmanufacturing of gelatin nanoparticies by a one-stepdesolvation procedure.

The mean molecular weight of gelatin sediment rangesclearly above the one of the Gelita batches, which maynot only be attributed to even more redu ced amounts ofpeptides < 65 kDa far below 20 in the sediment. Thus,the fractionation of gelatin bulk material during two-stepdesolvation s upposedly led to a depletion of molecularweight fractions bigger than 65 kDa,In the process of method development for the analysisof MA and MS gelatins, several ultrafiltration membranetypes were tested. After experiments w ith different materialsand cut-off values regenerated cellulose with a cut-off of10 kDa proved to be the most adequate. The results in

terms of recovery rate, repeatability and signai quality arepresented in Table 1. Regenerated cellulose Is a very lowprotein binder and therefore ideally suited for analyses thatrequire m aximum sam ple recovery. In addition the membranepossesses a go od solvent resistance with both aqueous andorganic solvents, and is able to work over a wide pH range.The derivated h ydrophobic prototype gelatins showed amolar mass distribution from 140-10000 kDa and200 -10 000 0 kDa for MS and MA, respectively. While theaverage molecular weight of MS was 218 kDa, MA showedan average molecular w eight of 395 kD a. The motar massdistribution of standard gelatin was found to be between

140-1000 kDa with an average molar m ass of 158 kDa.(Figure 5). This is in acc ordance with the prior s tudies, whereeven fractions up to 10000 kDa were d etected. The averagerecovery was about 97.4%.

Figure 8: Comparison of the calculated molecular weights withdisulphide b onds an d free suiphhydryl group s in the chitosansamples. The CS-NAC low-molecular-weight-sampie w as tooviscous for analysis.

Molecular weight vs thiol groups and disulpli idB bond

AF4 Measurements of ChitosansFor the analysis of ch itosan, the cumulative massdistribution was plotted for a better com parison with thegeiatin sampies. As seen in Figure 6 the distribution ofmolecular weight fractions in chitosan is much broaderthan for gelatin (Figure 7). Interestingly, chitosan Imwmodified with thiobutylamidine as a potentiai nanoparticlecrossiinker show ed an increased amount of low molecularweight fractions compared with the unmodified chitosanImw samples. A polymer crosslinking throughout the smallmolecular w eight range cou ld be the potential reason for thdata. In contrast, the thiobutylamidine mod ification of iowviscosity chitosan lead to higher molar mass profiles over thwhole range, which might be based on the natural origin ofchitosan and also on a partial depolymerization during thesuiphhydryl modification process.

Because the chitosans were mod ified with the potentiallycrosslinking suiphhydryl groups we compared the totalamount of free sulphydryl groups and the existing disulphidbonds to the calculated moiar masses. In Figure 8 thesethree parameters were correlated and it was show n, that aconstant amount of disulphide bends throughout the sampidoes not automatically acc ount for higher molar massesof the po lymers. The infiuence of these m odifications isgiven exemplariiy for the case of TBA-m odified chitosan inFigure 9, For the W-acetylcysteine modification of chitosanImw, the viscosity was so high, that a molar mass ca lculatiocould not be made. In all other cases the molar separationand molecular weight calculation was successful andreproduc ible. In conclusion, AF4 analysis was able to givea go od idea of what the m olecular weight distribution of thechitosan samples looks like and where mo difications of thebackbone lead to different retention behaviour.Automstic microviscosimetry: The molecular weight resufrom AF4 were in accordance with the calculated values froautomatic microviscosimetry via the Mark-Houwink equation{Table 2). A correlation factor of 1.0 stands for an optimumcompliance of the calculation with the measured values.Nanoparticle formulation and characterization: Theprototype gelatin nanoparticles were prepared from a1% solution of gelatin by a single desoivation methodincluding several modifications to the above mentionedtwo-step de solvation m ethod: the pH for negatively chargedMS gelatin nan oparticles had to be adjusted to 10 for ahomogeneous and small particie size, while MA geiatin withlong hydrophobic side chains lead to nanoparticles in thewhole pH range from 2.5-10. Paying tribute to the changedphysicochem ical p roperties of the protein, inert glassstirring beads had to be used for the preparation processto prevent aggrega tion during desolvation. Furthermore, theamount of acetone needed for desotvation and the amount glutaraldehyde as a crossiinker had to be optimized becausseveral changes in the functional groups of the protein hadoccurred during the prior modification.

Resulting MA nanoparticles were 193 nm 16 nm (mean SD; n = 3) in size with a polydispersity index (PDl) of 0.10while MS nanoparticles were larger, with 362 nm 22 nm(mean SD; n = 3) and a PDl of 0.053. Statistical analysisof the data was performed by a one way analysis of varianc


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However, their polydispersity index was much higher than forthe gelatin nanoparticles.ConclusionWith the precise determination of the moleoular weightdistribution of modified gelatin and chitosan prototypesby AE4, an important basis for further studies ccn cerntngthe development of small, uniform and stable gelatin andchitosan nanoparticles for drug delivery purposes wasachieved.

The analysis of gelatin bulk material by the combinationof asymmetrical flow field-flow fractionation and multianglelight scattering was accom plished as a continuation of earlierstudies frcm Fraunhofer.^ At first, their basic results obtainedfor gelatin bulk material app lied fcr the manufacturing ofgelatin nancparticles by two-step desolvation cculd beconfirmed, S eccndly, mean molecular weights of newcustomized ge latin quality c haracterized by the dep letion oflow molecular weight fractions during production could besuccessfully classified in between the mean molecular weightdetermined for gelatin bulk material from Sigma-A ldrich andgelatin sediment o btained from two-step d esolvation.

These results demonstrated the impact of the lowmolecular weight fraction of gelatin for the manufacturingof gelatin nanoparticles and contributed to furtherunderstanding and description of gelatin nanoparticlesynthesis by desolvation. This was at least feasible in a cne-step attempt using particular batches of the custom izedGelita materialJ^ The cne-step desolvation is not ontystraightforward in terms of technological aspe cts because itsimplifies the manufacturing procedure, but is also especiallyinteresting for regulatory considerations. A simplified

formulation process will increase the cha nce for regulatoryapproval and make post-approval changes easier andfaster to realize due tc the process being less complex.The simplicity of the whole gelatin nanoparticle formulationprocess makes them attractive for FDA approva l.One of the major drawbacks cf the two-step desolvationis that gelatin nancparticles are produce d w ith bulk materiaobtained from the first desolvation step that is not exactlydefined (i,e., varying iaboratcry equipment may lead todifferent fractionation outcomes in terms of m olecular weighThe first desolvation step w ithin the two-step desolvationmethod requires a manual discardin g of the supernatant,leaving the whole process with a formulator based variableJust recently we standardized this proce ss using a newlydeveloped instrument that reprcducibly fractionates thegelatins the same way each time. The successful applicatiocf the one-step desclvaticn for gelatin nan oparticle synthescircumvents this problem.It was further shown that hydropho bically mod ified largeproteins can be sized by AF4. As with ali quantitative analytools the ap plication of the correct separation material,in this case the right membrane type and cut-cff had tobe taken into close c onsideration during the process ofmethod deveiopment- We can state that the AF4 methoddescribe d provides a fast and reliable tool for the analysis oprcteins w ith a broad molecular weight distribution. This waconfirmed not only with prototype gelatin samples, normalgelatin, but also with m odified chitosan batches at differentthfol-bond crosslinking rates. The adapted desolvationprocess proved to be a reliable way of produc ingmono-modal and small-sized nanoparticles confirmed bystatic light scattering. With the negative zetapotential of the

Figure 9: Molar mass signals of chitosan sam plei1.0x108

1.0X10

1,0X106 -

f

Chitosan-low m olecular weightChitosan-TBA-low m olecular w eight

15 20


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Schul tes e t a l .prototype nanoparticies, these carriers can be used forelectrostatic attachment of positive charged drug molecules,such as methotrexate. Additionally, the new prototypenanoparticles possess different surface properties that holdpromise for a different body distribution pattern. Further invivo studies are in progress to analyse the properties of thosenew gelatin nanoparticles.

In the future AF4 w ill definitely be used to a greater extentfor the analysis of polymers, proteins and nancparticles. Newdevelopments in the fields of liposomes, cells, bacteria andespecially antibodies wiil raise the demand for a fast andreliable analysis method such as AF4, particularly when thestandard separation methods for polymeric and colloidalanalytes come to their limits, AF4 may be the new method ofchoice.

Stephan Schuites, Kathrin Mathis, Klaus Zwiorek an dJan Ziilies are scientists in the group of Dr Coester an dProfessor Winter, all involved besides their individual projectsin pharmaceutics in the development of AF4 applications.Conrad Coester is principal investigator at the LM U.Department of Pharmacy. He is specializing onnanoparticulate drug delivery, focusing on numerousapplications of gelatin nanoparticles.Gerhard Winter is a full professor for Pharm aceuticalTechnology and Bicpharmacy. After 12 years in the biotechindustry he now focuses his research at the LMU since 1999on parenteral dosage forms including depot systems, colloidsand especially protein drugs.

ReferencesI. J.C. Giddings, Science. 260(5113), 1456-1465 (1993).2 W. Fraunhofer and G. Winter, European Journal of Pharmaceutics an dBiopharmaceutics. 58(2), 369-383 (2004) .3 C. Augsten and K. Maeder, Light scattering for the Masses:Cha racterization of PolyD.L-lactide-co-glycoiie) Nan oparticles: WyatlTechnology Corporation Application Notes (2005).4. R. Lang, and G Winter, Light Scattering for the Masses:Characterization of Virus Uhe Particies by Asymmetrical FlowField-Flow Fractionation: Wyatt Technolog y Corpora tion ApplicationNotes (2006).5. M. Schimpf, K.D. Caldwell and J.C. Giddings (eds.); Field-FlowFractionation Handbook. John Wiley & Sons, Inc., New York (2000).6. W. Babef, Chemie in unserer Zeit, 30(2), 1-11 (1996).7. C. Coester, NewDrugs. (1), 14-17 (2003),8. R. Langer. Nature (London) 39 2 (6679, SuppL), 5-10 (1998).9. S.V. Vinagradov, T.K. Bronich and A.V. Kabanov. Adv. Drug Del. Rev.54, 223-23 3 (2002) .

10. G. Kaul. and M. Amiji, Pharmaceutical R esearch. 19(7), 1061-1067(2002)-II, C. Coester et al.. Journal of Microer]capsuition. 17(2). 187-193 (2000).12. A, Bernkop-Schnrch and Th,E, Hopf. Sei. Pharm.. 69(2}. 109-118(2001).13. A, Bernk op-Sch nrch. M. Hornof and T ZoidI, nt. I Pharm . 260(2),229-237 (2003) .14. DIN 53015 und ISO 12058.15. K. Zwiorek, Gelatin Nanoparticies as Delivery System for Nudeotide-based drugs. Dissertation, Ludw ig-Maximilians-U niversity fvlunich(2006).16. S. Schultes et a i, Novel Hydrophobic Prototype Geialins forNanoparticle Preparation Analyzed b y AF4 and MALS: 6th WorldMeeting on Pharmaceutics, Biopharmaceutics and PharmaceuticalTechnology, Ba rcelona, Spa in, April, 6th-10lh (20 08),17 W. Fraunhofer, G. Winter and C. Coester, Analytical Chemistry. 76(7)1909-1920(2004) .18. M. Meyer and B, Morgenstern, Biomacromolecules. 4(6), 1727-1732(2003).19. M.N. Myers, Journal of Microcoiumn Separations. 9{3), 151-162(1997).

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analysis of polymers and protein nanoparticles using asymmetrical flow field-flow fractionation

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