`
`Distribution of Silicone Oil in Prefilled Glass Syringes
`Probed with Optical and Spectroscopic Methods
`
`ZAI-QING WEN1*, AYLIN VANCE1, FABIAN VEGA2, XIAOLIN CAO1, BRUCE EU2,
`and ROBERT SCHULTHESIS2
`
`1Department of Formulation and Analytical Resources; and 2Department of Drug Delivery Engineering, Amgen Inc.,
`Thousand Oaks, CA 91320 ©PDA, Inc. 2009
`
`ABSTRACT: Prefilled glass syringes (PFSs) have become the most commonly used device for the delivery of
`recombinant protein therapeutics in parenteral formulations. In particular, auto-injectors preloaded with PFSs greatly
`facilitate the convenient and efficient self-administration of protein therapeutics by patients. Silicone oil is used as
`a lubricant in PFSs to facilitate the smooth motion of the plunger during injection. However, there have been few
`sophisticated analytical techniques that can qualitatively and quantitatively characterize in-situ the morphology,
`thickness, and distribution of silicone oil in PFSs. In this paper, we demonstrate the application of three optical
`techniques including confocal Raman microscopy, Schlieren optics, and thin film interference reflectometry to
`visualize and characterize silicone oil distribution in PFS. The results showed that a container coating process could
`produce unevenly distributed silicone oil on the glass barrel of PFSs. An insufficiency of the amount of silicone oil
`on the glass barrel of a PFS can cause stalling when the device is preloaded into an auto-injector. These analytical
`techniques can be applied to monitor the silicone oil distribution in PFSs.
`
`KEYWORDS: Silicone oil, Prefilled glass syringe, Raman microscope, Interference reflectometry, Zebra Schlieren,
`Protein formulation, Drug delivery device.
`
`Introduction
`
`Prefilled glass syringes (PFSs) have become the pri-
`mary container for protein therapeutics in parenteral
`formulation in recent years (1, 2). They offer several
`advantages over conventional primary containers such
`as glass vials. For example, PFSs eliminate several
`steps that are needed to inject a drug from the vial into
`a patient. They contain the exact dose to be injected,
`unlike in vials, which often are overfilled by up to 25%
`to ensure that the desired dose will be available for
`withdrawal and delivery, which may in turn lead to
`dosing errors. The precisely filled dose in PFSs also
`reduces the waste of precious protein therapeutics.
`The majority of protein therapeutics on the market is
`now contained in PFSs. Moreover, PFSs have been
`incorporated into auto-injectors for self administration
`of the drug by patients and for use in clinical centers
`for convenience and needle safety (3).
`
`* Author for correspondence: email: zwen@amgen.com
`
`In contrast to the many benefits PFSs present to pa-
`tients and physicians, the change of primary container
`from glass vials to PFSs poses great challenges for
`formulation scientists and device and packaging engi-
`neers in the biopharmaceutical industry. The degree of
`complexity of a PFS and the auto-injector pre-loaded
`with a PFS is significantly increased compared to that
`of simple glass vials. Protein therapeutics in particular
`pose potential interactions between the protein and the
`additional materials that comprise the PFS. We have
`observed visible particulates that have formed due to
`the interaction of protein products with foreign matters
`left in PFSs during the PFS manufacturing process (4).
`For instance, metals used to make the syringe needle
`may generate iron rust, which could be hazardous to
`protein products. Residual tungsten left in PFS inter-
`acts with the protein to form visible particles and
`generate defects. Silicone oil is an essential material
`used as a lubricant for proper movement of the plunger
`in PFS. In contrast, vials are essentially free of sili-
`cone oil except for an extremely small quantity around
`the vial stopper for assembly purposes. Silicone oil
`has been observed to induce turbidity and particulate
`formation (5, 6) in insulin and other proteins (7).
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`the performance of an auto-injector de-
`Moreover,
`pends on the reduction of friction in the PFS using
`silicone oil.
`
`It is, therefore, important to characterize the distribu-
`tion of silicone oil in PFSs and to understand the
`complex interaction between protein therapeutics and
`silicone oil used in PFSs. In the present study, we
`investigate silicone oil distribution and its correlation
`with the function of PFSs and auto-injectors by using
`a number of optical techniques including optical mi-
`croscopy, confocal Raman microscopy (8), Schlieren
`optics (9), and interference reflectometry (10). The
`results demonstrate that silicone oil could be unevenly
`distributed on the glass barrel of the PFS, or in ex-
`treme cases it could be completely absent in certain
`areas. The deficiency of and uneven distribution of
`silicone oil can result in the stalling of a PFS in an
`auto-injector. On the other hand, external force placed
`on excessive amounts of silicone oil localized in spe-
`cific areas on the PFS can generate silicone oil micro-
`droplets and contribute to particulate formation asso-
`ciated with proteins. These findings should be helpful
`to PFS manufacturers for improving the silicone oil
`coating process in PFSs and improving the overall
`quality of PFSs used in auto-injectors.
`
`Experimental Design
`
`Materials
`
`PFSs with 1.0-mL barrels were obtained from a vendor
`without further treatment. (BD, Franklin Lakes, NJ)
`Syringes from three lots were selected as samples.
`Two lots were coated with silicone oil. However, one
`lot coated with silicone oil showed poor performance
`when the syringes were loaded onto the auto-injector
`for auto-injection testing. The other lot exhibited good
`performance. The third lot of syringes was not coated
`with silicone oil and was used to provide control
`samples. None of the syringes in these three lots were
`filled with product unless it was otherwise noted that
`a product-filled syringe had stalled during auto-injec-
`tion testing. The product in the syringe was not emp-
`tied when it was examined using the Schlieren optical
`method.
`
`Methods
`
`We employed three optical techniques to visualize the
`silicone oil and measure in-situ the thickness of the
`silicone oil droplets. A confocal Raman microscope
`
`and Schlieren optics were used to observe the silicone
`oil droplets on the glass barrel. The Raman spectrum
`of silicone oil was used to confirm its identity. A
`visible reflectometry based on the light interference of
`transparent thin film (10) was applied to determine the
`thickness of silicone oil and its distribution along the
`longitudinal direction of the syringe barrel. The sili-
`cone oil coated on prefilled syringes could be treated
`as transparent thin film.
`
`Optical Microscope: A Carl-Zeiss stereomicroscope
`(Stemi 2000C, Carl-Zeiss, Thornwood, NY) was em-
`ployed to take micrographs of the empty glass sy-
`ringes.
`
`Confocal Raman Microscope: The optical micro-
`scope on the confocal Raman microscope (Senterra威,
`Bruker Optics, Billerica, MA) was employed to exam-
`ine the silicone oil distribution, and the Raman module
`was used to confirm the identity of the silicone oil.
`Three objective lenses attached to the nosepiece with
`20⫻, 50⫻ and 100⫻ magnification, corresponding to
`a spatial size of 5, 2, and 1 m, respectively. Lower
`magnification objective lenses have a larger field of
`view and working distance, which enables ease of
`observation and sample handling. The total magnifi-
`cation is determined by the product of the objective
`lens and binocular eyepieces (10⫻). The syringe was
`placed on a XY-stage, which is precisely controlled by
`micrometers on the microscope. The light beam pro-
`jected onto the sample syringe was vertically focused
`with a 4⫻ objective lens.
`
`The Raman spectrometer module accommodates two
`lasers for exciting Raman scattering: a red 785-nm
`diode laser and a green 532-nm green solid laser to
`facilitate the measurement of samples with different
`scattering natures. Variable laser power can be se-
`lected depending upon the sample signal. The maxi-
`mum laser power is 100 mW for the 785-nm laser and
`20 mW for the 532 nm laser. The spectral coverage is
`from 70 cm⫺1 to 3700 cm⫺1 with a spectral resolution
`of 4 cm⫺1. Two special functions were implemented
`on the Raman microscope: the FlexFocus™ for con-
`focal Raman detection and SureCal威 for continuous
`automated frequency calibration. Both play an impor-
`tant role in the microscope’s performance. The confo-
`cal function provides a significant
`improvement
`in
`both the contrast and the spatial resolution in the
`vertical (z) axis. The properly adjusted confocal opti-
`cal configuration offers the capability of “optical sec-
`tioning” and depth profiling of heterogeneous and thin
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`Zebra strip
`
`Syringe Lamps
`
`Optical Microscope Camera
`
`Figure 1
`
`Schematic of a Zebra Schlieren optic set up for visualization of silicone oil in PFS. The light emitted from lamps
`is projected toward the Zebra strip passing through the syringe standing on the holder. The reflected light from
`the syringe is collected by the microscopic lens and a picture is then taken by a digital camera.
`
`specimens, which is crucial for the successful obser-
`vation of silicone oil micro-droplets on the glass barrel
`of PFSs.
`
`Zebra Schlieren Visualization: A Zebra Schlieren
`optics technique was developed to visualize silicone
`oil on the glass syringe barrel. This technique was
`used to observe the optical inhomogeneity in transpar-
`ent media that
`the human eye cannot directly see
`because of an extremely small difference of refractive
`index (9). It exploits the Schlieren phenomena in solid,
`liquids, and gases that have refractive-index gradients
`in one, two, and three dimensions due to inhomoge-
`neity or resulting from temperature change, high-
`speed flow, or the mixing of dissimilar media. This is
`a qualitative visualization method to display the opti-
`cal
`inhomogeneity of a material system. Schlieren
`optics has been widely employed in many sciences and
`engineering fields,
`including the detection of gas
`leaks, jet stream, and liquid flow (9). However, to the
`best of our knowledge,
`this is the first
`time that
`Schlieren optics has been applied to view silicone oil
`droplets on PFSs. The PFSs coated with silicone oil
`
`have a refraction-index gradient along the glass sy-
`ringe barrel due to the presence of silicone oil drop-
`lets. Both silicone oil and the glass barrel are trans-
`parent
`to visible light; however,
`the density and
`refractive index of silicone oil and glass differ from
`each other in that the silicone oil used in PFSs as a
`lubricant has a refractive index of ⬃1.42 at 20 °C,
`while the refractive index of the glass barrel is close at
`⬃1.5. The PFS coated with silicone oil constitutes a
`Schliere. The small difference between the refractive
`index of the silicone oil coated on the boronsilica glass
`barrel makes it difficult to see the silicone oil with the
`naked eye or under a low-resolution optical micro-
`scope.
`
`The optical set-up to observe a Schliere is fairly sim-
`ple, yet it can visualize the silicon oil distribution over
`the PFS in a practical and dramatic manner. Figure 1
`shows a schematic of the optical configuration for the
`Schlieren visualization of silicone oil on the glass
`barrel. A zebra strip pattern was placed in front of a
`screen. This is the key component allowing the
`Schlieren optics to dramatically reveal micron-size
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`droplets of silicone oil on a glass barrel. A glass
`syringe was placed vertically on a syringe holder
`between the zebra strip and a high resolution digital
`optical microscope
`(model VHX-600, Keyence,
`Woodcliff Lake, NJ). Two projector fluorescence
`lamps were placed on the left and right sides of the
`digital microscope to illuminate the syringe. All other
`background light was minimized to reduce disruption.
`The images of the syringes on whose inner surfaces
`silicone oil had been distributed were taken with a
`digital camera attached to the optical microscope. All
`pictures were recorded on a computer without further
`processing.
`
`Interference Reflection Spectroscopy: A rap.ID
`Layer Explorer (rap.ID Particle Systems GmbH, Ber-
`lin, Germany), based on the principle of light interfer-
`ence of thin film and reflection spectrometry, was
`employed for the measurement of silicone oil thick-
`ness. The Layer Explorer employs a beam of white
`light that is projected onto the glass syringe, and the
`reflected light from the thin oil film on the syringe
`surface is collected with an optical fiber probe (10).
`The interference pattern generated by the interaction
`of white light with the thin silicone oil film was
`calculated to determine thickness. The instrument has
`a precisely controlled and automated XY-stage. The
`syringe was laid horizontally on the syringe holder,
`which is fixed on the XY-stage. The software auto-
`matically divides the length of the syringe along the
`longitudinal direction to the number of measurement
`points and moves automatically along the syringe and
`collects the data on each point. It takes only a few
`seconds to measure one point. The size of the spot
`measured was ⬃100 m. The detection limit of the
`thin film thickness was 50 nm. Below 50 nm the
`thickness was displayed as zero because the optical
`interference will not occur when the film thickness is
`much smaller than the wavelengths of visible light.
`
`Results
`
`Microscopic Image of Silicone Oil in PFS
`
`Figure 2 shows the layout of a glass syringe on the
`confocal Raman microscope’s XY-stage, this syringe
`having been selected from a “syringe lot” that showed
`low performance in the syringe injection test. The
`syringe was not filled with product solution. A total of
`30 images were taken of one syringe and are numbered
`accordingly from the plunger side to the needle end
`along the longitudinal direction. We selected 15 out of
`
`1, 2, 3
`Figure 2
`
`29, 30
`
`The layout of the syringe on the XY-stage of the
`Raman Microscope. Each number represents a
`1.5-mm length of a segment of the syringe barrel of
`which a micrograph was taken. A total of 30 mi-
`crographs were taken sequentially with 15 of them
`exhibited in Figure 3.
`
`the 30 images to show the silicone oil distribution on
`the barrel. Figure 3 shows the 15 individual images
`consecutively from the plunger side to the needle side
`of the glass barrel sections. Each picture corresponds
`to a 1.5-mm section of the syringe barrel. The silicone
`oil was seen as micro-droplets surrounded with a thin
`film layer, which under light illumination exhibited a
`typical thin film interference pattern. They resemble
`the Newton rings generated by a thin oil film on water
`under sunlight. These droplets and thin film layers
`were confirmed by Raman spectroscopy to be silicone
`oil. Figure 4 shows the Raman spectra of silicone oils
`on the syringe barrel and of a droplet of reference
`silicone oil on a glass slide. The Raman spectrum of
`the silicone oil droplets on the glass barrel was noisy,
`but the fingerprint pattern unambiguously confirmed
`that it was silicone oil.
`
`The most striking observation was that the distribution
`of silicone oil on the barrel syringe was unevenly
`spread from one end (plunger side) to the other (needle
`side) throughout the syringe barrel. For the syringe
`examined, the first few segments of the syringe barrel
`on the plunger side contained much more silicone oil
`than did segments in other areas. It can be seen that
`there are many small silicone oil droplets that are
`surrounded with the thin layer. However, towards the
`needle end, there were fewer oil droplets with no thin
`oil film surrounding them. The last few segments at
`
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`2
`
`4
`
`6
`
` 10
`
`12
`
`14
`
`16
`
` 18
`
`20
`
`22
`
`24
`
`Figure 3
`
`26
`
`28
`
` 30
`
`Micrographs of a glass syringe barrel obtained using the confocal Raman microscope. Each image from left to
`right represents a 1.5-mm section of the syringe barrel. An uneven distribution of silicone oil from the plunger
`side to the needle end can be seen.
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`Figure 4
`
`Raman spectra of silicone oil on the glass barrel of the PFS (top trace) and of a reference spectrum of silicone
`oil on a glass slide (bottom trace). The positive and negative spikes on the top trace are due to cosmic rays.
`
`the needle side contained the minimum amount of
`silicone oil, and some of the segments showed virtu-
`ally no silicone oil at all. This observation was con-
`sistent with the results of the toluene zone extraction
`of silicone oil obtained with size exclusion chroma-
`tography analysis (11) of syringes with poorly distrib-
`uted silicone oil, showing that the silicone oil is rich
`on the plunger section but poor at the needle-side
`section. They are also consistent with the results ob-
`tained using the gold nano-particle staining method,
`which demonstrated that, for unevenly coated PFSs,
`the amount of silicone oil was deficient at the needle
`end (12). A significant increase of extrusion force at
`the end of the injection was observed when unevenly
`coated PFSs were tested with an Instron Mechanical
`Testing Systems Model 5564 (Instron, Norwood,
`MA). This was attributed to the protein adsorption on
`bare glass surfaces that were deficient of silicone oil
`droplets (12).
`
`Zebra Schlieren Visualization of Silicone Oils
`
`Figure 5 presents images of three representative glass
`syringes that show different silicone oil distributions
`on the glass barrel as revealed by Schlieren optics. The
`N3 syringe was from the syringe lot
`that had no
`silicone oil coating, showing no silicone oil droplets at
`all. The P3 syringe represents the uniformly distrib-
`uted silicone oil on the barrel surface from the syringe
`
`lot that showed good performance in an auto-injection
`test. The A3 syringe shows an uneven distribution of
`silicone oil from the silicone oil-coated syringe lot that
`showed poor injection performance with an auto-in-
`jector. The silicone oil droplets can be clearly seen in
`the two syringes (A3 and P3) that have been coated
`with silicone oil. However, unusually large silicone oil
`droplets are seen (see arrow on A3 in Figure 5) on the
`syringe barrel that has uneven oil distribution. Some
`large silicone oil droplets are estimated to be a few
`millimeters in size, which is apparently too big for
`uniform coating. For the PFS with more uniform dis-
`tributions of silicone oil (P3), the droplets are smaller
`and more uniform. However, they are still seen as
`micro-droplets instead of as a uniform thin film. These
`results are consistent with the pictures obtained with
`the confocal Raman microscope. Schlieren optics,
`thus, provides a quick and dramatic visualization
`method for silicone oil distribution on glass barrels
`and could be developed into a screening technique for
`silicone oil distribution on PFSs for quality control
`and selecting PFSs for auto-injector assemblies. How-
`ever, Schlieren optics is not a quantitative analytical
`method that can determine the thickness of silicone
`oils. For that purpose, we resorted to interference
`reflectometry that can measure the thickness of sili-
`cone oil on a glass barrel, as described in the following
`section.
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`Figure 5
`
`Visualization of the silicone oil used in glass syringes. N3 is not coated with silicone oil. P3 is coated evenly with
`silicone oil. A3 is coated unevenly with silicone oil.
`
`Thickness of Silicone Oil
`
`Figure 6 shows the thickness distribution of silicone
`oil on two syringe barrels in the longitudinal direction
`as obtained using the rap.ID Layer Explorer. The two
`syringes were selected from the syringe lot
`that
`showed poor results in auto-injection testing. The
`thickness of the silicone oil on these two specific
`syringes varies from ⬃100 nm to ⬃700 nm, with a
`median thickness of about ⬃200 nm. However, some
`spots were as thick as 700 nm while other spots show
`no detectable silicone oil (the detection limit is 50
`nm). These quantitative data, in general, are consistent
`with the results obtained from the Schlieren visualiza-
`tion and confocal Raman microscopic data demon-
`strating that the silicone oil was unevenly distributed
`as droplets.
`
`The theoretical thickness of silicone oils on the barrel
`surface can be calculated with the assumption that a
`uniform layer was formed as follows:
`
`
`
`t ⫽ Ri ⫺ 共Ri2 ⫺ m/h兲1/ 2
`
`(1)
`
`where
`
`t is the thickness of silicone oils on the syringe barrel
`
`Ri is the inner diameter of the syringe barrel
`
` is the density of silicone oil
`
`h is the length of the syringe barrel
`
`For a glass syringe having a barrel length of 50 mm
`and an inner diameter of 6 mm that is coated uniformly
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`1
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`3
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`7
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`9 11 13 15 17 19 21 23 25 27 29 31 33 35
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`Position on Syringe
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`800
`
`700
`
`600
`
`500
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`400
`
`300
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`200
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`100
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`0
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`800
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`700
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`600
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`500
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`400
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`300
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`0
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`Thickness (nm)
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`Thickness (nm)
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`1
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`7
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`9 11 13 15 17 19 21 23 25 27 29 31 33 35
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`Position on Syringe
`
`Figure 6
`
`Silicone oil thickness profile along the longitudinal direction of two syringe barrels from plunger end (left) to
`needle end (right).
`
`with 0.4 mg of silicone oil (the thickness of silicone
`oil coating recommended by major PFS manufactur-
`ers), the mean thickness of the silicone oil would be
`⬃430 nm. This means that the silicone oil in some
`areas was nearly twice as thick as the mean value. On
`the other hand, few places showed a complete absence
`of silicone oil. The average measured thickness of 200
`nm was far less than the theoretical mean thickness of
`⬃430 nm. This again suggests that the silicone oil was
`not uniformly distributed. We also measured the thick-
`ness of silicone oil on the syringe barrel at four radial
`positions by rotating the syringe at 45, 90, 135, and
`180 degrees. The distribution pattern of silicone oil
`
`was found to be similar along the longitudinal direc-
`tion at these four angles (data not shown). However, if
`the syringe was placed horizontally for a period of
`time, the silicone oil migrated to the bottom of the
`syringe due to gravity. The thickness of the silicone oil
`at the bottom would be significantly greater than that
`at the glass barrel’s top and sides because the silicone
`oil was not covalently bound to the glass surface.
`
`It has been observed that some prefilled syringes pre-
`loaded onto the auto-injector can be stalled during
`auto-injection testing (12). The auto-injector testing
`for the spring force without loading the PFSs showed
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`consistent performance, which ruled out the malfunc-
`tion of
`the auto-injector. Furthermore, PFSs with
`fairly even distributions of silicone oil (as seen in P3
`in Figure 5) showed no stalling when they were as-
`sembled on an auto-injector and were tested for injec-
`tion. We surmised that the PFS stalling in auto-injec-
`tion was caused by the uneven distribution and
`deficiency of silicone oil on the syringe barrel. The
`stalling of PFSs preloaded onto the auto-injector was
`due to a lack of appropriate lubrication. Several lines
`of evidence support this hypothesis. First, the defi-
`ciency of silicone oils in certain sections on an un-
`evenly coated glass syringe, in particular towards the
`needle end, resulted in high friction force when they
`were tested with the Instron mechanical tester (12).
`Second, post-examination of a Protein X PFS where
`the auto-injector had stalled revealed that there are
`proteins adsorbed on the 1/5 section toward the needle
`end of the barrel. This constitutes indirect evidence of
`the deficiency of silicone oil on this specific section, as
`proteins would preferentially adsorb to glass surfaces
`that were not coated with silicone oil. Third, when two
`stalled PFSs preloaded on auto-injectors were disas-
`sembled and examined using the optical microscope
`and Schlieren visualization, they were found to have
`no silicone oil at all in the PFSs. Figure 7 shows such
`a picture of a stalled PFS that was filled with product
`and then removed from the auto-injector and disas-
`sembled. The picture was taken without removing the
`liquid content after disassembly. Examination using
`Zebra Schlieren optics clearly displays a minimum
`amount of silicone oil on the barrel. We also examined
`the syringe needle hole using a fine tungsten probe
`after the liquid content was removed and found that
`the needle hole was not clogged, this ruling out the
`possibility that a clogged needle had stalled the sy-
`ringe. Finally,
`the above evidence leads us to the
`conclusion that PFS without a silicone oil coating
`would not be able to function smoothly for injection.
`
`Concluding Remarks
`
`We have demonstrated three optical techniques, includ-
`ing confocal Raman microscopy, Schlieren optics, and
`interference reflectometery, as powerful tools to display
`silicone oil distribution, to confirm the identity of the
`coating, and to determine the thickness of silicone oil
`film on the glass barrel of PFS. The first two methods are
`qualitative yet very practical in visualizing silicone oil
`distribution on PFSs. In particular, Schlieren optics is
`very effective in directly observing the silicone oil drop-
`lets on PFSs. These methods may be utilized in a man-
`
`Figure 7
`
`A stalled syringe filled with protein-X solution dis-
`assembled from an auto-injector. Zebra Schlieren
`optics reveals an absence of silicone oil droplets on
`the glass barrel. The picture was taken with the
`liquid content in the syringe.
`
`ufacturing environment to pre-screen the quality of PFSs
`in regard to silicone oil uniformity and so may help to
`ensure the quality of PFSs intended for use in auto-
`injectors. Interference reflection spectrometry can pre-
`cisely measure silicone oil thickness and distribution on
`the glass barrel. All three techniques are non-destructive
`and easy to carry out, and together they offer a set of very
`effective optical tools to characterize silicone oil coat-
`ings on glass surfaces. All three techniques were able to
`show that the silicone oil coating on a syringe’s glass
`barrel was unevenly distributed, accounting for the poor
`performance of some glass syringes. A deficiency of
`silicone oil on the glass barrel can cause the plunger in a
`PFS to stall during injection using the auto-injectors. The
`availability of sophisticated analytical tools to character-
`ize the silicone oil on PFSs in situ can aid in creating a
`uniformly distributed silicone oil coating, which in turn
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`is expected to improve the performance of PFSs for
`protein pharmaceuticals.
`
`Acknowledgements
`
`We are grateful to rap.ID Particle Systems GmbH for
`the use of the Layer Explorer to measure silicone oil
`thickness; and to Drs. Robin Hwang and Ron Forster
`for constructive discussion during this work and Drs.
`Linda O. Narhi and David Brems for critically reading
`the manuscript.
`
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