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RESEARCH ARTICLE – Pharmaceutical Biotechnology

Protein Aggregation and Particle Formation in PrefilledGlass Syringes

ALANA GERHARDT,1 NICOLE R. MCGRAW,1 DANIEL K. SCHWARTZ,1 JARED S. BEE,2

JOHN F. CARPENTER,3 THEODORE W. RANDOLPH1

1Department of Chemical and Biological Engineering, University of Colorado – Boulder, Boulder, Colorado2Formulation Sciences, MedImmune, Gaithersburg, Maryland3Department of Pharmaceutical Sciences, University of Colorado – Denver, Aurora, Colorado

Received 2 January 2014; revised 10 March 2014; accepted 25 March 2014

Published online 11 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23973

ABSTRACT: The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of proteinmolecules to silicone oil–water interfaces and air–water interfaces. In addition, agitation, such as that experienced during transportation,may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study,surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed tosilicone oil–water interfaces (siliconized syringe walls), air–water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 �m diameter) were detected under all conditions. The highest particle concentrationswere found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oildroplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism ofparticle generation in PFS in which capillary forces at the three-phase (silicone oil–water–air) contact line remove silicone oil and gelledprotein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil–waterinterfaces, air–water interfaces, and agitation in the generation of particles in protein formulations. C© 2014 Wiley Periodicals, Inc. and theAmerican Pharmacists Association J Pharm Sci 103:1601–1612, 2014Keywords: PFS; silicone oil; microparticles; protein formulation; protein aggregation; image analysis; adsorption; monoclonal antibody

INTRODUCTION

Glass prefilled syringes (PFS) are often the packaging methodof choice for therapeutic protein products. Their many ben-efits include reduced contamination risk, minimal overfill,dose accuracy, ease of administration, and improved patientcompliance.1 Thus, it is not surprising that PFS are one of thefastest growing markets in the drug delivery sector, and theiruse is expected to increase further with the growing demandfor injectable biologic drugs.1

Prefilled syringes serve as both the delivery device and thestorage container for protein therapeutics. During a product’sshelf life (typically 2 years), therapeutic protein molecules areexposed to a variety of interfaces in PFS, including siliconeoil–water interfaces and air–water interfaces. The silicone oil–water interface is present because silicone oil is commonly usedas a lubricant to reduce and smooth the force required for injec-tion. In typical PFS, levels of siliconization are approximately0.4–1.0 mg silicone oil per 1 mL syringe.2 The air–water inter-face is introduced in the syringe because of an air bubble thatremains as a consequence of the syringe filling and stopperingprocess. Several recent studies have focused on how proteinsmay be affected by exposure to these interfaces. In particular,protein adsorption to both air–water and silicone oil–water in-

Correspondence to: Theodore W. Randolph (Telephone: +303-492-8592;Fax: +303-492-8425; E-mail: [email protected])

This article contains supplementary material available from the authors uponrequest or via the Internet at http://wileylibrary.com.

Journal of Pharmaceutical Sciences, Vol. 103, 1601–1612 (2014)C© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

terfaces has been shown to promote protein aggregation andparticle formation.3–9

The presence of particles of any kind in a protein formulationis a major concern. Both proteinaceous particles and particlesshed during manufacturing and storage (i.e. glass, stainlesssteel, and silicone oil) may be present in therapeutic proteinproducts.10–12 The acceptable number of particles >25 and >10 :m in an injectable drug product is outlined in USP.13 In addition, there is a growing concern about thenumber of particles between 0.1 and 10 :m in protein formula-tions, as particles in this size range may be the most immuno-genic and are considered a critical quality attribute.12 With newtechnologies, the ability to quantify and characterize particlesin the 0.1–10 :m size range has significantly improved,10,14–16

and as a result, several recent studies have focused on identi-fying the extent to which these particles arise in protein ther-apeutic products and their impact on product quality, safety,and immunogenicity.5,17–21 For example, Barnard et al.20 sug-gest a link between the presence of aggregates and particles ininterferon-$ products and the production of neutralizing anti-bodies in patients. In contrast, Lubiniecki et al.21 found thatalthough switching a protein drug product from vials to PFSresulted in a slight and statistically significant increase in thelevels of subvisible particles, there were no detectable differ-ences in patient responses in clinical trials between productsin vials versus products in PFS.

In addition to the exposure to interfacial stresses, protein for-mulations are also subject to various transportation-associatedstresses such as agitation. Studies of protein formulations thatwere agitated in the presence of a silicone oil emulsion have

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1602 RESEARCH ARTICLE – Pharmaceutical Biotechnology

shown large losses of monomeric protein due to the aggrega-tion and not simply adsorption to the interface.3,22 Other re-cent studies have detected significant particle formation in pro-tein formulations agitated in siliconized syringes.5,9 However,some researchers have suggested that the particles formed instressed PFS samples are composed of only silicone oil dropletsand that there is minimal impact on protein aggregation fromthe presence of silicone oil, in particular if the protein formu-lation contains surfactant.5,21,23 However, even particles com-posed only of silicone oil could be a product quality concern.

Previous studies suggest that the combination of silicone oil–water interfaces, air–water interfaces, and agitation may actsynergistically to adversely affect protein formulations,3,22,24

but the mechanism behind this observation is still uncertain.It is widely known that proteins readily adsorb to hydropho-bic interfaces, such as the silicone oil–water interface and theair–water interface.3,7,25,26 Upon adsorption to hydrophobic in-terfaces, many proteins form viscoelastic gel layers.27–29 Dis-ruption of the protein gel layer results in the release of proteinaggregates and particles into the bulk solution.6,7 Further workis needed to evaluate the role of the silicone oil–water interfacein generating protein aggregates and particulates and to deter-mine how interfaces and agitation combine synergistically tocompromise the quality of therapeutic protein formulations.

Therefore, the objective of this study was to determine themechanism by which silicone oil–water interfaces, air–waterinterfaces, and agitation work together to induce particle gener-ation in protein formulations. The effects of these three factorswere evaluated in two different formulations (containing eitherlysozyme or a monoclonal antibody) in PFS. Protein formula-tions were incubated quiescently or with end-over-end rotationin siliconized or unsiliconized glass syringes, both in the pres-ence and absence of an air bubble. Under certain conditions,glass beads were added to the syringes to add bulk shear forceswithout the addition of an air bubble. The number of particlesgenerated in each incubation condition was counted using flowmicroscopy, which also recorded images of the observed parti-cles. Because we wanted to observe the effect of the air–waterinterface and the silicone oil–water interface on the proteinformulation, the formulations were prepared without surfac-tant present. In addition, we measured the interfacial tensionsof the silicone oil–water interface and the air–water interfacewith protein adsorbed and the contact angle of a protein so-lution on a siliconized surface in order to estimate the forcesacting inside a PFS.

MATERIALS AND METHODS

Materials

Humanized IgG1 monoclonal antibody (molecular weight146 kDa), here denoted as “3M”, was provided by MedImmune(Gaithersburg, Maryland).30 The antibody was obtained at astock concentration of 150 mg/mL in 10 mM L-histidine at pH6. For consistency with previous work,3 3M formulations wereprepared in 10 mM L-histidine pH 5. Lysozyme from chickenegg white (molecular weight 14.3 kDa) with ≥90% purity waspurchased as a lyophilized powder from Sigma–Aldrich (St.Louis, Missouri). Silicone oil (Dow Corning 360, 1000 cSt) wasof medical grade and purchased from Nexeo Solutions (Denver,Colorado). All buffer salts were of reagent grade or higher. Brad-ford reagent (Brilliant Blue G in phosphoric acid and methanol)

was obtained from Sigma–Aldrich. All solutions were preparedin deionized water filtered with a 0.22 :m Millipore filter (Bil-lerica, Massachusetts). The syringes used in the incubationstudies were BD Hypak SCF 1 mL long 27G1/2 (BD Medical-Pharmaceutical Systems, Franklin Lakes, New Jersey).

3M Adsorption to Surfaces

To compare the surface coverage of 3M on glass with the pre-viously measured surface coverage of 3M on silicone oil,3 glassparticles were prepared from Type I glass vials (5cc, Type 1Glass, USP/PhEur, nontreated; Alcan Packaging, Syracuse, Ne-braska) as described in Bee et al.31 The particles were sievedthrough a 45 :m screen before use. A suspension of 100 mg/mLglass microparticles in 10 mM L-histidine buffer pH 5 was pre-pared for use in the adsorption studies. The particle surfacearea and the particle size distribution of the glass suspensionwere measured as previously described.31

A bulk depletion method similar to that described by Ger-hardt et al.3 was used to measure the amount of 3M adsorbedto glass surfaces. Aliquots of 3M stock solution (3M concen-tration 0.73 mg/mL) ranging in volume from 0.00 to 0.10 mLwere mixed with a suspension of glass microparticles in 10 mML-histidine to achieve a total volume of 0.5 mL. The sampleswere incubated end-over-end at 8 rpm for 1 hour at room tem-perature to allow the protein to adsorb to the suspended glassmicroparticles. After incubation, the vials were centrifuged at12,000g for 30 min to separate the glass microparticles from theaqueous phase, and approximately 300 :L of the supernatantwas removed and transferred to 0.6 mL microcentrifuge vials.

Following previously described methods, a modified Bradfordassay was performed to determine the bulk 3M concentrationof each sample and subsequently the amount of 3M adsorbed,and the data were fit to a Langmuir isotherm to provide anestimate of the monolayer surface coverage of 3M on glass.3

Removal of Silicone Oil from Syringes

As controls, some incubation studies were conducted in unsili-conized syringes. To remove the silicone oil coating, syringeswere cleaned as follows. A 1% solution of Micro-90 (Inter-national Products Corporation, Burlington, New Jersey) waspipetted in and out of the syringes four times. This was followedby a rinse with deionized water. Then, hexane (ACS grade;EMD, Billerica, Massachusetts) was pipetted in and out of thesyringes five times, and the syringes were allowed to air dryat room temperature. Finally, the syringes were submerged inpiranha solution (70% sulfuric acid:30% hydrogen peroxide) for1 hour (with the needle facing up and out of the solution) andthen rinsed with deionized water and dried with nitrogen gas.Caution: Piranha solution is extremely corrosive and should behandled with extreme care.

Contact angle measurements on treated glass slides wereused to verify that this cleaning procedure successfully removessilicone oil from glass. First, the contact angle of a 1 :L dropletof 0.2 :m-filtered deionized water on a bare glass slide wasmeasured using an Artcam-130MI-BW camera (Artray Com-pany, Ltd., Tokyo, Japan). The contact angle of water on theslide was calculated based on the shape of the droplet using theFTA32 Video 2.0 software (First Ten Angstroms, Portsmouth,Virginia). Then, slides were incubated overnight in a 3% solu-tion of DC 360 medical fluid (1000 cSt; Dow Corning, Midland,Michigan) in toluene to coat them with silicone oil. After excess

Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014 DOI 10.1002/jps.23973

RESEARCH ARTICLE – Pharmaceutical Biotechnology 1603

liquid was removed from the surface of the glass slide, the re-maining toluene was allowed to evaporate, leaving a thin, uni-form coating of silicone oil on the glass slide. Subsequently, theabove-described cleaning procedure was performed to removethe silicone oil coating, and the contact angle of a 1 :L dropletof deionized water was measured on the cleaned surface. Thecontact angles measured before and after coating and cleaningwere identical within experimental error (data not shown).

In addition, the difference between the hydrophobic sili-conized syringe surface and the hydrophilic cleaned bare glasssyringe surface was confirmed by filling each syringe with1 mL of deionized water and visually comparing the shape ofthe menisci (Supplemental Information Fig. S1).

Incubation of Protein Formulations in PFS

Two formulations of proteins were prepared for incubation insyringes: 1 mg/mL 3M in 10 mM L-histidine pH 5 and 1 mg/mLlysozyme in 10 mM potassium phosphate pH 7.2. For incu-bations with an air bubble, 1.26 mL of the formulation waspipetted into the syringe, and the syringe was stoppered, whichcreated a headspace containing 30 :L of air. For incubation con-ditions with no air bubble, the syringe was stoppered such thatno air bubbles remained in the syringe. For incubation con-ditions with beads, two 4 mm glass beads were added to thesyringe, which was then filled with protein formulation andstoppered such that no air bubbles remained in the syringe.Triplicate syringes were prepared for each incubation conditionat each time point. For incubation conditions with agitation, thesyringes were rotated end-over-end at 1.5 rpm at room temper-ature. For stationary incubation conditions, the syringes wereincubated horizontally on the bench top at room temperature.

Counting of Particles in Incubated Protein Formulations

At each time point, the syringes were unstoppered, and theformulations were removed from the flanged end using a trans-fer pipet. The protein formulation was not ejected using thesyringe needle to avoid the generation of particles due to theplunger movement along the syringe barrel. For each sample,particles between 2 :m and 2 mm (equivalent spherical diam-eter) were counted using a Fluid Imaging Technologies Bench-top FlowCAM R© (Scarborough, Maine). The FlowCAM was fittedwith a FC100 flow cell, a 10× objective and collimator, and a0.5 mL syringe. The gain and flash duration were set such thatthe average intensity mean of the image was consistently be-tween 180 and 200. A sample volume of 0.2 mL was analyzedfor each sample at a flow rate of 0.145 mL/min. Particle countswere normalized by dividing the number of particles per sampleby the total volume imaged per sample to obtain the particleconcentration (#/mL). In addition to the samples incubated insyringes, buffer solutions and protein formulations not incu-bated in syringes were also analyzed by FlowCAM R©.

Contact Angle and Interfacial Tension Measurements

The contact angle (measured through the liquid) of a 1 :Ldroplet of 1 mg/mL 3M formulation was measured on a sili-conized glass slide using the method described above. Measure-ments were made on two different slides with three dropletsmeasured on each slide. All measurements were averaged, andthe standard deviation was reported.

Interfacial tension measurements were conducted using thependant drop technique.32 A camera (described above) was used

to collect images of droplets of silicone oil (ca. 100 :L) in a1 mg/mL 3M formulation or droplets of 1 mg/mL 3M solution(ca. 10 :L) in air. The drop shape analysis software (describedabove) analyzed the shape of each droplet using the Young–Laplace equation in order to determine the interfacial tension ofthe droplet in each image. Images are collected approximatelyevery 10 s. Supplemental Figure S2 shows a representativeplot of the silicone oil–protein solution interfacial tension as afunction of time. Two samples were analyzed for each interface(silicone oil–water and air–water), and the interfacial tensionvalues measured at ca. 40 s (corresponding to the time requiredfor one full rotation of a syringe in the incubation experiments)were used in subsequent calculations.

RESULTS

3M Adsorption to Glass

The surface coverage of 3M on glass was plotted as a functionof the bulk 3M concentration and fit to a Langmuir isothermmodel (data not shown). The amount of adsorbed 3M to formmonolayer surface coverage on glass was 1.9 ± 0.4 mg/m2.

Particle Concentrations in 3M Formulations in PFS

Particles 2 :m or greater were detected in 3M formulationsand in a buffer solution incubated in PFS under all conditions(Fig. 1). A buffer solution incubated in agitated, siliconized sy-ringes, with or without an air bubble, contained low levels ofparticles that did not change appreciably over the course of 2weeks (Fig. 1a). The observed particles were spherically shapedand, thus, assumed to be silicone oil droplets (Fig. 2a). In addi-tion, a similarly low level of particles was observed in 3M formu-lations incubated in siliconized syringes that were not agitated(Fig. 1b), and the particle concentrations did not change overtime. Both silicone oil droplets and protein aggregates wereobserved in this condition (Fig. 2b).

The greatest number of particles was detected in 3M for-mulations incubated in agitated, siliconized syringes with anair bubble (Fig. 1d; closed symbols). In these formulations, theparticle concentrations increased steadily over time. Imagescollected during flow microscopy suggested that these particlestypically consisted of silicone oil droplets, aggregated protein,and agglomerates of protein aggregates and silicone oil droplets(Fig. 2d). In 3M formulations that were incubated in agitated,siliconized syringes in the absence of an air bubble, the result-ing particle concentrations were two orders of magnitude lowerthan those observed in siliconized syringes with an air bubble(Fig. 1d; open symbols), and the particle concentrations wereinvariant over time. In the absence of an air bubble, the num-ber of particles detected was not appreciably higher than thenumber of particles detected in the incubated buffer solutionsand in the absence of agitation.

In some siliconized syringes without air bubbles, glass beadswere added to introduce shearing in the syringes without theaddition of an air–water interface. In otherwise identical con-ditions (i.e. 3M formulations in agitated, siliconized syringes),particle counts were lower in the presence of glass beads thanin the presence of an air bubble (Fig. 1d; cross symbols). Inthe presence of the glass beads, the particle concentrations alsoincreased with time, and, the images of particles showed bothsilicone oil droplets and aggregated protein (Fig. 2e).

DOI 10.1002/jps.23973 Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014


Figure 1. Particle concentrations in 3M formulations and buffer solutions incubated in PFS as a function of time. Open symbols correspondto syringes incubated with no air bubble, closed symbols correspond to syringes incubated with an air bubble, and cross symbols correspond tosyringes incubated with no air bubble but with glass beads. The particle concentrations in a buffer solution (solid black line) and in a 3M solution(dashed black line) that were not incubated in syringes are also shown. The incubation conditions are as follows: (a) L-histidine buffer (no protein)in agitated, siliconized syringes, (b) 3M formulation in quiescent, siliconized syringes, (c) 3M formulation in agitated, unsiliconized syringes, and(d) 3M formulation in agitated, siliconized syringes.

The number of particles that were detected in 3M formula-tions incubated in agitated, unsiliconized syringes with no airbubble were comparable to those observed in 3M formulationsincubated in agitated, siliconized syringes with no air bubble(Figs. 1c and 1d; open symbols). The addition of an air bubble to3M formulations incubated in agitated, unsiliconized syringesslightly increased the number of particles detected (Fig. 1c;closed symbols). However, the particle concentrations in 3M for-mulations incubated in agitated, unsiliconized syringes with anair bubble remained an order of magnitude lower than the par-ticle concentrations in 3M formulations incubated in agitated,

siliconized syringes with an air bubble (Figs. 1c and 1d; closedsymbols). The images of particles in 3M formulations incu-bated in unsiliconized syringes showed only protein aggregates(Fig. 2c).

In summary, a baseline level of particles was observed underall incubation conditions. The particle concentrations increasedabove this baseline level in protein formulations incubated inagitated, unsiliconized syringes with an air bubble and in ag-itated, siliconized syringes with glass beads, but the largestparticle concentrations were observed in protein formulationsincubated in agitated, siliconized syringes with an air bubble.



Figure 2. An example of the particles observed after 1 day of incubation in: (a) L-histidine buffer (no protein) in agitated, siliconized syringeswith an air bubble, (b) 3M formulation in quiescent, siliconized syringes with an air bubble, (c) 3M formulation in agitated, unsiliconized syringeswith an air bubble, (d) 3M formulation in agitated, siliconized syringes with an air bubble, and (e) 3M formulation in agitated, siliconized syringeswith no air bubble but with glass beads. Some examples of agglomerates of protein aggregates and silicone oil droplets are highlighted in whitein panel (d).



Figure 3. Particle concentrations in lysozyme formulations and buffer solutions incubated in PFS as a function of time. Open symbols correspondto syringes incubated with no air bubble, closed symbols correspond to syringes incubated with an air bubble, and cross symbols correspond tosyringes incubated with no air bubble but with glass beads. The particle concentrations in a buffer solution (solid black line) and in a lysozymesolution (dashed black line) that were not incubated in syringes are also shown. The incubation conditions are as follows: (a) phosphate buffer(no protein) in agitated, siliconized syringes, (b) lysozyme formulation in quiescent, siliconized syringes, (c) lysozyme formulation in agitated,unsiliconized syringes, and (d) lysozyme formulation in agitated, siliconized syringes.

Particle Concentrations in Lysozyme Formulations in PFSParticles 2 :m or greater were detected in lysozyme formula-tions and in a buffer solution incubated in PFS under all condi-tions (Fig. 3). The trends in particle concentration were similarin lysozyme formulations as in 3M formulations, and the im-ages of particles in lysozyme formulations were similar to thosefor 3M formulations (Supplemental Information Fig. S3). Thelowest levels of particles were detected in a buffer solution inagitated, siliconized syringes (Fig. 3a), with slightly higher lev-els in lysozyme formulations incubated in siliconized syringesthat were not agitated (Fig. 3b). Lysozyme formulations in-cubated in agitated, unsiliconized syringes also contained low

levels of particles (Fig. 3c), but the particle concentrations didnot change over time.

Lysozyme formulations incubated in agitated, siliconized sy-ringes with an air bubble contained the greatest number ofparticles (Fig. 3d), and the particle concentrations increasedwith time. As with 3M formulations, lysozyme formulationsincubated in agitated, siliconized syringes with no air bub-ble exhibited particle concentrations 1–2 orders of magnitudelower than lysozyme formulations incubated in agitated, sili-conized syringes with an air bubble (Fig. 3d). The addition ofglass beads to lysozyme formulations incubated in agitated, sil-iconized syringes with no air bubble increased the number of



particles detected (Fig. 3d; cross symbols) in the solution, butthese particle concentrations remained lower than the particleconcentrations in lysozyme formulations incubated in agitated,siliconized syringes with an air bubble.

Contact Angle and Interfacial Tension Measurements

The contact angle of a 1 :L droplet of 1 mg/mL 3M formulationon a siliconized glass slide is 98◦ ± 1◦. The interfacial ten-sion of the silicone oil–water interface with 3M adsorbed is ca.30 mN/m after ca. 40 s (Supplemental Fig. S2). The interfa-cial tension of the air–water interface with 3M adsorbed is ca.65 mN/m after ca. 40 s (data not shown).

DISCUSSION

Particle Generation in Protein Formulations in PFS

Particles (≥2 :m) were detected in all formulations underall incubation conditions in PFS. Even protein-free buffer so-lutions incubated in agitated, siliconized syringes containeda baseline level of particles with a concentration of about1000 particles/mL. However, roughly three orders of magni-tude more particles were generated in protein formulations in-cubated in agitated, siliconized syringes in the presence of anair bubble. The increase in particle concentrations was seen inboth formulations tested.

There was a marked difference in the particle concentra-tions in protein formulations incubated with and without anair bubble in agitated, siliconized syringes. Without an air bub-ble, the particle concentrations remained between 1000 and10,000 particles/mL, but the particle concentrations were or-ders of magnitude higher when an air bubble was present. Onepotential explanation for the increases in particle concentrationin the presence of an air bubble is that bulk fluid shear forcesinduced by the movement of the air bubble displaced proteinaggregates and silicone oil droplets from the syringe wall intothe bulk solution. To test this hypothesis, we added glass beadsto the syringe, while carefully avoiding the presence of air–water interfaces. As the syringe was rotated, the beads insidethe syringe barrel fell, resulting in mixing and bulk fluid shearforces near the silicone oil–water interface but without contri-butions from the air–water interface to shear. Although it wasdifficult to quantify their magnitude, we expected that the bulkfluid shear forces exerted by the glass beads (density ca. 2.5) asthey passed by the syringe walls were similar to or larger thanthose exerted by rising air bubbles. Thus, if bulk fluid shearforces and/or mixing were the dominant mechanisms for theproduction of particles, we would expect that large numbers ofparticles would be formed when samples were agitated in thepresence of glass beads. As seen in Figures 1d and 3d, the ad-dition of glass beads to siliconized syringes did result in higherparticle concentrations than those observed in syringes withoutbeads, but the particle concentrations were still significantlylower than those observed in siliconized syringes agitated withan air bubble. Therefore, although bulk fluid shear forces andmixing resulting from air bubble movements may contribute toparticle generation they are not the major contributing factor.

The effect of bulk fluid shear induced by the motion of the airbubble was also observed in protein formulations incubated inagitated, unsiliconized syringes. Similar to samples that wereincubated in siliconized syringes with no air bubble, the parti-cle concentrations in unsiliconized syringes with no air bubble

remained less than 10,000 particles/mL. The presence of anair bubble in the unsiliconized syringes increased the particleconcentrations but not to the same extent that an air bubbleincreased the particle concentrations in siliconized syringes.Therefore, some particle generation can be attributed to thebulk fluid shear forces due to the air bubble movement in thesyringe. However, the highest particle concentrations observedin this study consistently occurred when the air–water inter-face and the silicone oil–water interface were both present.

It is possible that more particles were generated in sili-conized syringes with an air bubble than in unsiliconized sy-ringes with an air bubble because the protein adsorbed tothe different interfaces with varying degrees of coverage. Theamount of protein required to achieve monolayer surface cov-erage of 3M adsorbed on glass in 10 mM L-histidine pH 5, mea-sured in this study, is 1.9 ± 0.4 mg/m2. The amount required toachieve monolayer surface coverage of 3M adsorbed on siliconeoil in 10 mM L-histidine pH 5, measured in previous work,3 is2.7 ± 0.6 mg/m2. These values are comparable with literaturevalues for proteins adsorbed to solid and fluid interfaces.33,34

Thus, there is only a modest difference in the surface cover-age of 3M on siliconized and unsiliconized surfaces, and thesubstantial difference in particle concentrations between sili-conized and unsiliconized syringes with an air bubble must beattributed to other factors.

The higher particle concentrations observed in siliconizedsyringes with an air bubble than in unsiliconized syringes withan air bubble might also be due to silicone oil droplets alonesloughing off the syringe wall into the bulk solution. However,the images of particles captured by FlowCAM clearly showedthat the particles did not consist of silicone oil droplets only.

In addition to the presence of the silicone oil–water inter-face and the air–water interface, agitation was also requiredto yield the highest particle concentrations. In protein formu-lations incubated in siliconized syringes with no agitation, theparticle concentrations remained at the baseline level of 1000–10,000 particles/mL, and no increase was seen when an air bub-ble was added. Therefore, movement of the air bubble along thesiliconized syringe wall was integral to the generation of parti-cles in PFS. This was consistent with a previous study wherethe combination of agitation, air–water interfaces, and siliconeoil–water interfaces induced more protein aggregation any onestress alone.22

Composition of Particles Detected in PFS

It has been suggested that the particles detected in PFS pri-marily consist of silicone oil droplets and that silicone oil hasminimal impact on proteins within the formulations, in partic-ular if the formulation contains surfactant.5,21,23 Droplets of sil-icone oil characteristically appear in flow microscopy images asspherical shapes.14–16 Images with such spherical shapes wereobserved under all conditions for formulations incubated in sil-iconized syringes (Fig. 2 and Supplemental Fig. S3). However,in all of the formulations that contained protein, the imagescollected during flow microscopy analysis also clearly showednonspherical particles characteristic of protein aggregates. Themost interesting images came from protein formulations agi-tated in siliconized syringes in the presence of an air bubble(Fig. 2d). In addition to the particle images that appeared to re-flect primarily spherically shaped silicone oil droplets or non-spherical protein aggregates, there were images of particles



that consisted of silicone oil droplets apparently coated withaggregated protein. Furthermore, several images showed largeagglomerations of protein-coated silicone oil droplets (whitehighlights in Fig. 2d). These images are similar to the agglom-erates of protein aggregates and silicone oil droplets observedin an abatacept formulation incubated in PFS9 and in an IFN-$-1a product formulated in PFS.20 Thus, we conclude that thehigh particle count observed after agitation of protein formu-lations in siliconized syringes cannot be ascribed solely to thesloughing off of silicone oil droplets; some particles consist ofaggregated protein as well.

The presence of aggregated protein in a therapeutic productis a major concern because protein aggregates have been shownto elicit an immune response.35–38 Therefore, it is essential toknow if the particles detected in a therapeutic product consistof protein aggregates. Several recent studies14–16 have proposeddigital filtering techniques based on image analysis to differen-tiate between particles composed of silicone oil droplets andparticles composed of protein aggregates. In each of these stud-ies, pre-formed silicone oil droplets were mixed with pre-formedprotein aggregates. These mixtures of silicone oil droplets andprotein aggregates were analyzed by microflow imaging, andthe resulting images were sorted according to various shapeand image intensity criteria, allowing separate populations ofsilicone oil droplets and protein aggregates to be distinguishedand counted. In contrast, the samples in the current study wereformed during incubation of protein formulations in siliconizedPFS. Particles composed of both silicone oil droplets and ag-gregated protein can clearly be seen in the microflow images.However, the images also suggest that many particles consist ofagglomerates of aggregated protein and silicone oil droplets inaddition to particles that are predominantly composed of justsilicone oil or just protein. Therefore, current digital filteringtechniques are not sufficient to classify the particles detectedin this study, and further refinement of these techniques isnecessary to accurately categorize particles consisting of bothsilicone oil and protein.

Proposed Mechanism of Particle Generation in Siliconized PFS

In this study, the highest particle concentrations in proteinformulations in PFS occurred when three elements were allpresent: (1) silicone oil–water interfaces, (2) air–water inter-faces, and (3) agitation. Agitation, in the form of end-over-endrotation, caused the air bubble to move inside the syringe. How-ever, the air bubble movement was not the same in siliconizedand unsiliconized syringes (Fig. 4). In unsiliconized syringes,the air bubble moved from one end of the syringe to the otherend in less than 1 s when the syringe was rotated. In sili-conized syringes, the air bubble moved slowly, requiring about5 s to move along the syringe wall when the syringe was ro-tated. Movies of the air bubble movement are included in Sup-plemental Information. Movement of the air bubble along thesiliconized syringe wall is where the three key elements cometogether, and we hypothesize that this movement is the pri-mary cause of particle generation in siliconized syringes withan air bubble.

Proteins readily adsorb to hydrophobic surfaces,26,32,39 suchas silicone oil, and are known to form a viscoelastic gel at thesesurfaces.27–29 When a protein formulation (without surfactant)is filled into a siliconized syringe, the protein immediately be-gins to adsorb to the siliconized syringe wall. At the protein

Figure 4. In an unsiliconized syringe, the air bubble moves quicklythrough the center of the syringe (a). In a siliconized syringe, the airbubble moves slowly along the syringe wall (b).

concentration used in this work, the adsorbed protein layershould form a gel within seconds (Fig. 5).40 Because an air bub-ble is introduced into the syringe during the filling and stop-pering process, a three-phase contact line is created where theair–water interface, the silicone oil–water interface, and thesilicone oil–air interface meet (Fig. 6). The contact angle at thisthree-phase contact line is determined by a balance of the in-terfacial tensions of each of the three interfaces present.41 Thevalue of the interfacial tension at each interface depends onhow much protein is adsorbed to that interface; more adsorbedprotein decreases the interfacial tension.

When the syringe is subjected to agitation (end-over-end ro-tation in this study), the air bubble moves along the siliconizedsyringe wall. During this motion, the gelled protein layer atthe silicone oil–water interface is disrupted. Capillary forcesat the three-phase (silicone oil–water–air) contact line are hy-pothesized to pull on the gelled protein layer causing the gel tofragment and release gelled protein particles from the siliconeoil–water interface into the bulk solution (Fig. 5). In addition,as seen in Figure 2, not only are gelled protein aggregates re-moved from the interface, but silicone oil is displaced as wellbecause the protein is adsorbed to silicone oil. Therefore, pro-tein aggregates, silicone oil droplets, and agglomerates of pro-tein aggregates and silicone oil droplets are all detected in thebulk formulation. In addition, the particles detected in pro-tein formulations in agitated, siliconized syringes with an airbubble do not change size over time (data not shown). This isconsistent with an interfacial mechanism of particle generationwhere particles are removed from the interface already aggre-gated rather than a mechanism where protein aggregates arenucleated from an aggregation-prone species and then grow inthe bulk.

Upon the removal of some of the adsorbed protein from thesilicone oil–water interface, the interfacial tension at that in-terface increases. Thus, the interfacial tension at the siliconeoil–water interface at the receding edge of the bubble is ex-pected to be larger than the interfacial tension at the advancingedge of the bubble, where gelled protein is being removed. Thisis consistent with observations that the advancing contact an-gle (measured through the liquid) is smaller than the recedingcontact angle because of the difference in the amount of pro-tein adsorbed at the two edges (Fig. 6). Typically, differencesbetween advancing and receding contact angles are attributed



Figure 5. Proposed mechanism of particle generation in protein for-mulations in agitated, siliconized syringes with an air bubble. Whenthe syringe is filled, the protein molecules adsorb to the silicone oil–water interface and gel (1). Agitation of the syringe causes the airbubble in the syringe to move. When the air bubble moves, gelled pro-tein aggregates and silicone oil are removed from the interface to thebulk due to the capillary forces at the advancing edge three-phase con-tact line (2). The removal of protein and silicone oil leaves space onthe interface for more protein molecules to adsorb and gel (3), and theprocess repeats every time the air bubble moves along a section of theinterface.

to surface roughness at the sub-microscopic level.41 However,in this system, silicone oil presents an essentially defect-free,smooth liquid interface. Thus, the differences in contact anglebetween the advancing and receding edges of the bubble cannotbe the result of surface roughness. Instead, they are due to adifference in interfacial tension over the length of the bubble,which we ascribe to corresponding differences in the concentra-tions of adsorbed protein along the interface.

Figure 6 shows the balance of the interfacial tensions paral-lel to the syringe wall at the three-phase contact lines at theadvancing and receding edges of the air bubble. This balanceof interfacial tensions in the parallel direction is determinedby applying the Young equation at the advancing edge of thebubble (indicated by subscript a) and at the receding edge ofthe bubble (indicated by subscript r):

γ SO−air − γ SO−liq,a = γ air−liq,a · cos(2a) (1a)

γ SO−air − γ SO−liq,r = γ air−liq,r · cos(2r) (1b)

The silicone oil–air interfacial tension ((SO−air) is20.9 mN/m.42 At the silicone oil–water and the air–water in-terfaces, the interfacial tensions will depend on how much pro-tein is adsorbed to those interfaces. The interfacial tensions atthe advancing edge of the bubble where protein is adsorbed areestimated based on the values measured by the pendant droptechnique after ca. 40 s of adsorption time (corresponding tothe time required for one syringe rotation): ca. 65 mN/m forthe air–water interfacial tension ((air−liq,a) and ca. 30 mN/m forthe silicone oil–water interfacial tension ((SO−liq,a). Using theseinterfacial tension values, the contact angle at the advancingedge (θa) is calculated to be 98◦. An approximate value of thiscontact angle also can be measured using the contact angleat the three-phase contact line of a 1 :L droplet of 1 mg/mL3M solution on a siliconized glass slide. This measured angle(measured through the liquid) is 98◦ ± 1◦, consistent with thecalculated value of θa. The interfacial tensions at the recedingedge of the bubble are estimated based on their values whenno protein is adsorbed: 72 mN/m for the air–water interface((air−liq,r)41 and 45.5 mN/m for the silicone oil–water interface((SO−liq,r).42 Thus, the receding contact angle at the trailing edgeof the bubble (2r, measured through the liquid) is calculatedfrom the Young equation to be 110◦ if all adsorbed protein isremoved from that interface. The exact values of the interfa-cial tensions and the contact angle at the receding edge of thebubble will depend on how much protein is actually removedfrom that interface, which cannot be measured directly insidethe syringe. However, visual observations of the air bubble inthe syringe are consistent with a receding contact that is largerthan the advancing contact angle.

At the three-phase contact line, there is also a componentof the air–liquid interfacial tension that is perpendicular tothe syringe wall (Fig. 6). Using the approximate diameter ofa monoclonal antibody (dprotein = 12 nm), we can estimate theforce F perpendicular to the syringe wall acting on one proteinmolecule at the advancing edge three-phase contact line:

F = γ air−liq,a · sin(2a) · dprotein (2)

Using the estimated air–water interfacial tension of65 mN/m and a value θa = 98◦, we obtain a value for F of



Figure 6. Schematic of the contact angles at the advancing and receding edges of the air bubble (top), and schematic of the interfacial tensionsat the three-phase contact lines at the advancing and receding edges of the air bubble (bottom). The balance of the interfacial tensions parallelto the syringe wall at the advancing and receding edges of the bubble are determined by the Young Equation (Eq. 1a). The interfacial tensionand contact angle values are defined in the text. The subscript a corresponds to the advancing edge of the bubble. The subscript r corresponds tothe receding edge of the bubble. The three phase contact line at each edge is indicated by the black dot in each image. There is also a componentof the air–liquid interfacial tension that is perpendicular to the syringe wall. The force acting perpendicular to the syringe wall pulling on oneprotein molecule at the advancing edge three-phase contact line is ca. 770 pN (calculations shown in the text).

770 pN, which is on the order of the magnitude of the force re-quired to mechanically unfold a protein as measured by atomicforce microscopy.43 Therefore, it is likely that a force of thismagnitude is strong enough to fragment a gelled protein layerand pull gelled protein particles off the interface.

After the air bubble passes over a section of the siliconizedsyringe wall and removes some of the adsorbed protein, spacebecomes available on the silicone oil interface for more proteinto adsorb from the bulk. This newly adsorbed protein gels, andwhen the air bubble passes by again, more protein aggregatesand silicone oil droplets are removed from the interface andinto the bulk. Thus, the particle concentration in siliconizedsyringes with an air bubble continues to increase with time,as was observed in this study (Figs. 1d and 3d). This interfa-cial mechanism of particle generation in siliconized syringesis summarized in Figure 5. In unsiliconized syringes, particlegeneration does not occur in this manner because the air bub-ble does not move along the syringe wall and interact with theadsorbed protein layer. Instead, the air bubble moves quicklythrough the center of the syringe. Thus, the gelled protein layeris not disrupted.

Interfaces have often been suggested to play a major rolein protein aggregation and particle generation in protein

formulations.44–46 However, it is not simply the presence of aninterface that causes particle generation. In the PFS used inthis study, the silicone oil–water interfacial area available forprotein to adsorb is about 10 cm2/syringe. Based on the surfacecoverage of the antibody 3M on silicone oil,3 less than 3 :gof 3M can adsorb to this interfacial area. This is a negligibleamount of protein compared with the bulk protein concentra-tion in the syringe (1 mg/mL). Furthermore, in the absence ofagitation, any protein aggregates that form on the silicone oil–water interface would be expected to stay at the interface andnot desorb into the bulk. Single molecule tracking experimentsusing total internal reflectance fluorescence microscopy showthat the surface residence times of protein aggregates on thesilicone oil–water interface increase with increasing aggregatesize.47,48 However, based on the mechanism proposed above,during agitation, adsorbed protein will be removed from theinterface multiple times, and the subsequent replenishment ofthe adsorbed layer by protein from the bulk will eventually leadto significantly more than just 3 :g of protein interacting withthe silicone oil–water interface.

Therefore, although the presence of an interface is necessaryfor a protein gel layer to form, the disruption of the interfacialprotein gel layer is the key factor in generating particles. The



effect of disrupting this layer is apparent in the 1–2 orders ofmagnitude difference between the particle concentrations inprotein formulations with an air bubble in quiescently incu-bated syringes and in agitated, siliconized syringes (Figs. 1b,1d, 3b, and 3d; closed symbols). The presence of an air bub-ble has minimal effect on the particle concentrations when itdoes not move within the syringe. In addition, there are twoorders of magnitude less particles in protein formulations inagitated, siliconized syringes with no air bubble than in agi-tated, siliconized syringes with an air bubble (Figs. 1d and 3d;open and closed symbols). Without movement of the air bubble,the protein gel layer at the silicone oil–water interface is notdisrupted, and the particle concentrations do not increase.

A disruption of the interfacial protein gel layer has beenshown to generate particles in other systems, as well. Tearingand detachment of a gelled protein layer was observed whena Langmuir trough was used to expand and compress the air–water interfacial area.49 In Bee et al.,6 a protein formulationwas exposed to an air–water interface that was repeatedly ex-panded and compressed. Because of the continual expansionand compression of the air–water interface, the gelled proteinlayer at that interface was disrupted which released particlesinto the bulk protein solution.6 In another air–water interfacestudy,7 a needle was inserted through the air–water interfaceof a protein solution. This mechanical disturbance disruptedthe gelled protein layer at the air–water interface and releasedparticles into the protein solution. Therefore, we conclude thata major cause of particle generation in protein formulations isa disruption of the protein layer that adsorbs and gels at aninterface present in the system. In the current study, the gelledprotein layer is disrupted due to the air bubble movement in-duced by end-over-end rotation of the syringe. However, airbubble movement in the syringe may be induced by a variety oftransportation-associated stresses, and it is not necessary thatthe air bubble move along the entire length of the syringe inorder to generate particles.

CONCLUSIONS

This study measured the particle concentrations in two differ-ent protein formulations after rotation and quiescent incuba-tion in siliconized and unsiliconized glass syringes in the pres-ence and absence of an air bubble. Agitated, siliconized syringeswith an air bubble induced the greatest particle generation inboth protein formulations tested, and the particles observedin this condition consisted of protein aggregates, silicone oildroplets, and agglomerates of protein aggregates and siliconeoil droplets. Although bulk shear forces due to the rotation ofthe air bubble caused some particle formation, they were notthe primary source of particle generation in the agitated, sili-conized syringes with an air bubble. Instead, capillary forces atthe three-phase contact line caused particle generation and ex-plained the synergistic effects of silicone oil–water interfaces,air–water interfaces, and agitation on particle generation inPFS.

Consistent with the mechanism proposed above, there aretwo options to consider for reducing the number of particles insiliconized syringes with an air bubble. One option is to add asurfactant (such as polysorbate 20) to the protein formulation.Because they are surface active, surfactants can lower the in-terfacial tension and are commonly used in the pharmaceutical

industry to minimize protein adsorption to interfaces.45,50 Inaddition, surfactants have also been shown to inhibit gelationof adsorbed protein layers.29 The second option involves the sil-icone oil coating on the syringe wall. The coating should containthe least amount of silicone oil necessary to provide the desiredlubrication and should be strongly adhered to the syringe wallto minimize the amount of silicone oil that can slough off. Fu-ture experiments are planned to investigate these two optionsfor mitigating the number of particles in protein formulationsin PFS.

ACKNOWLEDGMENTS

The authors gratefully acknowledge support from MedIm-mune, Inc. and the National Science Foundation award #CBET-1133871.

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32. Aggregation and Particle Formation of Therapeutic Proteins in Contact With a Novel Fluoropolymer Surface Versus Siliconized Surfaces: Effects of Agitation in Vials and in Prefilled Syringes

33. Characterization of Protein Aggregating Tungstates: Electrospray Mass Spectrometry Analysis of Extracts from Prefilled Syringes and from Tungsten Pins Used in the Manufacture of Syringes

34. Competition between protein aggregation and protein complex formation

35. Differential effects of glycation on protein aggregation and amyloid formation

36. Particle-based simulation of ellipse-shaped particle aggregation as a model for vascular network formation

37. Impact of Sterilization Method on Protein Aggregation and Particle Formation in Polymer-Based Syringes

38. INOSITOL DERIVATIVES AND THEIR USES IN THE TREATMENT OF DISEASES CHARACTERIZED BY ABNORMAL PROTEIN FOLDING OR AGGREGATION OR AMYLOID FORMATION, DEPOSITION, ACCUMULATION OR PERSISTENCE

39. Bio-nanoimaging || Polymorphism in Casein Protein Aggregation and Amyloid Fibril Formation

40. Protein Aggregation and Pore-Formation of a Neurodegenerative Protein Fragment

41. IgG\r 1\r aggregation and particle formation induced by silicone-water interfaces on siliconized borosilicate glass beads: A model for siliconized primary containers

42. Effects of Phosphate Buffer in Parenteral Drugs on Particle Formation from Glass Vials

43. Impact of Sterilization Method on Protein Aggregation and Particle Formation in Polymer-Based Syringes

44. Characterization of antibody aggregation: Role of buried, unpaired cysteines in particle formation

45. Detecting Protein Aggregation on Cells Surface: Concanavalin A Oligomers Formation

46. From Protein Structure to Function with Bioinformatics || Prediction of Protein Aggregation and Amyloid Formation

47. Mechanistic complexity of subvisible particle formation: Links to protein aggregation are highly specific

48. Differential effects of glycation on protein aggregation and amyloid formation

49. Aggregation drives “misfolding” in protein amyloid fiber formation

50. Inhibition of protein aggregation and amyloid formation by small molecules
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